Cell Journal (Yakhteh)

261-272

Authors: Parisa Sahranavardfard, Zahra Madjd, Amirnader Emami Razavi, Alireza Ghanadan, Javad Firouzi, Pardis Khosravani, Saeid Ghavami, Esmaeil Ebrahimie, Marzieh Ebrahimi

Objective Epithelial-mesenchymal transition (EMT) and the stemness potency in association with BRAF mutation are in dispensable to the progression of melanoma. Recently, microRNAs (miRNAs) have been introduced as the regulator of a multitude of oncogenic functions in most of tumors. Therefore identifying and interpreting the expression patterns of these miRNAs is essential. The present study sought to find common miRNAs regulating all three important pathways in melanoma development. Materials And Methods In this experimental study, 18 miRNAs that importantly contribute to EMT and have a role in regulating self-renewal and the BRAF pathway were selected based on current literature and cross-analysis with available databases. Subsequently, their expression patterns were evaluated in 20 melanoma patients, normal tissues, serum from patients and control subjects, and melanospheres. Pattern discovery and integrative regulatory network analysis were used to find the most important miRNAs in melanoma progression. Results Among 18 selected miRNAs, miR-205, -141, -203, -15b, and -9 were differentially expressed in tumor samples than normal tissues. Among them, miR-205, -15b, and -9 significantly expressed in serum samples and healthy donors. Attribute Weighting and decision trees (DT) analysis presented evidence that the combination of miR-205, -203, -9, and -15b can regulate self-renewal and EMT process, by affecting CDH1, CCND1, and VEGF expression. Conclusion We suggested here that miR-205, -15b, -203, -9 pattern as the key miRNAs linked to melanoma status, the pluripotency, proliferation, and motility of malignant cells. However, further investigations are required to find the mechanisms underlying the combinatory effects of the above mentioned miRNAs.

288-293

Authors: Saber Khazaei, Abbasali Khademi, Mohammad Hossein Nasr Esfahani, Mozafar Khazaei, Mohammad Hossein Nekoofar, Paul M.h. Dummer

Objective The aim of present study was to isolate and differentiate human adipose-derived stem cells (ASCs) into odontoblast-like cells. Materials And Methods In this experimental study, human adipose tissues were taken from the buccal fat pad of three individuals (mean age: 24.6 ± 2.1 years). The tissues were transferred to a laboratory in a sterile culture medium, divided into small pieces and digested by collagenase I (2 mg/mL, 60-90 minutes). ASCs were isolated by passing the cell suspension through cell strainers (70 and 40 µm), followed by incubation at 37ºC and 5% CO2in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS 5%) and penicillin/streptomycin (P/S). After three passages, the ASCs were harvested. Subsequently, flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect expression levels of NANOG and OCT4 to evaluate stemness. Then, a differentiation medium that included high-glucose DMEM supplemented with 10% FBS, dexamethasone (10 nM), sodium β-glycerophosphate (5 mM) and ascorbic acid (100 µM) was added. The cells were cultivated for four weeks, and the odontogenic medium was changed every two days. Cell differentiation was evaluated with Alizarin red staining and expressions of collagen I (COL1A1), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1). Results The ASCs were effectively and easily isolated. They were negative for CD45 and positive for the CD105 and CD73 markers. The ASCs expressed OCT4 and NANOG. Differentiated cells highly expressed DSPP, COL1A1 and DMP1. Alizarin red staining revealed a positive reaction for calcium deposition. Conclusion ASCs were isolated successfully in high numbers from the buccal fat pad of human volunteers and were differentiated into odontoblast-like cells. These ASCs could be considered a new source of cells for use in regenerative endodontic treatments.

303-312

Authors: Maesumeh Eslami, Shahrbanoo Oryan, Mehdi Rahnema, Mohammad Reza Bigdeli

Objective Choroid plexus epithelial cells (CPECs) have the epithelial characteristic, produce cerebrospinal fluid, contribute to the detoxification process in the central nervous system (CNS), and are responsible for the synthesis and release of many nerve growth factors. On the other hand, studies suggest that normobaric hyperoxia (HO) by induction of ischemic tolerance (IT) can protect against brain damage and neurological diseases. We examined the effect of combination therapy of encapsulated CPECs and HO to protect against ischemic brain injury. Materials And Methods In this experimental study, six groups of adult male Wistar rats were randomly organized: sham, room air (RA)+middle cerebral artery occlusion (MCAO), HO+MCAO, RA+MCAO+encapsulated CPECs, HO+MCAO+encapsulated CPECs, RA+MCAO+empty capsules. RA/HO were pretreatment. The CPECs were isolated from the brain of neonatal Wistar rats, cultured, and encapsulated. Then microencapsulated CPECs were transplanted in the neck of the animal immediately after the onset of reperfusion in adult rats that had been exposed to 60 minutes MCAO. After 23 hours of reperfusion, the neurologic deficit score (NDS) was assessed. Next, rats were killed, and brains were isolated for measuring brain infarction volume, blood-brain barrier (BBB) permeability, edema, the activity of superoxide dismutase (SOD), and catalase (CAT) and also, the level of malondialdehyde (MDA). Results Our results showed that NDS decreased equally in HO+MCAO, RA+MCAO+encapsulated CPECs, and HO+MCAO+encapsulated CPECs groups. Brain infarction volume decreased up 79%, BBB stability increased, edema decreased, SOD and CAT activities increased, and MDA decreased in the combination group of HO and transplantation of encapsulated CPECs in the ischemic brain as compared with when HO or transplantation of encapsulated CPECs was applied alone. Conclusion The combination of HO and transplantation of encapsulated CPECs for stroke in rats was more effective than the other treatments, and it can be taken into account as a promising treatment for ischemic stroke.

319-328

Authors: Mojtaba Eslami, Sahar Esfandyari, Marzieh Aghahossini, Zahra Rashidi, Shirzad Hosseinishental, Samane Brenjian, Aligholi Sobhani, Fardin Amidi

Objective Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes. Materials And Methods In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2O2) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity. Results This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05). Conclusion Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

335-340

Authors: Amir Hossein Jalali, Hossein Mozdarani, Hossein Ghanaati

Objective To evaluate the effect of contrast enhanced abdominopelvic magnetic resonance imaging (MRI), using a 3 Tesla scanner, on expression and methylation level of ATM and AKT genes in human peripheral blood lymphocytes. Materials And Methods In this prospective in vivo study, blood samples were obtained from 20 volunteer patients with mean age of 43 ± 8 years (range 32-68 years) before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced abdominopelvic 3 Tesla MRI. After separation of mononuclear cells from peripheral blood, using Ficoll-Hypaque, we analyzed gene expression changes of ATM and AKT genes 2 hours and 24 hours after MRI using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also evaluated methylation percentage of the above mentioned genes in before, 2 hours and 24 hours after MRI, using MethySYBR method. Results Fold change analysis, in comparison with the baseline, respectively showed 1.1 ± 0.7 and 0.8 ± 0.5 mean of gene expressions in 2 and 24 hours after MRI for ATM, while the results were 1.4 ± 0.6 and 1.4 ± 1 for AKT (P>0.05). Methylation of the ATM gene promoter were 8.8 ± 1.5%, 9 ± 0.6% and 9 ± 0.8% in before contrast enhanced MRI, 2 and 24 hours after contrast enhanced MRI, respectively (P>0.05). Methylation of AKT gene promoter in before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced MRI was 5.4 ± 2.5, 5 ± 3.2, 4.9 ± 2.9 respectively (P>0.05). Conclusion Contrast enhanced abdominopelvic MRI using 3 Tesla scanner apparently has no negative effect on the expression and promoter methylation level of ATM and AKT genes involved in the repair pathways of genome.

349-354

Authors: Nasrin Sereshki, Alireza Andalib, Mohadeseh Toghyani, Hossein Motedayyen, Mohammad Sadegh Hesamian, Abbas Rezaei, David Wilkinson

Objective The maternal immune response to paternal antigens is induced at insemination. We believe that pregnancy protective alloantibodies, such as anti-paternal cytotoxic antibody (APCA), may be produced against the paternal antigens in the context of stimulated immunity at insemination and that they increase during pregnancy. APCA is necessary for pregnancy. It is directed towards paternal human leucocyte antigens (HLAs) and has cytotoxic activity against paternal leucocytes. The present study aims to determine whether APCA is produced by the maternal peripheral blood mononuclear cells (PBMCs) in contact with the husband’s spermatozoa and to evaluate the relation of APCA production with HLA class I and II expressions by spermatozoa in fertile couples. Materials And Methods This cross-sectional study included 30 fertile couples with at least one child. The maternal PBMCs were co-cultured with the husband’s spermatozoa and the supernatant was assessed for the presence of IgG by ELISA. Cytotoxic activity of the supernatant on the husband’s PBMCs was assessed by the complement-dependent cytotoxicity (CDC) assay. Results IgG was produced in all co-cultures, and the mean level of supernatant IgG was 669 ng/ml. The cytotoxic activity of the supernatant was observed in all the supernatant obtained from the co-cultures. The mean percentage of APCA in supernatant was 73.93%. Conclusion Based on the results of this study it can be concluded that APCA may be a natural anti-sperm antibody (ASA), which can be produced during exposure to spermatozoa and may have some influence before pregnancy. Further research is required to determine the role of APCA before pregnancy.

366-366

Authors: Yan Gu, Zhanzhan Xiao, Jianlie Wu, Mingjin Guo, Ping Lv, Ning Dou

This article published in Cell J (Yakhteh), Vol 23, No 2, 2021, on pages 191-198, corresponding author and corresponding address were changed based on authors’ request. The authors would like to apologies for any inconvenience caused

382-388

Authors: Maryam Hezavehei, Bahare Shokoohian, Mohammad Hossein Nasr-Esfahani, Anastasia Shpichka, Peter Timashev, Abdolhossein Shahverdi, Massoud Vosough,

Coronavirus disease 2019 (COVID-19), as a severe respiratory disease, affects various tissues and organs. The specific SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), is highly expressed in male gonads. Thus, male reproductive tissues could be a potential target for virus colonization. We performed a comprehensive search in PubMed and Google Scholar to retrieve relevant articles published till 15 April 2021. The keywords used were: male fertility, male reproductive health, semen parameters, sex hormones, SARS-CoV-2, and COVID-19. Validated evidence about the adverse effects of the SARS-CoV-2 infection on the male reproductive system is limited and few studies have reported semen analysis results or presence of viral RNA in semen samples of infected men. Nevertheless, alterations in reproductive hormones such as decreased level of testosterone (T) with raised luteinizing hormone (LH) have been reported in some patients. Although the impact of SARS-CoV-2 infection on the male reproduction health remains unclear, evidence suggests that male gonads may be potentially vulnerable to SARS-CoV-2 infection. In this article, we discussed the possible impacts of COVID-19 on male gonads, sex hormones, and semen quality and suggested preventive solutions.

397-405

Authors: Esmat Ghalkhani, Mohammad Taghi Akbari, Pantea Izadi, Habibollah Mahmoodzadeh, Fatemeh Kamali

Objective: Metastasis might be latent or occur several years after primary tumor removal. Currently used methods for detection of distant metastasis have still some limitations. Blood tests may improve sensitivity and specificity of currently used screening procedures. The present study was designed to investigate promoter methylation status of DAPK1 and CAVIN3 genes in plasma circulating free DNA (cfDNA) samples in Iranian invasive ductal carcinoma (IDC) patients. We also investigated association of two gene promoter methylations with breast cancer (BC) and metastatic BC was also assessed. Materials and Methods: In this case-control study, MethySYBR assay was performed to determine DAPK1 and CAVIN3 promoter methylation status in breast IDC from 90 patients and 30 controls. Based on clinicopathological information, patient samples subdivided into stage I, II/III and IV groups (each group contained 30 individuals). Results: According to the results an increased promoter methylation level of the DAPK1 gene in BC patients was observed. It was found that as disease progressed, the percentage of methylation was changed while it was not significant. Methylation changes in metastatic and non-metastatic BC revealed that methylation levels were significantly increased in metastatic than non-metastatic group. Analysis revealed that promoter methylation of CAVIN3 gene in BC patients was significantly increased. The observed methylation changes from less to more invasive stages were not significant in the CAVIN3 gene. Moreover, promoter methylation was changed in metastatic rather than non-metastatic condition, although it was not significant. Conclusion: Promoter hypermethylation of DAPK1 and CAVIN3 genes in plasma are associated with the risk of BC and they can be potential diagnostic biomarkers along with current methods. Additionally, association of aberrant DAPK1 promoter methylation with metastasis suggests its potential usage as a non-invasive strategy for metastatic BC diagnosis.

414-420

Authors: Sayed Rasoul Zaker, Kamran Ghaedi

Objective: Breast cancer is one of the most frequent types of cancer with a gradually increasing incidence in developing countries. The aim of this study was to assess modulation of LINC02615 levels in breast cancer progress, using pairwise breast cancer and healthy control tissue samples with regard to the obesity and other conditions, as estrogen receptor (ER) expression. Materials and Methods: In this cohort study, the genes, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in several important pathways of chromosomal instability, apoptosis and proliferation were analyzed through in silico studies pinpointing the important genes which were responsible for the breast cancer incidence. Then, the respective miRNAs and lncRNAs were selected by relevant databases. At the next step, Lncbase was used for interaction analysis of selected miRNAs and LncRNAs, which resulted in final selection of LINC02615. Total RNA was isolated from 24 pairwise breast cancer and healthy control tissue samples. Expression profile of LINC02615 was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Correlation between LINC02615 expression and clinicopathological characteristics were analyzed using Pearson’s Chi-square test in breast cancer patients. Results: Data demonstrated that expression of LINC02615 was significantly downregulated in breast cancer tissues compared to the healthy controls (P=0.046). In particular, the relative LINC02615 expression was significantly different in breast cancer tissues especially in obese patients compared to those persons without obesity (P=0.047). Furthermore, a significant difference in LINC02615 level was found between the high and low ER expressions (P=0.014). However, the aberrant expression of LINC02615 was significantly related to physical activity and diabetes disease as well as the stress and age at menopause (P=0.028, P=0.046, P=0.047 and P=0.025, respectively). Conclusion: Taken together, we suggest that LINC02615 downregulation may be related to the risk of breast cancer in Iranian patients. Thus, it may serve as a novel biomarker for identification of breast cancer tissues.

429-434

Authors: Şahabettin Can Özyarım, Funda Karabağ Çoban

Objective: Colorectal cancer is one of the most prevalent consequences of cancer-bound decease worldwide and it remains one of the leading outcomes of cancer-bound decease. Boron is an important mineral that acts significant function in various biological courses. Some important chemical properties of boric acid support its utility in the treatment of cancer. The aim of this study is to evaluate the antiproliferative effects of boric acid in colon cancer. Materials and Methods: This experimental study effect of different concentrations of boric acid on the CCl-233 human colon adenocarcinoma cell lines was investigated, by analyzing proliferation assay (proliferation was applied to the cells for 24, 48 and 72 hours). Proliferation assay was performed using CCK8 Assay Kit. Vascular endothelial growth factor (VEGF) and poly (ADP-) ribose polymerase (PARP) analyses were performed using Sun-Red Human (VEGF) ELISA Kit and Sun-Red Human (PARP) ELISA Kit, respectively. Results: As a result of the studies, analysis of the cell viability showed that 50 mM boric acid decreased cell proliferation after 24, 48 and 72 hours. The maximal decrease in cell proliferation was found to occur at 48 hours. Therefore, PARP and VGEF analyses were performed at 48 hours. PARP values were significantly higher in cisplatin (P<0.05). In contrast, PARP levels were significantly lower (P<0.05) at two concentrations of boron (50-100 mM). In VEGF, analysis showed that boron levels were significantly different from cisplatin, but there was no significant difference between control groups. Conclusion: It is proposed that the molecular mechanisms leading to this type of cancer as well as the effect of boric acid on colon cancer should be clarified in more detailed ways for the early diagnosis and treatment of colon cancer.

445-450

Authors: Sajedeh Yadegari-Dehkordi, Seyed Mohammad Firoozabadi, Mehdi Forouzandeh Moghadam, Zeinab Shankay

Objective: The cell membrane is a major barrier for delivery of hydrophilic drugs and molecules into the cells. Although low voltage and high frequency electric fields (LVHF) are proposed to overcome the cell membrane barrier, the mechanism of membrane permeabilization is unclear. The aim of study is to investigate endocytosis pathways as a possible mechanism for enhancing uptake of bleomycin by LVHF. Materials and Methods: In this experimental study, MCF-7 cells were exposed to bleomycin or to electric fields with various strengths (10-80 V/cm), frequency of 5 kHz, 4000 electric pulse and 100 μs duration in the presence and absence of three endocytosis inhibitors-chlorpromazine (Cpz), amiloride (Amilo) and genistein (Geni). We determined the efficiency of these chemotherapeutic agents in each group. Results: LVHF, depending on the intensity, induced different endocytosis pathways. Electric field strengths of 10 and 20 V/cm stimulated the macropinocytosis route. Clathrin-mediated endocytosis was observed at electric field intensities of 10, 30, 60 and 70 V/cm, whereas induction of caveolae-mediated endocytosis was observed only at the lowest electric field intensity (10 V/cm). Conclusion: The results of this study imply that LVHF can induce different endocytosis pathways in MCF-7 cells, which leads to an increase in bleomycin uptake.

457-464

Authors: Mingyu Zhai, M.D., Mingming Han, Xiang Huang, Fang Kang, Chengwei Yang, Juan Li

Objective: The study was aimed to investigate the effects and potential mechanisms of Dexmedetomidine (Dex) on hypoxia/reoxygenation (H/R) injury in human renal tubular epithelial HK-2 cells. Materials and Methods: In this experimental study, HK-2 cells were divided into four groups: control group, Dex group, H/R group, and Dex+H/R group. The cells in control group received no treatment, and cells in Dex group were only treated with 0.1 nmol/L Dex. The cells in H/R group and Dex+H/R group were all treated with H/R (hypoxia for 24 hours and normoxia for 4 hours), and only the cells in Dex+H/R group were pre-administrated with 0.1 nmol/L Dex. Following treatments at 37˚C for 28 hours, cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Also, the expressions of hypoxia-inducible factor 1 (HIF-1α), glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP), caspase-12 and cleaved caspase-3 were determined by western blot. Results: The cell viability was significant decreased in H/R group compared with control group (P<0.05), while was significantly increased in Dex+H/R group compared with that in H/R group (P<0.05). However, the change tendency of the cell apoptosis was opposite to that of cell viability. Compared with H/R group, the expression of HIF-1α was evidently up-regulated, while GRP78, CHOP, capase-12 and cleaved caspase-3 expressions were all obviously downregulated in Dex+H/R group (P<0.05). In addition, the concentrations of malondialdehyde (MDA) in H/R group and Dex+H/R group were 1.68 ± 0.22 nmol/mgprot and 0.85 ± 0.16 nmol/mgprot, respectively. The superoxide dismutase(SOD) activity was higher in Dex+H/R group (121 ± 11 U/L), which which was more than twice larger than that in H/R group (57 ± 10 U/L). Conclusion: Dex could promote cell viability and inhibit apoptosis through up-regulating HIF-1α, reducing endoplasmic reticulum (ER) stress and mediating oxidative stress, thus ameliorating the H/R injury.

474-484

Authors: Hongchang Yang, Jing Zhou, Junlei Wang, Luoning Zhang, Quzhi Liu, Jing Luo, Hongyan Jia, Li Liu, Qiang Zhou

Objective: This study aimed to characterize the circulating exosomal microRNA (miRNA) profiles associated with acute soft tissue injury. Materials and Methods: In this experimental study, a total of 12 rats were randomly divided into control group and model group (n=6 for each group). The rats in the model group were used to establish an acute soft tissue injury following the mechanical injury of the leg. The exosomes from the peripheral blood of all the rats were isolated and then characterized by Nanosight NS300 particle size analyser (NTA), transmission electron microscopy (TEM) and western blot. Next, the exosomal miRNAs in the control and model groups were sequenced, and the differentially expressed miRNAs (DE-miRNAs) were identified using the DESeq algorithm. Functional analyses were performed using Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. Finally, quantitative reverse-transcription polymersa chain reaction (qRT-PCR) was used to verify the expression of the DE-miRNAs. Results: TEM, NTA and western blot results showed that the exosomes were approximately 100 nm in size and exhibited cup-shaped morphology. A total of 628 miRNAs were obtained by sequencing. After that, 28 DE miRNAs (DEmiRNAs) were identified, including seven down-regulated miRNAs and 21 up-regulated miRNAs. These DE-miRNAs were linked to 7539 target genes with GO. Also, KEGG analyses demonstrated that these genes were enriched for phosphorylation, VEGF signaling pathway, and MAPK signaling pathway. Additionally, the consistency rate between the qRT-PCR and sequencing results was 83.33%, which showed a high relative reliability of the sequencing results. Conclusion: These findings suggest that these 28 exosomal miRNAs may be involved in the regulation of acute soft tissue injury, by one of critical biological processes (BP), phosphorylation. The findings provide valuable clues by utilizing exosomes as therapeutic targets for the effective treatment of acute soft tissue injury.

145-153

Authors: Caroline Mathen, Wilfrid Dsouza

Objective: The past decade has witnessed a rapid growth in harnessing the potential of adult stem cells for regenerative medicine. An investigational new drug (IND) or a regenerative medicine advanced therapy (RMAT) product must fulfil many requirements, such as stability studies, after cryopreservation. Such studies are important to ascertain the utility of off-the-shelf allogeneic cells for clinical applications. The present work describes a complete characterisation of xenofree human Wharton’s Jelly mesenchymal stromal cells (hWJ-MSCs) before and up to 28 months post-cryopreservation. Materials and Methods: In this experimental study, culture methods that involved plasma derived human serum and recombinant trypsin were used to develop clinical grade cells. Complete cell characterisation involved flow cytometry studies for viability, positive and negative markers, colony forming unit (CFU) potential, population doubling time (PDT), soft agar assay to evaluate in vitro tumourigenicity, karyotype analysis and differentiation studies which were performed before and at 6, 12, 18 and 28 months post-cryopreservation. Results: Our data showed consistency in the flow cytometry, CFU assay, PDT, soft agar assay, karyotyping and differentiation studies. Conclusion: Using our protocols for extended xeno-free culture and cryopreservation of hWJ-MSCs, we could establish the shelf life of the cell-based product for up to 28 months.

164-173

Authors: Ali Moghimi Khorasgani, Reza Moradi, Farnoosh Jafarpour, Faezeh Ghazvinizadehgan, Somayyeh Ostadhosseini, Alireza Heydarnezhad, Ali Akbar Fouladi-Nashta, Mohammad Hossein Nasr-Esfahani

Objective: Alpha-lipoic acid (ALA) as a strong antioxidant has a protective effect. This study was designed to assess whether supplementation of maturation medium with ALA during in vitro maturation (IVM) can attenuate the toxic effect of ethanol. Materials and Methods: In this experimental study, to assess the antioxidant capacity of ALA challenged by 1% ethanol during in vitro maturation, immature ovine oocytes were exposed to 1% alcohol in the presence or absence of 25 μM ALA during oocyte maturation. The cumulus expansion index, intracellular reactive oxygen species (ROS), and thiol content levels were assessed in matured oocytes of various treatment groups. Consequently, the blastocyst formation rate of matured oocytes in various treatment groups were assessed. In addition, total cell number (TCN), cell allocation, DNA fragmentation, and relative gene expression of interested genes were assessed in resultant blastocysts. Results: The results revealed that alcohol significantly reduced cumulus cells (CCs) expansion index and blastocyst yield and rate of apoptosis in resultant embryos. Addition of 25 μM ALA to 1% ethanol during oocyte maturation decreased ROS level and elevated Thiolcontent. Furthermore, supplementation of maturation medium with ALA attenuated the effect of 1% ethanol and significantly increased the blastocyst formation and hatching rate as compared to control and ethanol groups. In addition, the quality of blastocysts produced in ALA+ethanol was improved based on the low number of TUNEL positive cells, the increased expression level of mRNA for pluripotency, and anti-oxidant markers, and decreased expression of apoptotic genes. Conclusion: The current findings demonstrate that ALA can diminish the effect of ethanol, possibly by decreasing the ROS level and increasing Thiolcontent during oocyte maturation. Using the ALA supplement may have implications in protecting oocytes from alcohol toxicity in affected patients.

184-190

Authors: Ying Liu, Yi Song, Siyuan Li, Li Mo

Objective: Quercetin (Que) is a plant-derived polyphenolic compound, that was shown to possess anti-inflammatory activity in myocardial ischemia/reperfusion (I/R) models in vivo; however, detailed mechanisms of its anti-inflammatory effects remain unclear. This study aimed to examine the effects of quercetin postconditioning (QPC) on I/R-induced inflammatory response in a rat model and evaluate the role of the mitochondrial K-ATP (mitoKATP) channels and NO system in this regard. Materials and Methods: In this experimental study, hearts of male Wistar rats (250 ± 20 g) perused by Langendorff apparatus, were subjected to 30 minutes of global ischemia followed by 55 minutes reperfusion, and Que was added to the perfusion solution immediately at the onset of reperfusion. Creatine kinase (CK) levels in the coronary effluent were measured by spectrophotometry. Interleukin-1 (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) levels were analyzed by an enzyme-linked immunosorbent assay (ELISA) rat specific kit to assess the inflammatory condition of the myocardial tissue. Results: Our results showed that QPC significantly improved left ventricular developed pressure (LVDP) (P<0.05), and decreased the CK release into the coronary effluent vs. control group (P<0.01). The levels of IL-1β (P<0.01), TNF-α (P<0.01), and IL-6 (P<0.05) were significantly diminished in Que-treated groups when compared to the control group. Inhibiting mitoKATP channels by 100 μM 5-hydroxydecanoate and blocking NO system by 100 μM L-NAME reversed the cardioprotective effects of Que. Conclusion: The findings of this study suggested that QPC exerts cardioprotective effects on myocardial I/R injury (MIRI) through inhibition of inflammatory reactions and improvement of contractility potential. Also, mitoKATP channels and NO system might be involved in this anti-inflammatory effect.

199-210

Authors: Qing-Hua Yin,Yuan Zhou,,Zhi-Yuan Li

Objective: Our study aimed to investigate function and mechanism of miR-373 in proliferation and apoptosis of pancreatic cancer (PC) cells by regulating NAD+-dependent histone deacetylase sirtulin 1 (SIRT1). Materials and Methods: This experimental study included two PC cell lines AsPC-1 and PANC-1 in which expression levels of miR-373 and SIRT1 were manipulated. The level of miR-373 was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Expression levels of SIRT1, BCL-2, BAX, cleaved CASPASE-8/9/3, PARP, PGC-1α, NRF2, eNOS and iNOS were examined via RT-qPCR and western blotting, respectively. The binding sites of miR-373 on the SIRT1 were examined via dual-luciferase assay. Cell proliferation and apoptosis were examined by MTT assay, colony formation assay, Annexin-V/PI staining and TUNEL assay. The oxidative metabolic changes were monitored by reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) detection. Results: miR-373 could specifically target the 3’-UTR of SIRT1 and reduce its expression in PC cells. Either elevated expression of miR-373 or partial loss of SIRT1 inhibited cell proliferation and induced cell apoptosis. Accumulation of BAX and cleaved CASPASE-8/9/3, inhibition of PGC-1α/NRF2 pathway, increase oxidative stress and reduction of BCL-2 as well as uncleaved PARP were found in the presence of miR-373 or the absence of SIRT1. Overexpression of SIRT1 could reduce anti-proliferative and pro-apoptotic effects of miR-373. Conclusion: Overall, this study concluded that miR-373-dependent SIRT1 inhibition displays anti-proliferative and proapoptotic roles in PC cells via PGC-1α/NRF2 pathway, which highlights miR-373 as a potential target for PC treatment.

218-224

Authors: Zhiliang Guo, Yuanyuan Liu, Yu Gao, Xiumei Guan, Hong Li, Min Cheng

Objective: The present study aimed to screen the differentially expressed (DE) circular RNAs (circ-RNAs) between lumbar intervertebral disc degeneration (IVDD) and normal tissues. Material and Methods: In this experimental study, microarray hybridization was performed to evaluate circ-RNA expression, and the DE circ-RNAs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Host genes of DE circ-RNAs were predicted, and their functions were evaluated. Further, a competitive endogenesis (ce) RNA network among 4 DE circ-RNAs-miRNA-mRNA was constructed by Cytoscape. Results: A total of 2636 circ-RNAs were detected in all samples; among them, 89.23% were exonic circ-RNAs. There were 138 DE circ-RNAs, including 134 up-regulated circ-RNAs and 4 downregulated circ-RNAs in IVDD samples. qRT-PCR validation experiments showed that expression trends of hsa_circ_0003239, hsa_circ_0003162, hsa_circ_0005918, and hsa_circ_0005556 were in line with the microarray analysis results. Functional enrichment analysis showed that host genes of DE circ-RNAs significantly disturbed pathways of regulation of actin cytoskeleton, propanoate metabolism, and ErbB signaling pathway. The four DE circ-RNAs related ceRNA network was constructed. Conclusions: Our results revealed that circ-RNAs can function as miRNA sponges and regulate parent gene expression to affect IVDD.

238-246

Authors: Ran Wu, Fei Guo, Chen Wang,, Baohua Qian, Fuming Shen, Fang Huang, Weidong Xu

Objective: Circular RNA (circRNA) is of significant interest in genetic research. The aim of this study was to assess global trends in circRNA research production in order to shed new light on future research frontiers. Materials and Methods: In this retrospective study, we conducted a literature search using the Web of Science Core Collection (WoSCC) database on March 21, 2019 to retrieve publications from 2007 to 2018. Excel 2013, CiteSpace V, and VOSviewer were used to evaluate bibliometric features that included publication output, countries/regions, institutions, journals, citation frequency, H-index, and research hotspots. Results: Global cumulative publication output on circRNA consisted of 998 papers with a total citation of 28 595 during 2007-2018. China, the US, and Germany were the most prolific countries. China ranked first in H-index (60 times) and citations (13 333 times). The most productive institution was Nanjing Medical University with 73 papers. Biochemical and Biophysical Research Communications (impact factor [IF]2017:2.559) ranked first among journals in the number of publications (64 papers). The keywords shifted from "sequence", "intron", and "splice-site" to "transcriptome", "microRNA sponge", "exon circularization", and "circRNA biogenesis" overtime. The burst keywords "transcriptome", "microRNA sponge", "exon circularization", and "circRNA biogenesis" were the latest frontiers by 2018. Conclusion: This is a relatively novel bibliometric analysis to inspect research related to circRNA. The results show that publications have continuously increased in the past decade. China, the US, and Germany were the leading countries/regions in terms of quantity. Recent studies on topics related to circRNA biogenesis and function should be closely followed in this field.

261-272

Authors: Parisa Sahranavardfard, Zahra Madjd, Amirnader Emami Razavi, Alireza Ghanadan, Javad Firouzi, Pardis Khosravani, Saeid Ghavami, Esmaeil Ebrahimie, Marzieh Ebrahimi

Objective: Epithelial-mesenchymal transition (EMT) and the stemness potency in association with BRAF mutation are in dispensable to the progression of melanoma. Recently, microRNAs (miRNAs) have been introduced as the regulator of a multitude of oncogenic functions in most of tumors. Therefore identifying and interpreting the expressionpatterns of these miRNAs is essential. The present study sought to find common miRNAs regulating all three important pathways in melanoma development. Materials and Methods: In this experimental study, 18 miRNAs that importantly contribute to EMT and have a role in regulating self-renewal and the BRAF pathway were selected based on current literature and cross-analysis with available databases. Subsequently, their expression patterns were evaluated in 20 melanoma patients, normal tissues, serum from patients and control subjects, and melanospheres. Pattern discovery and integrative regulatory network analysis were used to find the most important miRNAs in melanoma progression. Results: Among 18 selected miRNAs, miR-205, -141, -203, -15b, and -9 were differentially expressed in tumor samples than normal tissues. Among them, miR-205, -15b, and -9 significantly expressed in serum samples and healthy donors. Attribute Weighting and decision trees (DT) analysis presented evidence that the combination of miR-205, -203, -9, and -15b can regulate self-renewal and EMT process, by affecting CDH1, CCND1, and VEGF expression. Conclusion: We suggested here that miR-205, -15b, -203, -9 pattern as the key miRNAs linked to melanoma status, the pluripotency, proliferation, and motility of malignant cells. However, further investigations are required to find the mechanisms underlying the combinatory effects of the above mentioned miRNAs.

288-293

Authors: Saber Khazaei, Abbasali Khademi, Mohammad Hossein Nasr Esfahani, Mozafar Khazaei, Mohammad Hossein Nekoofar, Paul M. H. Dummer

Objective: The aim of present study was to isolate and differentiate human adipose-derived stem cells (ASCs) into odontoblast-like cells. Materials and Methods: In this experimental study, human adipose tissues were taken from the buccal fat pad of three individuals (mean age: 24.6 ± 2.1 years). The tissues were transferred to a laboratory in a sterile culture medium, divided into small pieces and digested by collagenase I (2 mg/mL, 60-90 minutes). ASCs were isolated by passing the cell suspension through cell strainers (70 and 40 μm), followed by incubation at 37ºC and 5% CO2 in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS 5%) and penicillin/streptomycin (P/S). After three passages, the ASCs were harvested. Subsequently, flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect expression levels of NANOG and OCT4 to evaluate stemness. Then, a differentiation medium that included high-glucose DMEM supplemented with 10% FBS, dexamethasone (10 nM), sodium β-glycerophosphate (5 mM) and ascorbic acid (100 μM) was added. The cells were cultivated for four weeks, and the odontogenic medium was changed every two days. Cell differentiation was evaluated with Alizarin red staining and expressions of collagen I (COL1A1), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1). Results: The ASCs were effectively and easily isolated. They were negative for CD45 and positive for the CD105 and CD73 markers. The ASCs expressed OCT4 and NANOG. Differentiated cells highly expressed DSPP, COL1A1 and DMP1. Alizarin red staining revealed a positive reaction for calcium deposition. Conclusion: ASCs were isolated successfully in high numbers from the buccal fat pad of human volunteers and were differentiated into odontoblast-like cells. These ASCs could be considered a new source of cells for use in regenerative endodontic treatments.

303-312

Authors: Maesumeh Eslami, Shahrbanoo Oryan, Mehdi Rahnema, Mohammad Reza Bigdeli

Abstract Objective: Choroid plexus epithelial cells (CPECs) have the epithelial characteristic, produce cerebrospinal fluid, contribute to the detoxification process in the central nervous system (CNS), and are responsible for the synthesis and release of many nerve growth factors. On the other hand, studies suggest that normobaric hyperoxia (HO) by induction of ischemic tolerance (IT) can protect against brain damage and neurological diseases. We examined the effect of combination therapy of encapsulated CPECs and HO to protect against ischemic brain injury. Materials and Methods: In this experimental study, six groups of adult male Wistar rats were randomly organized: sham, room air (RA)+middle cerebral artery occlusion (MCAO), HO+MCAO, RA+MCAO+encapsulated CPECs, HO+MCAO+encapsulated CPECs, RA+MCAO+empty capsules. RA/HO were pretreatment. The CPECs were isolated from the brain of neonatal Wistar rats, cultured, and encapsulated. Then microencapsulated CPECs were transplanted in the neck of the animal immediately after the onset of reperfusion in adult rats that had been exposed to 60 minutes MCAO. After 23 hours of reperfusion, the neurologic deficit score (NDS) was assessed. Next, rats were killed, and brains were isolated for measuring brain infarction volume, blood-brain barrier (BBB) permeability, edema, the activity of superoxide dismutase (SOD), and catalase (CAT) and also, the level of malondialdehyde (MDA). Results: Our results showed that NDS decreased equally in HO+MCAO, RA+MCAO+encapsulated CPECs, and HO+MCAO+encapsulated CPECs groups. Brain infarction volume decreased up 79%, BBB stability increased, edema decreased, SOD and CAT activities increased, and MDA decreased in the combination group of HO and transplantation of encapsulated CPECs in the ischemic brain as compared with when HO or transplantation of encapsulated CPECs was applied alone. Conclusion: The combination of HO and transplantation of encapsulated CPECs for stroke in rats was more effective than the other treatments, and it can be taken into account as a promising treatment for ischemic strok.

319-328

Authors: Mojtaba Eslami, Sahar Esfandyari, Marzieh Aghahosseini, Zahra Rashidi, Shirzad Hosseinishental, Samane Brenjian, Aligholi Sobhani, Fardin Amidi

Objective: Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes. Materials and Methods: In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2O2) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity. Results: This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05). Conclusion: Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

335-340

Authors: Amir Hossein Jalali, Hossein Mozdarani, Hossein Ghanaati

Objective: To evaluate the effect of contrast enhanced abdominopelvic magnetic resonance imaging (MRI), using a 3 Tesla scanner, on expression and methylation level of ATM and AKT genes in human peripheral blood lymphocytes. Materials and Methods: In this prospective in vivo study, blood samples were obtained from 20 volunteer patients with mean age of 43 ± 8 years (range 32-68 years) before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced abdominopelvic 3 Tesla MRI. After separation of mononuclear cells from peripheral blood, using Ficoll-Hypaque, we analyzed gene expression changes of ATM and AKT genes 2 hours and 24 hours after MRI using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also evaluated methylation percentage of the above mentioned genes in before, 2 hours and 24 hours after MRI, using MethySYBR method. Results: Fold change analysis, in comparison with the baseline, respectively showed 1.1 ± 0.7 and 0.8 ± 0.5 mean of gene expressions in 2 and 24 hours after MRI for ATM, while the results were 1.4 ± 0.6 and 1.4 ± 1 for AKT (P>0.05). Methylation of the ATM gene promoter were 8.8 ± 1.5%, 9 ± 0.6% and 9 ± 0.8% in before contrast enhanced MRI, 2 and 24 hours after contrast enhanced MRI, respectively (P>0.05). Methylation of AKT gene promoter in before contrast enhanced MRI, 2 hours and 24 hours after contrast enhanced MRI was 5.4 ± 2.5, 5 ± 3.2, 4.9 ± 2.9 respectively (P>0.05). Conclusion: Contrast enhanced abdominopelvic MRI using 3 Tesla scanner apparently has no negative effect on the expression and promoter methylation level of ATM and AKT genes involved in the repair pathways of genome.

349-354

Authors: Nasrin Sereshki, Alireza Andalib, Mohadeseh Toghyani, Hossein Motedayyen, Mohammad Sadegh Hesamian, Abbas Rezaei, David Wilkinson

Objective: The maternal immune response to paternal antigens is induced at insemination. We believe that pregnancy protective alloantibodies, such as anti-paternal cytotoxic antibody (APCA), may be produced against the paternal antigens in the context of stimulated immunity at insemination and that they increase during pregnancy. APCA is necessary for pregnancy. It is directed towards paternal human leucocyte antigens (HLAs) and has cytotoxic activity against paternal leucocytes. The present study aims to determine whether APCA is produced by the maternal peripheral blood mononuclear cells (PBMCs) in contact with the husband’s spermatozoa and to evaluate the relation of APCA production with HLA class I and II expressions by spermatozoa in fertile couples. Materials and Methods: This cross-sectional study included 30 fertile couples with at least one child. The maternal PBMCs were co-cultured with the husband’s spermatozoa and the supernatant was assessed for the presence of IgG by ELISA. Cytotoxic activity of the supernatant on the husband’s PBMCs was assessed by the complement-dependent cytotoxicity (CDC) assay. Results: IgG was produced in all co-cultures, and the mean level of supernatant IgG was 669 ng/ml. The cytotoxic activity of the supernatant was observed in all the supernatant obtained from the co-cultures. The mean percentage of APCA in supernatant was 73.93%. Conclusion: Based on the results of this study it can be concluded that APCA may be a natural anti-sperm antibody (ASA), which can be produced during exposure to spermatozoa and may have some influence before pregnancy. Further research is required to determine the role of APCA before pregnancy.

1-13

Authors: Fattah Sotoodehnejadnematalahi, Reza Moghadasali, Mostafa Hajinasrollah, Ehsan Ehsani, Ensiyeh Hajizadeh-Saffar, Niloofar Sodeifi, Reza F. Saidi, Morteza Zarrabi, Mohammad Farzanehkhah, Bahareh Sadeghi, Hossein Baharvand, Nasser Aghdami

Objective: In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation. Materials and Methods: In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H&E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses. Results: A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05). Conclusion: It seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

21-31

Authors: Shang-Gan Zeng, Jian-Hong Xie, Qun-Ying Zeng, Shao-Hua Dai, Yun Wang, Xue-Mei Wan, Ji-Chun Liu

Objective: Although growing evidences have showed that long non-coding RNA (lncRNAs) plasmacytoma variant translocation 1 (PVT1) plays a critical role in the progression of non-small cell lung cancer (NSCLC), there are still many unsolved mysteries remains to be deeply elucidated. This study aimed to find a new underlying mechanism of PVT1 in regulating the tumorigenesis and development of NSCLC. Materials and Methods: In this experimental study, Quantitative reverse transcription polymerase chain reaction (qRTPCR) was used to profile the expression of PVT1 in NSCLC tissues and cells. The effects of PVT1 on cell growth, migration and invasion were detected by colony formation assay, Matrigel-free transwell and Matrigel transwell assays, respectively. Changes of the key protein expression in Hippo and NOTCH signaling pathways, as well as epithelialmesenchymal transition (EMT) markers, were analyzed using western blot. Interaction of PVT1 with enhancer of zeste homolog 2 (EZH2) was verified by RNA pull-down, and their binding to the downstream targets was detected by Chromatin Immunoprecipitation (ChIP) assays. Results: These results showed that PVT1 was up-regulated in NSCLC tissue and cell lines, promoting NSCLC cell proliferation, migration and invasion. Knockdown of PVT1 inhibited the expression of Yes-associated protein 1 (YAP1) and NOTCH1 signaling activation. Further, we have confirmed that PVT1 regulated expression of YAP1 through EZH2-mediated miR-497 promoter methylation resulting in the inhibition of miR-497 transcription and its target YAP1 upregulation, and finally NOTCH signaling pathway was activated, which promoted EMT and invasion and metastasis. Conclusions: These results suggested that lncRNA PVT1 promotes NSCLC metastasis through EZH2-mediated activation of Hippo/NOTCH1 signaling pathways. This study provides a new opportunity to advance our understanding in the potential mechanism of NSCLC development.

40-50

Authors: Simin Nafian Dehkordi, Farzaneh Khani, Seyedeh Nafiseh Hassani, Hossein Baharvand, Hamid Reza Soleimanpour-Lichaei, Ghasem Hosseini Salekdeh

Objective: Sexual dimorphism in mammals can be described as subsequent transcriptional differences from their distinct sex chromosome complements. Following X inactivation in females, the Y chromosome is the major genetic difference between sexes. In this study, we used a male embryonic stem cell line (Royan H6) to identify the potential role of the male-specific region of the Y chromosome (MSY) during spontaneous differentiation into embryoid bodies (EBs) as a model of early embryonic development. Materials and Methods: In this experimental study, RH6 cells were cultured on inactivated feeder layers and Matrigel. In a dynamic suspension system, aggregates were generated in the same size and were spontaneously differentiated into EBs. During differentiation, expression patterns of specific markers for three germ layers were compared with MSY genes. Results: Spontaneous differentiation was determined by downregulation of pluripotent markers and upregulation of fourteen differentiation markers. Upregulation of the ectoderm markers was observed on days 4 and 16, whereas mesoderm markers were upregulated on the 8th day and endodermic markers on days 12-16. Mesoderm markers correlated with 8 MSY genes namely DDX3Y, RPS4Y1, KDM5D, TBL1Y, BCORP1, PRY, DAZ, and AMELY, which were classified as a mesoderm cluster. Endoderm markers were co-expressed with 7 MSY genes, i.e. ZFY, TSPY, PRORY, VCY, EIF1AY, USP9Y, and RPKY, which were grouped as an endoderm cluster. Finally, the ectoderm markers correlated with TXLNGY, NLGN4Y, PCDH11Y, TMSB4Y, UTY, RBMY1, and HSFY genes of the MSY, which were categorized as an ectoderm cluster. In contrast, 2 MSY genes, SRY and TGIF2LY, were more highly expressed in RH6 cells compared to EBs. Conclusion: We found a significant correlation between spontaneous differentiation and upregulation of specific MSY genes. The expression alterations of MSY genes implied the potential responsibility of their gene co-expression clusters for EB differentiation. We suggest that these genes may play important roles in early embryonic development.

61-69

Authors: Qian Zhang, Yiwen Pan, Xiaochun Ma, Hao Yang, Jun Chang, Ling Hong, Huiwen Yan, Shubing Zhang

Objective: Atherosclerosis (AS) is one of the most common causes of human death and disability. This study is designed to investigate the roles of aldosterone (Aldo) and oxidized low-density lipoprotein (Ox-LDL) in this disease by clinical data and cell model. Materials and Methods: In this experimental study, clinical data were collected to investigate the Aldo role for the patients with primary aldosteronism or adrenal tumors. Cell viability assay, fluorescence-activated cell sorting (FACS) assay, apoptosis assay, cell aging analysis, and matrigel tube formation assay were performed to detect effects on human umbilical vein endothelial cells (HUVECs) treated with Aldo and/or Ox-LDL. Quantitative polymerase chain reaction (qPCR) and Western blot analysis were performed to figure out critical genes in the process of endothelial cells dysfunction induced by Aldo and/or Ox-LDL. Results: We found that the Aldo level had a positive correlation with the TG/HDL-C ratio. Endothelial cell growth, angiogenesis, senescence, and apoptosis were significantly affected, and eNOS/Sirt1, the value of Bcl-2/Bax and Angiopoietin1/2 were significantly affected when cells were co-treated by Aldo and Ox-LDL. Conclusion: Elevated Aldo with high Ox-LDL together may accelerate the dysfunction of HUVEC, and the Ox-LDL, especially for those patients with high Aldo should be well controlled. The assessment of the role of Aldo may provide a theoretical basis for the effective prevention and investigation of a new treatment of AS.

75-84

Authors: Bin Liu, Guanglei Qiao, Wen Cao, Chenlu Li, Shaojun Pan, Lirui Wang, Yanlei Liu, Lijun Ma, Daxiang Cui

Objective: We aimed to identify the differentially expressed proteins (DEPs) and functional differences between exosomes derived from mesenchymal stem cells (MSCs) derived from umbilical cord (UC) or adipose tissue (AD). Materials and Methods: In this experimental study, the UC and AD were isolated from healthy volunteers. Then, exosomes from UC-MSCs and AD-MSCs were isolated and characterized. Next, the protein compositions of the exosomes were examined via liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by evaluation of the DEPs between UC-MSC and AD-MSC–derived exosomes. Finally, functional enrichment analysis was performed. Results: One hundred and ninety-eight key DEPs were identified, among which, albumin (ALB), alpha-II-spectrin (SPTAN1), and Ras-related C3 botulinum toxin substrate 2 (RAC2) were the three hub proteins present at the highest levels in the protein-protein interaction network that was generated based on the shared DEPs. The DEPs were mainly enriched in gene ontology (GO) items associated with immunity, complement activation, and protein activation cascade regulation corresponding to 24 pathways, of which complement and coagulation cascades as well as platelet activation pathways were the most significant. Conclusion: The different functions of AD- and UC-MSC exosomes in clinical applications may be related to the differences in their immunomodulatory activities.

93-98

Authors: Shirin Azizidoost, Hossein Babaahmadi-Rezaei, Zahra Nazeri, Maryam Cheraghzadeh, Alireza Kheirollah

Objective: Dysregulation of cholesterol metabolism in the brain is responsible for many lipid storage disorders, including Niemann-Pick disease type C (NPC). Here, we have investigated whether cyclodextrin (CD) and apolipoprotein A-I (apoA-I) induce the same signal to inhibit cell cholesterol accumulation by focusing on the main proteins involved in cholesterol homeostasis in response to CD and apoA-I treatment. Materials and Methods: In this experimental study, astrocytes were treated with apoA-I or CD and then lysed in RIPA buffer. We used Western blot to detect protein levels of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and ATP-binding cassette transporter A1 (ABCA1). Cell cholesterol content and cholesterol release in the medium were also measured. Results: ApoA-I induced a significant increase in ABCA1 and a mild increase in HMGCR protein level, whereas CD caused a significant increase in HMGCR with a significant decrease in ABCA1. Both apoA-I and CD increased cholesterol release in the medium. A mild, but not significant increase, in cell cholesterol content was seen by apoA-I; however, a significant increase in cell cholesterol was detected when the astrocytes were treated with CD. Conclusion: CD, like apoA-I, depletes cellular cholesterol. This depletion occurs in a different way from apoA-I that is through cholesterol efflux. Depletion of cell cholesterol with CDs led to reduced protein levels of ABCA1 along with increased HMGCR and accumulation of cell cholesterol. This suggested that CDs, unlike apoA-I, could impair the balance between cholesterol synthesis and release, and interfere with cellular function that depends on ABCA1.

109-118

Authors: Elham Fazeli, Ahmad Hosseini, Mohammad-Hasan Heidari, Fattaneh Farifteh-Nobijari, Mohammad Salehi, Hojjat-Allah Abbaszadeh, Hamid Nazarian, Zahra Shams Mofarahe, Saman Ayoubi, Sara Hosseini, Mona Shayeghpour, Mojgan Bandehpour, Marefat Ghaffari Novin

Objective: In vitro maturation (IVM) of human oocytes is used to induce meiosis progression in immature retrieved oocytes. Calcium (Ca2+) has a central role in oocyte physiology. Passage through meiosis phase to another phase is controlled by increasing intracellular Ca2+. Therefore, the current research was conducted to evaluate the role of calcium ionophore (CI) on human oocyte IVM. Materials and Methods: In this clinical trial study, immature human oocytes were obtained from 216 intracytoplasmic sperm injection (ICSI) cycles. After ovarian stimulation, germinal vesicle (GV) stage oocytes were collected and categorized into two groups: with and without 10 μM CI treatment. Next, oocyte nuclear maturation was assessed after 24–28 hours of culture. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to assess the transcript profile of several oocyte maturation-related genes (MAPK3, CCNB1, CDK1, and cyclin D1 [CCND1]) and apoptotic-related genes (BCL-2, BAX, and Caspase-3). Oocyte glutathione (GSH) and reactive oxygen species (ROS) levels were assessed using Cell Tracker Blue and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye staining. Oocyte spindle configuration and chromosome alignment were analysed by immunocytochemistry. Results: The metaphase II (MII) oocyte rate was higher in CI‐treated oocytes (73.53%) compared to the control (67.43%) group, but this difference was not statistically significant (P=0.13). The mRNA expression profile of oocyte maturation-related genes (MAPK3, CCNB1, CDK1, and CCND1) (P<0.05) and the anti-apoptotic BCL-2 gene was remarkably up-regulated after treatment with CI (P=0.001). The pro-apoptotic BAX and Caspase-3 relative expression levels did not change significantly. The CI‐treated oocyte cytoplasm had significantly higher GSH and lower ROS (P<0.05). There was no statistically significant difference in meiotic spindle assembly and chromosome alignment between CI treatment and the control group oocytes. Conclusions: The finding of the current study supports the role of CI in meiosis resumption of human oocytes. (Registration Number: IRCT20140707018381N4)

129-136

Authors: Zahra Shams, Babak Akbari, Sarah Rajabi, Nasser Aghdami

Objective: Functional cardiac tissue engineering holds promise as a candidate approach for myocardial infarction. Tissue engineering has emerged to generate functional tissue constructs and provide an alternative means to repair and regenerate damaged heart tissues. Materials and Methods: In this experimental study, we fabricated a composite polycaprolactone (PCL)/gelatine electrospun scaffold with aligned nanofibres. The electrospinning parameters and optimum proportion of the PCL/ gelatine were tested to design a scaffold with aligned and homogenized nanofibres. Using scanning electron microscopy (SEM) and mechanophysical testes, the PCL/gelatine composite scaffold with a ratio of 70:30 was selected. In order to simulate cardiac contraction, a developed mechanical loading device (MLD) was used to apply a mechanical stress with specific frequency and tensile rate to cardiac progenitor cells (CPCs) in the direction of the aligned nanofibres. Cell metabolic determination of CPCs was performed using real-time polymerase chain reaction(RT-PCR). Results: Physicochemical and mechanical characterization showed that the PCL/gelatine composite scaffold with a ratio of 70:30 was the best sample. In vitro analysis showed that the scaffold supported active metabolism and proliferation of CPCs, and the generation of uniform cellular constructs after five days. Real-time PCR analysis revealed elevated expressions of the specific genes for synchronizing beating cells (MYH-6, TTN and CX-43) on the dynamic scaffolds compared to the control sample with a static culture system. Conclusion: Our study provides a robust platform for generation of synchronized beating cells on a nanofibre patch that can be used in cardiac tissue engineering applications in the near future.

138-139

Authors: Fazel Sahraneshin Samani, Marzieh Ebrahimi, Tahereh Zandieh, Reyhaneh Khoshchehreh, Mohamadreza Baghaban Eslaminejad, Nasser Aghdami, Hossein Baharvand

In this article which was published in Cell J, Vol 17, No 2, Summer 2015, on pages 211-220, the authors found that Figures 3 and 4 had some errors that accidentally happened during organizing figures as well as because of mislabeling of some images and saving them in an incorrect folder. The following figures are corrected. The authors would like to apologies for any inconvenience caused.

143-144

Authors: Zahra Azhdari Tafti, Mehdi Mahmoodi, Mohamad Reza Hajizadeh, Vahid Ezzatizadeh, Hossein Baharvand, Massoud Vosough, Abbas Piryaei

In this article which was published in Cell J, Vol 20, No 3, Autumn 2018, on pages 377-387, the scale bars in Figures 5-A missed unintentionally during production. The following figure is corrected. The authors would like to apologies for any inconvenience caused.

401-405

Authors: Sima Aghajari, Sayed Mohammad Javad Mortazavi, Mehdi Kalani, Samaneh Nematolahi, Parham Habibzadeh, Shirin Farjadian

Objective: Astronauts are exposed to a wide range of environmental stresses during spaceflights that reduce their immune responses and make them more susceptible to infections and malignancies. Exposure to a low dose of a certain stress induces an adaptive response, which leads to resistance to higher doses of the same or other types of stress. We designed this study to investigate the effect of radiofrequency electromagnetic field (RF-EMF)-induced adaptive response on immune system modulation in a mouse model of hindlimb unloading (HU) as a ground-based animal model of spaceflight conditions. Materials and Methods: In this experimental study, serum levels of T helper (Th)-mediated cytokines were determined by the multiplex cytometric bead assay in four groups of mice (n=10 per group): HU mice, RF-EMF-treated mice, HU mice pre-exposed to RF-EMF; and untreated controls. Mice were exposed to 2450 MHz RF-EMF with SAR 0.478 W/ kg for 12 hours/day for three successive days. Results: Tumor necrosis factor-alpha (TNF-α), interleukin-9 (IL-9) and IL-22 were significantly decreased in HU mice. Comparison between HU mice and RF-EMF-treated mice showed an opposite change in IL-6, while IL-9, IL-22, IFN-γ and TNF-α decreased in both groups. However, just interferon gamma (IFN-γ) was significantly decreased in HU mice that were pre-exposed to RF-EMF compared to the control group. Conclusion: The effect of RF-EMF in elevating IL-6 and reducing IL-9 in opposite directions in HU mice suggest a modulating effect of RF-EMF on HU-induced changes in these cytokines, as Th2 and Th9 eventually returned to normal levels and balances in cytokine ratios were also restored in HU mice pre-exposed to RF-EMF.

415-424

Authors: Florina Popovska-Perčinić, Milica Manojlović-Stojanoski, Lazo Pendovski, Suzana Dinevska Kjovkarovska, Biljana Miova, Jasmina Grubin, Verica Milošević, Vladimir Ajdžanović

Objective: As a consequence of global warming, the increase in the average annual temperature is observed, while the living organisms actively adapt to these changes. High environmental temperature initiates numerous physiological, autonomic, and behavioral responses, and activates the stress response. Thus, the aim of the study was to investigate effect of a moderate increase in ambient temperature on the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis by determining histological changes in adrenal glands and hormonal levels in adult male rats. Material and Methods: In this experimental study, the morpho-functional state of adrenal glands was estimated by stereological evaluation of parameters, including the adrenal volume, adrenocortical cell/nuclear size and number, and the volume density of vascular tissues after four days of exposure to a moderate increase in ambient temperature of 35 ± 1˚C. Novelli histochemical and vascular endothelial growth factor (VEGF) immunohistochemical staining provided insight into the adrenal gland vascular network. Additionally, the adrenal levels of aldosterone, corticosterone, and pituitary adrenocorticotropic hormone (ACTH) were determined as crucial indicators of the hypothalamic-pituitaryadrenocortical (HPA) axis activity. Results: Prolonged exposure to a moderate increase in ambient temperature for four days resulted in a significant increase in ACTH level up to 24%, which altered adrenal glands both structurally and functionally. The adrenocortical volume and number of cells in all cortical zones were markedly increased (P<0.05). A statistically significant increase was shown in the level of aldosterone (16%) and corticosterone (25%) in serum levels of individuals. Conclusion: Increased activity of the HPA axis reflects the response to a moderate increase in ambient temperature during four days, showing the capacity of the HPA axis to adapt the organism to daily temperature changes.

431-436

Authors: Zahra Fazeli, Masoumeh Rajabibazl, Sepideh Faramarzi, Mir Davood Omrani, Sayyed Mohammad Hossein Ghaderian, Niloufar Safavi Naini

Objective: In the recent years, mesenchymal stem cells (MSCs) were considered as the suitable source of cells for transplantation into the damaged tissues in regenerative medicine. There was low number of these cells in different organs and this characteristic was the main drawback to use them in treatment of diseases. Cellular senescence of the stem cells has been demonstrated to be dependent to the telomerase activity. The aim of present experimental study was to evaluate correlation of the expression of telomerase components and WNT signaling pathway in MSCs derived from human peripheral blood (PB-MSCs). Materials and Methods: In this experimental study, following the isolation of MSCs from peripheral blood mononuclear cells, RNA was extracted from these cells in the early culture (8-9th days) and late culture (14-17th days). Then, expression of TERT, TERC, TCF4, GSK and CTNNB1 was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) based on SYBR Green. Results: Our data indicated that there was a significantly reduced expression of TERT in the late culture of human MSCs derived from peripheral blood (P<0.05). Although a negative correlation was observed between GSK and TERC expression levels in the early culture of MSCs, spearman analysis showed that there was no significant correlation between the expression of telomerase components (TERC and TERT) and WNT signaling pathway (P>0.05). Conclusion: The obtained results suggested that WNT signaling pathway likely plays a minor role in the maintenance of telomere length and proliferation potential of MSCs.

450-456

Authors: Zahra Chekini, Maryam Shahhoseini, Reza Aflatoonian, Parvaneh Afsharian

Objective: Endometriosis is a common gynecological and inflammatory disorder. Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that is secreted by accumulated active macrophages in ectopic endometrial tissues. Two promoter polymorphisms of MIF [-794(CATT)5–8 /-173G/C] were identified to susceptibility and severity of several immune and inflammatory diseases. We aimed to evaluate the possible association between MIF promoter polymorphisms and susceptibly to endometriosis and its corolation with mRNA level. Materials and Methods: This case-control study was performed in Royan Institute from 2015 to 2017. Polymorphisms were evaluated in 106 endometriosis patients and 110 controls. For 17 endometrioma tissues, gene expression studies were conducted during secretory phase of menstrual cycle. Restriction fragment length polymorphism (RFLP) analysis was performed to determine -173G/C polymorphism and -794(CATT)5-8 were detected by sequencing. Quantitative polymerase chain reaction (Q-PCR) was carried out to determine MIF expression level. Results: Homozygote of CATT7 was observed only in endometriosis whilst we did not detect the significant allele and genotype variation in both groups. The homozygotes for -794(CATT)5–8 and -173G/C polymorphisms were obtained to estimate the haplotype frequencies. Significantly higher haplotype frequencies were observed for CATT5/G in controls [global P value=0.044]. Additionally, the CATT5/C and CATT7/G haplotypes were not detected in any groups. Expression level of mRNA in ectopic tissue of endometriosis patients with CATT6,7/CC haplotype, were significantly higher compared to other haplotypes including CATT5,5/GG (2.91 fold, P=0.007), CATT5,5/GC (2.48 fold, P=0.047) and CATT6,6/GG (2.08 fold, P=0.046). Conclusion: We report, for the first time, a strong linkage between the decreased repetition of CATT and G allele in control and CATT6/C and CATT7/C haplotypes in endometriosis patients. Increased MIF expression is affected by genetic variants in the MIF promoter in ectopic endometrial tissues. This promoter haplotype might play an important role in the development and establishment of endometriosis.

467-475

Authors: Mehdi Totonchi, Babak Babaabasi, Hadi Najafi, Mojtaba Rezazadeh Valojerdi, Poopak Eftekhari-Yazdi, Lila Karimian, Navid Almadani, Anahita Mohseni Meybodi, Morteza Kimiai, Mehri Mashayekhi, Tahereh Madani, Hamid Gourabi

Objective: In vitro fertilization (IVF) is one of the most efficient approaches within the context of assisted reproductive technology (ART) to treat infertility. High pregnancy rates have become the major index of successful IVF in clinical studies. It is not clear yet which factors are certainly responsible for IVF success, as various outcomes were obtained in different IVF centers with different settings. In this study, we aimed to address controversies in the interpretation of promising results of IVF with respect to preimplantation genetic screening (PGS). Materials and Methods: In this retrospective case series study, we built a dataset containing data from 213 IVF patient candidates for PGS (654 embryos) with blastomere biopsy at day 3 and trophectoderm biopsy in day 5, referred to Royan Institute, Tehran, Iran from 2015 to 2018. Next, the data were analyzed to find influential factors affecting success rate of ART cycles. Results: Data analyses showed that regardless of PGS indications (ART failures, recurrent miscarriage, chromosomal abnormalities, etc.), the pregnancy rate is influenced by maternal and embryonic factors such as the age of mother as well as quantity and quality of transferred embryos. Furthermore, genotyping of embryos using array comparative genomic hybridization (aCGH) depicted the highest rate of chromosomal aberrations for chromosomes 1, 16 and 19 while the lowest frequency for chromosomes 11 and 17. Similarly, we detected 463 genetically abnormal embryos by aCGH, among which only 41.9% could be detected by classical fluorescent in situ hybridization (FISH) method. Conclusion: This study not only highlighted the advantages of aCGH over the FISH method in detection of chromosomal abnormalities, but also emphasized the importance of genetic abnormality as an indication for determination of IVF success rate.

482-490

Authors: Atefeh Rezaeian, Mohammad Karimian, Abasalt Hossienzadeh Colagar

Objective: In this study, we evaluated the effects of promoter methylation of MTHFR on oligozoospermia risk, followed by an in silico analysis. Materials and Methods: In a case-control study, semen samples were collected from infertile and healthy control men. MTHFR promoter region was amplified by methylation-specific polymerase chain reaction (PCR). Finally, the promoter region of MTHFR was analyzed by bioinformatics software. Results: Our data revealed significant associations of CpG island promoter methylation with oligozoospermia in a case-control study. In silico analysis showed that promoter contains a strong nucleosome exclusion region, a bonafide CGIs, six PROSITE motifs without a defined TATA box and 14 transcription factor (TF) binding sites, which are directly involved in spermatogenesis Conclusion: Based on our findings, methylation of the MTHFR gene promoter region may be a risk factor for oligozoospermia. However, this is a preliminary report representing data for future comprehensive studies to make a clinical conclusion on the potential biomarker role of methylation of this promoter in elevating susceptibility to oligozoospermia.

502-513

Authors: Alireza Rajabzadeh, Fatemeh Rahbarizadeh, Davoud Ahmadvand, Maryam Salmani, Amir Ali Hamidieh

Objective: Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospect in cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety, which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of less immunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likely overcome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a third generation lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation and cytotoxicity of redirected human T cells. Materials and Methods: In this experimental study, we established a second generation novel CAR (VHH-based anti- MUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembrane domain and signalling endodomains of CD28 and CD3ζ. Next, we constructed lentiviral vectors that contained this CAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cell surface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3+ T cells. Results: The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CAR showed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxic activity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) compared with A431 (MUC1-negative) or untransduced T cells. Conclusion: Our results suggested that, given the unique properties of VHHs to prevent immunogenic responses and tonic signalling, our novel VHH-based anti-MUC1 CAR might be effective for clinical purposes in cancer immunotherapy.

523-531

Authors: Xiangjing Ming, Yanling Guo, Yishan Zhou, Xiuping Chen

Objective: Ulcerative colitis (UC) is a long-lasting inflammatory disease of the colon. Epidemiological studies showed that the prevalence and incidence of UC are increasing worldwide in recent years. Neferine is a natural alkaloid isolated from Nelumbo nucifera Gaertn that exerts a variety of biological activities. This study was designed to evaluate the protective effect of neferine on dextran sulfate sodium (DSS)-induced experimental UC in mice. Materials and Methods: In this experimental study, 4% DSS was used to induce a mice model of UC. Neferine (5 and 10 mg/kg) was administered by intraperitoneal injection (ip). Clinical symptoms and disease activity index (DAI) scores were recorded and calculated. Pathological changes of colon tissues were detected by Hematoxylin and Eosin (H&E) staining. The levels of inflammatory mediators were detected by ELISA kits. Western blotting and immunohistochemical analysis were used for the evaluation of protein expressions. Results: Neferine treatment significantly alleviated DSS-induced UC by inhibiting weight loss, decreasing DAI scores, and alleviating the pathological changes in colon tissues. Furthermore, neferine significantly decreased serum levels of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 and increased serum levels of anti-inflammatory cytokine IL-10. The increased myeloperoxidase (MPO) activity and nitric oxide (NO) in colon tissues were also inhibited. In addition, neferine significantly down-regulated inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and intercellular cell adhesion molecule-1 (ICAM-1) expression in colon tissues. Conclusion: These results provided evidence that neferine could protect against DSS-induced UC symptoms in an experimental mice model. This effect might be mediated through inhibition of inflammation.

542-548

Authors: Ali Rajabi, Zohreh Sharifi, Fatemeh Yari, Mohammadreza Deyhim, Mohammadali Jalili

Objective: MicroRNAs (miRNAs) are short, noncoding RNAs that play vital roles in gene regulation. It has been shown that storage has an effect on platelet miRNAs. MiR-16 is highly expressed in platelets and it appears to target the genes involved in cell death. It has been shown that platelets could be stored in Composol for a longer period of time. The aim of the present study was to assess and compare the expression pattern of miR-16 in platelet concentrates (PCs) in plasma and Composol media. Materials and Methods: In an experimental study, ten PC bags were collected and each bag was divided into two separate bags, one with the 70% Composol and the other with only plasma. Both bags were stored for 7 days at 22˚C and tested on days 1, 3, 5, and 7 of storage. For each sample, we performed quantitative real-time polymerase chain reaction (qRT-PCR). The water-soluble tetrazolium salt-1 (WST-1) test was used to assess platelet viability in all of the samples. Statistical analysis was done by SPSS and REST software. A P<0.05 was considered statistically significant. Results: miR-16 was significantly elevated during the storage days, with fold changes of 3.47 (plasma) and 2.77 (Composol). The Composol group had significantly decreased miR-16 expression compared with the plasma group. Results of the WST-1 test showed less decrease in optical density (OD) in the Composol group (0.93 ± 0.4) during the storage days compared with the plasma group (0.75 ± 0.3). Conclusion: Our finding supported results from previous studies that reported an increase in miR-16 expression during platelet storage. In addition, miR-16 down-regulation in Composol medium implied that Composol might be a good solution for long-term platelet storage because it has the potential to elevate the shelf-life of platelets stored at 22˚C.

556-564

Authors: Samaneh Sharif Mashadi, Mohammad Hossein Ghahremani, Masoud Soleimani

Objective: Neuroblastoma (NB) is one of the frequently observed malignant solid tumors of childhood and infancy, accounting for 15% of pediatric cancer deaths. Recently, the approach of differentiation therapy has shown considerable promise in effective treatment of NB patients. MiR-124 belongs to the nervous system-specific miRNAs that is increased during neuronal differentiation and may be one of the potential therapeutic targets for the treatment of NB. However, despite its well-established therapeutic potential, its efficient delivery to the targeted tumor cells is a challenging task. Mesenchymal stem cells (MSCs) are multipotent adult progenitor cells that have antitumor properties, and they can migrate to cancer cells and tumors. This study aimed to assess whether human adipose tissue-derived MSCs (hADMSCs) have the potential to deliver exogenous miRNAs to NB cells to induce differentiation and decrease proliferation of cancer cells. Materials and Methods: In this experimental study, hAD-MSCs were isolated, cultured, and differentiated. The M17 human NB cell line were also cultured. A specific type of miRNAs, i.e., miR-124 was successfully delivered to M17 NB cells with the aid of hAD-MSCs using the direct or indirect (exosome-based) contacts. Results: It was shown that indirect delivery of miR-124 considerably decreased the proliferation of NB cells and induced their differentiation. Conclusion: The results suggest the use of delivered exogenous miRNAs by the derived exosomes from hAD-MSCs as a novel cell-free stem cell-based therapy for NB cancer.

572-574

Authors: Jose Luis Royo

Recently, it has been proposed the association of a common deletion affecting toll-like receptor 2 promoter (-196 to -177) to type 2 diabetes mellitus risk. However, genotyping results show a significant deviation from the Hardy- Weinberg Equilibrium (HWE). The law of Hardy-Weinberg shows that for an autosomal biallelic marker with allele frequencies fA=p and fa=q, the proportion of subjects with genotypes AA, Aa, and aa should follow the following: fAA=p2, fAa=2pq, and faa=q2. Departure from HWE or Hardy-Weinberg Disequilibrium (HWD) in a human control population can be caused by natural factors such as selective pressure against a certain genotype. However their prevalence is scarce and magnitude of effect over the HWE are small. Other factors such as inbreeding caused by consanguinity, population stratification, or technical problems in genotyping are more usual. Nevertheless, if the control population follows a perfect HWE, the presence HWD among patients might be explained by the genetic association and evidencing a real link between the locus and the trait under study. However, HWD affecting both cases and controls, such as the one reported might be explained by one of the aforementioned issues.

1-10

Authors: Reza Koochaki, Bahareh Abd-Nikfarjam, Amirhosein Maali, Saeid Abroun, Farshad Foroughi, Sasan Ghaffari, Mehdi Azad

Severe combined immunodeficiency (SCID) is classified as a primary immunodeficiency, which is characterized by impaired T-lymphocytes differentiation. IL2RG, IL7Ralpha, JAK3, ADA, RAG1/RAG2, and DCLE1C (Artemis) are the most defective genes in SCID. The most recent SCID therapies are based on gene therapy (GT) of hematopoietic stem cells (HSC), which are faced with many challenges. The new studies in the field of stem cells have made great progress in overcoming the challenges ahead. In 2006, Yamanaka et al. achieved "reprogramming" technology by introducing four transcription factors known as Yamanaka factors, which generate induced pluripotent stem cells (iPSC) from somatic cells. It is possible to apply iPSC-derived HSC for transplantation in patients with abnormality or loss of function in specific cells or damaged tissue, such as T-cells and NK-cells in the context of SCID. The iPSC-based HSC transplantation in SCID and other hereditary disorders needs gene correction before transplantation. Furthermore, iPSC technology has been introduced as a promising tool in cellular-molecular disease modeling and drug discovery. In this article, we review iPSC-based GT and modeling for SCID disease and novel approaches of iPSC application in SCID.

19-28

Authors: Abbas Heidari-Moghadam, Vahid Bayati, Mahmoud Orazizadeh, Mohammad Rashno

Objective Researchers have been interested in the creation of a favorable cellular model for use in vascular-muscle tissue engineering. The main objective of this study is to determine the myogenic effects of vascular endothelial growth factor (VEGF) and human umbilical vein endothelial cells (HUVECs) on adipose-derived stem cells (ADSCs) to achieve an in vitro vascular-muscle cellular model. Materials And Methods The present experimental research was conducted on two primary groups, namely ADSCs monoculture and ADSCs/HUVECs co-culture that were divided into control, horse serum (HS), and HS/VEGF differentiation subgroups. HUVECs were co-cultured by ADSC in a ratio of 1:1. The myogenic differentiation was evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence in different experimental groups. The interaction between ADSCs and HUVECs, as well as the role of ADSCs conditional medium, was investigated for endothelial tube formation assay. Results Immunofluorescence staining indicated that Tropomyosin was positive in ADSCs and ADSCs and HUVECs co-culture groups on HS and HS/VEGF culture medium. Furthermore, the MyHC2 gene expression significantly increased in HS and HS/VEGF groups in comparison with the control group (P<0.001). More importantly, there was a significant difference in the mRNA expression of this gene between ADSCs and ADSCs and HUVECs co-culture groups on HS/VEGF culture medium (P<0.05). Current data revealed that the co-culture of ADSCs and HUVECs could develop endothelial network formation in the VEGF-loaded group. Also, the ADSCs-conditioned medium improved the viability and formation of the endothelial tube in the HS and VEGF groups, respectively. Conclusion It was concluded that ADSCs/HUVECs co-culture and dual effects of VEGF can lead to the formation of differentiated myoblasts in proximity to endothelial network formations. These in vitro cellular models could be potentially used in vascular-muscle tissue engineering implanted into organ defects where muscle tissue and vascular regeneration were required.

38-48

Authors: Azadeh Aminzadeh, Neda Tekye Maroof, Mehrnaz Mehrabani, Kobra Bahrampour Juybari, Ali Mohammad Sharifi

Objective This study aimed to investigate the reliability of diabetic adipose-derived stem cells (ADSCs) for autologous cell-based therapies by exploring the functionality of signalling pathways involved in regulating oxidative stress and apoptosis. Materials And Methods In this experimental study, ADSCs were isolated from streptozotocin (STZ)-induced diabetic rats (dADSCs) and normal rats (nADSCs). The colonies derived from dADSCs and nADSCs were compared by colony-forming unit (CFU) assay. Reactive oxygen species (ROS) formation and total antioxidant power (TAP) were also measured. Furthermore, the expression of antioxidant enzymes, including catalase (Cat), superoxide dismutase (Sod)-1 and -3, glutathione peroxidase (Gpx)-1, -3 and -4 was measured at mRNA level by semi-quantitative reverse transcriptase polymerase chain reaction assay. The expression of Bax, Bcl2, caspase-3, total and phosphorylated c-Jun N-terminal kinase (JNK) and P38 Mitogen-Activated Protein Kinase (MAPK) at protein level was analyzed by western blotting. Results The results of this study indicated that viability and plating efficiency of dADSCs were significantly lower than those of nADSCs. ROS generation and TAP level were respectively higher and lower in dADSCs. The gene expression of antioxidant enzymes, including Cat, Sod-1, Gpx-3 and Gpx-4 in dADSCs was significantly greater than that in nADSCs. However, Sod-3 and Gpx-1 mRNA levels were decreased in dADSCs. Moreover, Bax/Bcl-2 protein ratio, caspase-3 protein expression and phosphorylation of JNK and P38 proteins were increased in dADSCs compared to nADSCs. Conclusion Taken together, diabetes might impair the cellular functions of dADSCs as candidates for autologous cell- based therapies. This impairment seems to be mediated by JNK, P38 MAPKs, and mitochondria pathway of apoptosis and partly by disruption of antioxidant capacity.

61-67

Authors: Marziyeh Feyzi, Mohammad Reza Tabandeh, Mehrdad Shariati, Mohammad Amin Edalatmanesh

Objective Adiponectin has a crucial role in the function, proliferation and viability of β-cell via action of two receptors: AdipoR1 and AdipoR2. Nevertheless, age related change of Adiponectin system genes in pancreas is unclear or controversial. This study sought to investigate the effects of aging process on serum Adiponectin levels, Adiponectin and its receptor expression in the rat pancreas. Materials And Methods In this experimental study, insulin resistance markers including serum insulin and glucose concentrations, homeostatic model assessment of insulin resistance (HOMA-IR), oral glucose tolerance test (OGTT), glucose induced insulin secretion (GIIS), serum Adiponectin levels, pancreatic expression of Adiponectin and its receptors were studied in male Sprague-Dawley rats at the age of 2, 5, 10, 18, 52 and 72 weeks of age. Results We found that aging triggered signs of insulin resistance characteristics in rats at 72 age weeks including marked insulin reduction, hyperglycemia and increased HOMA-IR. Circulating Adiponectin as well as pancreatic expression of Adiponectin and AdipoR1 was gradually decreased with age, while the opposite expression pattern of AdipoR2 was observed in the old rats. Conclusion Because Adiponectin and Adiponectin signaling have crucial role in β-cell function and viability, we concluded that reduction of Adiponectin signaling may be involved in aging induced β-cell dysfunction. As a result, manipulation of Adiponectin signaling may be a beneficial approach for improvement of β-cell function in the old people.

74-80

Authors: Sahar Gharanfoli, Abdolhoseein Shahverdi, Azam Dalman, Pooneh Ghaznavi, Hiva Alipour, Poopak Eftekhari-Yazdi

Objective The Hippo pathway plays an important role in embryo development, and separation of trophectoderm (TE) and inner cell mass (ICM) cell lines. Therefore, this study investigated effect of maternal age on activity of Hippo pathway in human embryos. Materials And Methods In this experimental study, the developed up embryos to the blastocyst stage and the embryos whose growth stopped at the morula stage were collected from women aged 20-30 years old (young group, 94 embryos) and >37 years (old group, 89 embryos). Expression of OCT4, SOX2, CDX2, GATA3, YAP genes and the relevant proteins, in the both groups were evaluated using respectively quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence methods. Results There was no significant difference in the expression level of OCT4, SOX2, CDX2, GATA3 and YAP genes in blastocyst and morula stages, between the two groups. However, SOX2 and CDX2 gene expressions in morula stage embryos of the old group was statistically lower than that of the young group (P=0.007 and P=0.008, respectively). Additionally, in the embryos collected from women with >37 years of age, at the blastocyst stage, phospho-YAP (p-YAP) protein was found to be accumulated in the TE, but it was almost disappeared from the ICM. Additionally, in the old group, contrary to the expectation, YAP protein was expressed in the ICM, rather than TE. Conclusion The results of this study showed that YAP and P-YAP among the Hippo signalling pathway may be altered by increasing age.

89-100

Authors: Haipeng Feng, Bo Lin, Yifei Zheng, Junnv Xu, Ying Zhou, Kun Liu, Mingyue Zhu, Mengsen Li

Objective Explore the effect of GATA5 expression on Paclitaxel inhibiting growth of hepatocellular carcinoma (HCC) cells. Materials And Methods In the experimental study, HCC cell lines (HLE, Bel7402 and PLC/PRF/5) were treated with different concentrations of Paclitaxel (5-20 mg/ml) for 24 hours. HLE cells were transfected with GATA5-siRNA vector, while Bel7402 and PLC/PRF/5 cells were transfected with overexpressed GATA5 vector for 24 hours, followed by treatment of the cells with Paclitaxel (10 mg/ml) for 24 hours and subsequently 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay to detect growth of HCC cells. Soft agar cultured was used to analyze formation of colony. Apoptosis of HCC cells were detected by Flow cytometer. Migration of HCC cells was observed by trawell assays. Western blotting and laser confocal microscopy were utilized to detect expression and location of the proteins. Results Inhibiting expression of GATA5 reduced sensitivity of HLE cells to Paclitaxel, while overexpression of GATA5 increased sensitivity of Bel7402 cells and PLC/PRF/5 cells to Paclitaxel. Overexpression of GATA5 played a role in stimulating Paclitaxel to inhibit growth, colony formation and migration, as well as enhance apoptosis in HCC cells. Overexpression of GATA5 also promoted Paclitaxel to inhibit expression of reprogramming genes, such as Nanog, EpCAM, c-Myc and Sox2 in Bel7402 and PLC/PRF/5 cells. Inhibited expression of GATA5 led to enhancement of the expression of CD44 and CD133, in HLE cells. Overexpression of GATA5 was not only alone but also synergized with Paclitaxel to inhibit expression of CD44 and CD133 in Bel7402 or PLC/PRF/5 cells. Conclusion Overexpression of GATA5 played a role in enhancing Paclitaxel to inhibit the malignant behaviors of HCC cells. It was involved in suppressing expression of the reprogramming genes and stemness markers. Targeting GATA5 is an available strategy for applying paclitaxel to therapy of patients with HCC.

110-116

Authors: Nadia Pourmoshir, Gholamreza Motalleb, Sadeq Vallian

Objective Thirteen million cancer deaths and 21.7 million new cancer cases are expected in the world by 2030. Breast cancer is considered as the main cause of cancer mortality in women aged 20-59 years. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and they are highly expressed in malignancies, including breast cancer. The role of miRNAs in the pathogenesis of breast cancer is not fully understood. In the present study, for the first time, the impact of hsa-miR-423 rs6505162 on breast cancer risk was investigated in the central province of Iran, Isfahan. Materials And Methods This case-control study was conducted on 153 clinicopathological proven breast cancer patients and 153 sex-matched healthy women with no history of any cancer type and relative patients. The patients and controls were genotyped and association of their clinical characteristics with hsa-miR-423 rs6505162 genotype was analyzed. Results The findings indicated that CC genotype of hsa-miR-423 rs6505162 was associated with the increased risk of breast cancer [odds ratio (OR)=2.37, 95% confidence interval (CI)=1.29-4.35 and P=0.0023, CC vs. AA]. Conclusion The data suggested that hsa-miR-423 rs6505162 could be considered as a novel risk factor in breast cancer pathogenesis in Isfahan province of Iran.

125-132

Authors: Davod Ilbeigi, Mitra Nourbakhsh, Parvin Pasalar, Reza Meshkani, Hajar Shokri Afra, Ghodratollah Panahi, Mohammad Borji, Roya Sharifi

Objective Nicotinamide phosphoribosyltransferase (NAMPT), which is responsible for biosynthesis of nicotinamide adenine dinucleotide (NAD), has a regulatory role in cellular metabolism and thus, might be implicated in non-alcoholic fatty liver disease (NAFLD). This study aimed to show how NAMPT down-regulation in liver cells influences lipid metabolism and sirtiun 1 (SIRT1), as the main NAD-dependent deacetylase enzyme. Materials And Methods In this experimental study, HepG2 cells were transfected with NAMPT siRNA and hepatic triglyceride (TG) content and SIRT1 deacetylase activity were measured by colorimetric and fluorometric methods, respectively. Gene expression of fatty acid synthase (FAS) and sterol regulatory element-binding protein-1c (SREBP- 1c) was evaluated by real-time polymerase chain reaction (PCR). Total protein level and the phosphorylated form of acetyl-CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK) were also investigated by western blotting. Results Knockdown of NAMPT significantly promoted the accumulation of TG in HepG2 cells, accompanied by a remarkable decline in SIRT1 deacetylase activity. A significant rise in the gene expression of two key lipogenic factors, FAS and SREBP-1c was also observed. These effects were also accompanied by decreased phosphorylation of ACC and AMPK. On the other hand, treatment of transfected cells with either NAD, as the SIRT1 substrate or resveratrol, as the SIRT1 activator reversed the outcomes. Conclusion These results demonstrated a protective role for NAMPT against NAFLD and its involvement in the regulation of de novo lipogenesis through the SIRT1/AMPK pathway.

141-147

Authors: Sina Habibi, Gholamreza Khamisipour, Narges Obeidi, Saeedeh Zare Jaliseh

Objective The aim of this study was to investigate the possibility of producing safe hematopoietic stem cells without the use of viral infectious agents that can be used in stem cell transplantation. Materials And Methods In this experimental study, after single layer cell formation, human primary fibroblast cells were treated with static electromagnetic fields of 10 and 15 milli Tesla (mT) for 20 minutes each day for seven consecutive days. On the seventh day and immediately after the last radiation, the cells were added to the wells containing specific hematopoietic stem cell expansion media. After 21 days and colony formation, the cells belonging to each group were evaluated in terms of the expression of CD34, CD38, and GATA-1 genes using quantitative real-time polymerase chain reaction (PCR), as well as surface marker expression of CD34 by flow cytometry. Results Exposure to 10 mT and 15 mT electromagnetic field increased the expression of CD34 and CD38 genes (P<0.05). This increase in gene expression levels were 2.85 and 1.84 folds, respectively, in the 10mT group and 6.36 and 3.81 folds, respectively, in the 15 mT group. The expression of the GATA-1 gene in the 10 mT and 15 mT groups was not significantly different from that of the control group (P<0.05). Electromagnetic waves caused a marked increase in the expression of the CD34 marker at the surface of reprogrammed cells. The rate of expression was about 42.3% in the 15 mT group and 23.1% in the 10 mT group. Conclusion The presence of human primary fibroblasts exposed to electromagnetic fields can increase the expression of specific hematopoietic genes. This method can be suitable for reprogramming cells differentiated into hematopoietic- like stem cells and does not pose the risks of retroviral use.

151-154

Authors: Shaghayegh Ghavami, Shabnam Shahrokh, Nikoo Hossein-Khannazer, Anastasia Shpichka, Hamid Aghdaei, Peter Timashev, Massoud Vosough

Inflammatory bowel diseases (IBDs) are chronic disorders of the gastrointestinal tract. The goal of IBD treatment is to reduce the inflammation period and induce long-term remission. Use of anti-inflammatory drugs including corticosteroids, immunosuppressants and biologicals, is often the first step in the treatment of IBD. Therefore, IBD patients in pandemic of infectious diseases are considered a high-risk group. The public believes that IBD patients are at a higher risk in the current coronavirus 2 pandemic. Nevertheless, these patients may experience mild or moderate complications compared to healthy people. This might be because of particular anti-TNF-α treatment or any immunosuppressant that IBD patients receive. Moreover, these patients might be silent carrier for the virus.

166-168

Authors: Alireza Asgari

Mankind is witnessing economic uncertainty due to a health crisis as never before. In the era of industrialization where the emergence of invisible enemies of humans is causing a great death toll, "nothing seems more universal than health", the old proverb in nearly all human cultures is once again rebirthed by the current COVID-19 pandemic. Nevertheless what is distinctive is that the SARS-CoV-2 seems to be unequally targeting a particular sector of the populations with risk factors for preventable diseases. Comorbidities, mainly non-communicable diseases (NCDs), seem to be the primary contributors of the current pandemic and not the SARS-CoV-2 per se. The present letter attempts to underscore the converging pattern of communicable (CDs) and NCDs in human toll. For the tens of thousands of lives coming to an end since the turn of the year, we are all truly sad, but thankful to the virus for unearthing the grave need of the mankind to improve his life style and behaviors. It directs us to revisit the values and ultimately save millions of lives in future.