Cell Journal (Yakhteh)

603-612

Authors: Mohammad Hossein Abnosi; Javad Sargolzaei; Farshid Nazari

Objective: We previously reported that cadmium (Cd) inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In addition, gallic acid (GA) improves BMSC differentiation. Here, we aim to study the ability of GA to prevent osteogenic inhibition induced by Cd. Materials and Methods: In this experimental study, BMSCs were extracted and purified from Wistar rats and their viability was determined in the presence of Cd and GA. The results indicated that 1.5 μM Cd and 0.25 μM of GA were appropriate for further investigation. After 20 days in osteogenic medium, matrix production was analysed by alizarin red, calcium content, and alkaline phosphatase (ALP) activity. Osteogenic-related genes and collagen 1A1 (COL1A1) protein expressions were investigated. The preventive effect of GA on oxidative stress and metabolic change induced by Cd was estimated. Results: GA counteracted the inhibitory effect of Cd on matrix production and significantly (P=0.0001) improved the osteogenic differentiation ability of BMSCs. Also, GA prevented the toxic effect of Cd on osteogenic-related gene expressions and nullified the reducing effect of Cd on COL1Al and ALP activity. A significant reduction (P=0.0001) in malondialdehyde and lactic acid concentration showed that GA counteracted both oxidative stress and metabolic changes caused by Cd. Conclusion: GA prevented the toxic effect of Cd, an environmental pollutant and a factor in osteoporosis.

625-632

Authors: Liguo Wang; Yi Zhou; Hui Lin; Kezhu Hou

Objective: This study aims to investigate the potential role of relaxin, a peptide hormone, in preventing cellular deterioration and death in gastric carcinoma cells under hypoxic conditions. It explores the effects of recombinant relaxin 2 (RLXH2) on growth, cell differentiation, invasive potential, and oxidative damage in these cells. Materials and Methods: In this experimental study, the NCI-N87 cell line was cultured under normal conditions and then subjected to hypoxia using cobalt chloride (CoCl2). The cells were treated with RLXH2, and various assays were performed to assess cellular deterioration, death, and oxidative stress. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to measure the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1, and the translocation of Nrf2 to the nucleus was confirmed through Western blot analysis. Results: This study demonstrates, for the first time, that RLXH2 significantly reduces the formation of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH) in gastric cancer cells under hypoxic conditions. RLXH2 also enhances the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT), leading to a decrease in hypoxia-induced oxidative damage. RLXH2 promotes the translocation of Nrf2 to the nucleus, resulting in HO-1 expression. Conclusion: Our findings suggest that RLXH2 plays a significant protective role against hypoxia-induced oxidative damage in gastric carcinoma cells through the Nrf2/HO-1 signalling pathway. This research contributes to a better understanding of the potential therapeutic applications of RLXH2 in gastric cancer treatment.

645-654

Authors: Gözde Aydoğan Kılıç; Mojahed Alsafi

Objective: The present study aims to investigate the role of breast cancer-susceptibility gene 1 (BRCA1) protein in the β-Glucan (βG) molecule mediated regulation of lipopolysaccharide (LPS)-induced liver genotoxicity. Materials and Methods: In this experimental study, totally, 32 male Swiss Albino mice were randomly divided into 4 equal groups: control (C), LPS-administered (LPS), βG-administered (βG) and βG-pre-administered/LPS-administered (βG+LPS). The βG was injected at the dose of 150 mg/kg/day intraperitoneally (i.p.) for 3 days. A single dose of 4 mg/ kg (i.p.) LPS was administered 24 hours after the last βG injection. BRCA1 expression was determined by western blot analysis and confirmed by quantitative immunofluorescence. Proliferating cell nuclear antigen (PCNA), nuclear factor erythroid 2–related factor (Nrf2) and 8-OHdG protein levels were also determined by the immunofluorescence analysis. The alkaline comet assay was performed. superoxide dismutase (SOD), catalase (CAT) and membrane lipid peroxidation were biochemically measured, and light microscopic histology was evaluated. Results: The BRCA1 expression level was significantly decreased in the LPS group. However, in the βG+LPS group, expression of BRCA1 protein was over 2 folds higher than the control. After the LPS induction, the DNA strand breaks, oxidative DNA lesions and abnormal proliferation of the liver cells were almost entirely suppressed in βG preadministrated animals, indicating the BRCA1 mediated ubiquitination of PCNA and activation of the DNA damage repair pathways. Activation of Nrf2 in the βG+LPS group resulted in an increase in the levels of Nrf2 pathway dependent antioxidant enzymes SOD and CAT, prevented the peroxidation of membrane lipids and maintained the histological architecture of the liver. Conclusion: The results manifested that the βG is a strong inducer of the BRCA1 protein expression in the LPSinduced hepatic stress and the protein constitutes the key component of a βG mediated liver protection against an LPS-induced genotoxic and pathological damage.

660-664

Authors: Kyung-Ah Cho; Jiyun Kwon; Hyeon Ju Kim; So-Youn Woo

One of the most affected aspects of the aging process is immunity, with age-related immune system decline being responsible for an increase in susceptibility to infectious diseases and cancer risk. On the other hand, the aging process is accompanied with low-grade pro-inflammatory status. This condition involves a persistent rise in cytokine levels that can activate both innate and adaptive immune systems. Finally, despite the fact that immunological responses to antigenic stimulations decrease with age, the incidence and prevalence of many common autoimmune diseases increase in the elderly population. Overall, the co-existence of a prolonged, low-grade inflammatory status and declining immune activity appears to be a paradoxical phenomenon. This study characterized skin inflammation in mouse dermatitis model of various ages to monitor possible changes of inflammatory responses during aging.

524-535

Authors: Shukoofeh Torabi; Morteza Zarrabi; Nikoo Hossein-Khannazer; Majid Lotfinia; Masoumeh Nouri; Roberto Gramignoli; Moustapha Hassan; Massoud Vosough

Objective: Macrophages are multifunctional immune cells widely used in immunological research. While autologous macrophages have been widely used in several biomedical applications, allogeneic macrophages have also demonstrated similar or even superior therapeutic potential. The umbilical cord blood (UCB) is a well-described source of abundant allogenic monocytes and macrophages that is easy to collect and can be processed without invasive methods. Current monocyte isolation procedures frequently result in heterogenous cell products, with limited yields, activated cells, and high cost. This study outlines a simple isolation method that results in high yields and pure monocytes with the potential to differentiate into functional macrophages. Materials and Methods: In the experimental study, we describe a simple and efficient protocol to isolate highpurity monocytes. After collection of human UCB samples, we used a gradient-based procedure composed of three consecutive gradient steps: i. Hydroxyethyl starch-based erythrocytes sedimentation, followed by ii. Mononuclear cells (MNCs) isolation by Ficoll-Hypaque gradient, and iii. Separation of monocytes from lymphocytes by a slight hyperosmolar Percoll gradient (0.573 g/ml). Then the differentiation potential of isolated monocytes to pro- and antiinflammatory macrophages were evaluated in the presence of granulocyte colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF), respectively. The macrophages were functionally characterized as well. Results: A high yield of monocytes after isolation (25 to 50 million) with a high purity (>95%) could be obtained from every 100-150 ml UCB. Isolated monocytes were defined based on their phenotype and surface markers expression pattern. Moreover, they possess the ability to differentiate into pro- or anti-inflammatory macrophages with specific phenotypes, gene/surface protein markers, cytokine secretion patterns, T-cell interactions, and phagocytosis activity. Conclusion: Here we describe a simple and reproducible procedure for isolation of pure monocytes from UCB, which could be utilized to provide functional macrophages as a reliable and feasible source of allogenic macrophages for biomedical research.

546-553

Authors: Yongqiang Lai; Junhao Liu; Xiao Hu; Xiancheng Zeng; Peng Gao

Objective: Owing to the lethality of liver cancer, it is considered as one of the devastating types of cancers across the globe. Consistently, the study was designed to elucidate the role and to explore the therapeutic implications of miR-145 in human liver cancer. Materials & methods: In the current experimental study, gene expression was determined by RT-PCR analysis. Transfection of cancer cells was carried out using Lipofectamine 2000. The cell proliferation of liver cancer cells was estimated by MTT assay. Clonogenic assay was performed for analysis of colony forming potential of cancer cells. Flow cytometry was done to analyze the cell cycle phase distribution of cancer cells. Transwell chamber assay was performed to assess the motility of cancer cells. Western blotting was done to estimate the expression levels of proteins. Dual luciferase assay was performed for interaction analysis of miR-145 with CDCA3. Result: The miR-145 expression was found to be downregulated in liver cancer cells. The transfection mediated overexpression of miR-145 inhibited the cancer cell proliferation and when miR-145 inhibitor was transfected, cancer cells showed higher proliferation rates. Enrichment of miR-145 levels led to cell cycle arrest at G2/M phase by inhibiting cyclin B1. miR-145 also restricted the migration and invasion of cancer cells. CDCA3 was shown to be the intracellular target of miR-145 and it was found that the inhibitory effects of miR-145 were modulated through CDCA3, intracellularly. Conclusion: The current study clearly revealed that there is a need to investigate the regulatory role of different molecular entities like microRNAs in cancer development to better understand mechanics behind this pathogenesis and design more effective combating strategies against cancer.

564-569

Authors: Nasim Alipour; Somaye Fallahnezhad; Javad Bagheri; Hamideh Babaloo; Fatemeh Tahmasebi; Ghasem Sazegar; Hossein Haghir

Objective: Diabetes in pregnancy is a prevalent disease that can affect the central nervous system of the fetus by hyperglycemia. This study aimed to investigate the impact of maternal diabetes on neuronal apoptosis in the superior colliculus (SC) and the lateral geniculate nucleus (LGN) in male neonates born to diabetic mothers. Materials and Methods: In this experimental study, female adult rats were separated into three groups: control, diabetic (induced using an intraperitoneal injection of streptozotocin), and insulin-treated diabetic [diabetes controlled by subcutaneous neutral protamine hagedorn (NPH)-insulin injection]. Male neonates from each group were euthanized on 0, 7, and 14 postnatal days (P0, P7, and P14, respectively), and apoptotic cells were identified using TUNEL staining. Results: The numerical density per unit area (NA) of apoptotic cells was significantly higher in SC and the dorsal LGN (dLGN) in neonates born to the diabetic rats compared to the control group at P0, P7, and P14. However, insulin treatment normalized the number of apoptotic cells. Conclusion: This study demonstrated that maternal diabetes increased apoptosis in dLGN and SC of male neonates at P0, P7, and P14.

579-590

Authors: Elham Sadeghi; Mojtaba Rezazadeh Valojerdi; Mojdeh Salehnia

Objective: This study evaluates the interaction of mouse blastocysts as a surrogate embryo on a recellularized endometrial scaffold by seeding human endometrial mesenchymal cells (hEMCs). Materials and Methods: In this experimental study, prepared decellularized human endometrial tissues were characterized by morphological staining, DNA content analysis, and scanning electron microscopic (SEM) analysis. The scaffolds were subsequently recellularized by hEMCs. After seven days of cultivation, the mouse blastocysts were co-cultured on the recellularized scaffolds for 48 hours. Embryo attachment and implantation within these scaffolds were evaluated at the morphological, ultrastructural, molecular, and hormonal levels. Results: There was no morphological evidence of cells and nuclei in the decellularized scaffold. DNA content significantly decreased by 89.92% compared to the control group (P<0.05). Both decellularized and native tissues had similar patterns of collagen bundles and elastin fibers, and glycosaminoglycan (GAGs) distribution in the stroma. After recellularization, the hEMCs attached to the scaffold surface and penetrated different parts of these scaffolds. In the co-cultured group, the embryo attached to the surface of the scaffold after 24 hours and penetrated the recellularized endometrial tissue after 48 hours. We observed multi-layered organoid-like structures formed by hEMC proliferation. The relative expressions of epithelial-related genes, ZO-1 and COL4A1, and SSP1, MMP2, and PRL, as decidualizationrelated genes, were significantly higher in the recellularized group on day 9 in the presence of the embryo compared to the other groups (P<0.05). Beta human chorionic gonadotropin (β-hCG) and prolactin were statistically increased in the recellularized group on day 9 group (P<0.05). Conclusion: hEMCs and mouse embryo co-cultured on a decellularized endometrial scaffold provides an alternative model to study embryo implantation and the earlier stage of embryo development

447-454

Authors: Junming Zhang; Li Liu; Yuexia Li; Jinglei Wu; Xiangxin Lou

Objective: Previous reports showed that mouse embryonic fibroblasts (MEFs) could support pluripotent stem cell selfrenewal and maintain their pluripotency. The goal of this study was to reveal whether the decellularized extracellular matrix derived from MEFs (MEF-ECM) is beneficial to promote the proliferation of inner ear-derived cells. Materials and Methods: In this experimental study, we prepared a cell-free MEF-ECM through decellularization. Scanning electron microscope (SEM) and immunofluorescent staining were conducted for phenotype characterization. Organs of Corti were dissected from postnatal day 2 and the inner ear-derived cells were obtained. The identification of inner ear-derived cells was conducted by using reverse transcription-polymerase chain reaction (RT-PCR). Cell counting kit-8 (CCK-8) was used to evaluate the proliferation capability of inner ear-derived cells cultured on the MEFECM and tissue culture plate (TCP). Results: The MEF-ECM was clearly observed after decellularization via SEM, and the immunofluorescence staining results revealed that MEF-ECM was composed of three proteins, including collagen I, fibronectin and laminin. Most importantly, the results of CCK-8 showed that compared with TCP, MEF-ECM could effectively facilitate the proliferation of inner ear-derived cells. Conclusion: The discovery of the potential of MEF-ECM in promoting inner ear-derived cell proliferation indicates that the decellularized matrix microenvironment may play a vital role in keeping proliferation ability of these cells. Our findings indicate that the use of MEF-ECM may serve as a novel approach for expanding inner ear-derived cells and potentially facilitating the clinical application of inner ear-derived cells for hearing loss in the future.

461-469

Authors: Pakhshan Rahimpour; Dariush Sheikholeslami-Vatani; Mohammad Raman Moloudi; Saeed Ghaeeni

Objective: This study aimed to compare the effect of different physical training on the mechanism of ceramidedependent insulin resistance in the flexor hallucis longus (FHL) muscle of diabetic rats. Materials and Methods: In this experimental study, 7 healthy as a healthy control (HC) group, and 21 diabetics (55 mg/ kg Streptozotocin) Wistar rats (200-220 g; 8-10 weeks old) divided into the diabetic control (DC), moderate continuous training (MCT), and moderate intensity interval training (MIIT) groups. Both MCT (55-70% of maximal oxygen uptake (VO2max), and MIIT (85% VO2max) groups trained for 10-25 minutes at a speed of 10-20 m/minutes. The changes in the expression of blood glucose, insulin, insulin resistance, lipid profile and total ceramide were measured as well as ceramide synthase-1, Glucose transporter type 4 (GLUT4), Protein kinase B known as Akt, phosphorylated protein kinase B known as pAkt, protein kinase C (PKC), and tumour necrosis factor α (TNFα). Results: Blood glucose, triglyceride (TG) and ceramide synthase-1 (CS1) expression levels in the MCT group decreased in comparison with the DC group. FHL protein expression of GLUT4 in the MCT group was higher than the DC group. FHL expression of GLUT4, pAKT, AKT/pAKT, PKC, CS1 and total ceramide in the MIIT group were higher than the DC group. Cholesterol, low-density lipoprotein (LDL), TG, and TNF-α protein expression in the MIIT group were lower than the DC group. GLUT4, PKC, pAKT, AKT/pAKT in the MIIT group were higher, and total ceramide and TNF-α were lower in the MIIT group than the MCT group. Conclusion: It seems that both training plan MIIT and MCT have favorable effects on the metabolism of glucose, insulin, lipids, and the decrease of TNFα level in the diabetes, but in connection with the improvement of the ceramides mechanism, it seems that the MIIT training plan is more optimal than MCT training plan.

483-495

Authors: Elham Ghanbari; Mozafar Khazaei; Ahmad Mehdipour; Alibaradar Khoshfeterat; Behrooz Niknafs

Objective: The use of biocompatible scaffolds with appropriate characteristics to treat large bone defects has attracted significant attention. The main objective of the current study is to fabricate a 3D nanocomposite structure that contains green synthesized magnesium oxide nanoparticles (MgONPs) and bacterial cellulose (BC) nanofibres, as a bioscaffold for bone regeneration. Materials and Methods: In this experimental study, Camellia sinensis extract was used as the green method to synthesize MgONPs. The synthesized hydrogels were evaluated for their porosity, morphology, degradation rate, mechanical features, cell attachment, and cytocompatibility. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, real-time reverse transcription-polymerase chain reaction (RT-PCR), and alizarin red staining. Results: MgONPs significantly increased both mechanical strength (P=0.009) and porosity (P=0.01) of the BC hydrogels. Human MG-63 osteoblast proliferation significantly increased in the MgONP-BC group compared to the pure BC group (P=0.003). Expression rates of both the ALP (P=0.001) and osteocalcin (OCN) genes were significantly enhanced in cells seeded on the MgONP-incorporated BC. MG-63 cells had significantly greater calcium deposition and ALP activity (P=0.002) on the MgONP-BC scaffold compared to the BC at day 21. Conclusion: The MgONP-BC scaffold can promote the osteogenic activity of osteoblast-like cells, which indicates its therapeutic potential for bone tissue regeneration.

505-512

Authors: Hanieh Mahi; Zahra Yousefi; Fatemeh Toufan; Maryam Yarmohammadi; Moslem Jafarisani; Nahid Eskandari; Mohammad- Hossein Shams; Reza Jafari

Objective: Rheumatoid arthritis (RA) is a common progressive autoimmune disorder that causes chronic inflammation of the joints and damage to other organs. Previous studies have reported the important role of miRNA-146a in the pathogenesis of RA. In addition, the anti-inflammatory and modulatory effects of oleuropein (OLEU) on the expression pattern of microRNAs (miRNAs) have been shown in different diseases. Therefore, this study aimed to evaluate both the sensitivity and specificity of miRNA-146a and determine the potential effects of OLEU on the expression levels of miRNA-146a and tumour necrosis factor-alpha (TNF-α) in RA patients. Materials and Methods: The participants in this experimental study were divided into 2 groups: RA (n=45) and healthy controls (n=30). The isolated peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of OLEU; and the level of TNF-α expression, anti-citrullinated protein, and miRNA-146a were determined using enzyme-linked immunoassay and real-time polymerase chain reaction, respectively. In addition, the receiver operating characteristic (ROC) curve analysis evaluated the sensitivity and specificity of miRNA-146a in RA patients. Results: Results revealed a positive correlation between the levels of miRNA-146a expression with the serum levels of C-reactive protein (CRP) and rheumatoid factor (RF) in RA patients. In addition, OLEU treatment decreased the levels of TNF-α and miRNA-146a expression in treated PBMCs samples compared with untreated cells. The ROC curve analysis showed an 85% sensitivity and 100% specificity of miRNA-146a in RA patients. Conclusion: Therefore, miRNA-146a can be used as a useful biomarker for RA diagnosis, particularly for early detection. In addition, OLEU could suppress inflammation in RA patients through the regulation of miRNA-146a.

372-382

Authors: Afshin Noori; Mohammad Reza Mokhber Dezfouli; Sareh Rajabi; Fatemeh Ganji; Zahra Ghezelayagh; Elie El Agha; Hossein Baharvand; Sirous Sadeghian Chaleshtori; Yaser Tahamtani

Objective: Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells. Materials and Methods: This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments. Results: We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker) genes. Conclusion: Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

391-398

Authors: Xiaoqi Xiong; Jun Li; Zhen Chen; Changjun Luo; Wei Wei; Bing Li; Yi Kang; Xiuhong Nong; Fen Ai; jing Zhang

Objective: Peroxiredoxin-3 (Prx-3) is widely acknowledged as an antioxidant that protects against mitochondrial reactive oxygen species. Nonetheless, its role in cardiac fibrosis has not been elucidated. We aim to explore the role and mechanism of Prx-3 in cardiac fibrosis. Materials and Methods: In this experimental study, mice received subcutaneous injections of isoproterenol (ISO) for 14 consecutive days (10 mg/kg/d for three days, followed by 5 mg/kg/d for 11 days) to establish a cardiac fibrosis model. The mice were subsequently injected with adenovirus-Prx-3 (ad-Prx-3) to enable Prx-3 overexpression. Echocardiography was used to evaluate cardiac function. Mice heart fibroblasts were isolated and stimulated with transforming growth factor β1 (TGFβ1) to induce fibrosis in vitro. Cells were also transfected with ad-Prx-3 for overexpression of Prx-3. Results: Echocardiographic diameters and fibrosis markers indicated that Prx-3 could inhibit ISO-induced cardiac dysfunction and fibrosis. Fibroblasts with Prx-3 overexpression exhibited reduced activation, proliferation, and collagen transcription. We found that Prx-3 reduced the expression of NADPH oxidase 4 (NOX4) and reduced P38 levels. After treatment with a P38 inhibitor, the Prx-3 overexpression-induced anti-fibrosis effect was mitigated. Conclusion: Prx-3 could protect against ISO-induced cardiac fibrosis by inhibiting the NOX4-P38 pathway.

407-417

Authors: Melika Zangeneh Motlagh; Nazanin Mahdavi; Zohreh Miri-Lavasani; Pouyan Aminishakib; Mahsa Khoramipour; Mandana Kazem Arki; Zohreh Mirlavasni; Niloufar Rezaei; Nikoo Hossein-Khannazer; Massoud Vosough

Objective: Surgery and chemotherapy are the most common therapeutic strategies proposed for oral squamous cell carcinoma (OSCC). However, some of the disadvantages associated with the current methods like unwanted side effects and poor drug response lead the scientist to seek for novel modalities and delivery approaches to enhance the efficacy of treatments. The study aimed to assess the effectiveness of disulfiram (DSF)-loaded Niosomes on cancerous phenotypes of the OSCC cells. Materials and Methods: In this experimental study, an optimum formulation of DSF-loaded Niosomes was developed for the treatment of OSCC cells to reduce drug doses and improve the poor stability of DSF in the OSCC environment. The design expert software was utilized to optimize the particles in terms of size, polydispersity index (PDI), and entrapment efficacy (EE). Results: Acidic pH increased the release rate of DSF from these formulations. The size, PDI, and EE of Niosomes were more stable at 4°C compared to 25°C. The results indicated that DSF-loaded Niosomes could induce apoptosis (P=0.019) in the OSCC cells compared to the control group. Moreover, it could reduce colony formation ability (P=0.0046) and also migration capacity of OSCC cells (P=0.0015). Conclusion: Our findings indicated that the application of proper dose of DSF-loaded Niosomes (12.5 μg/ml) increases apoptosis, decreases colony formation capacity and declines the migration ability of OSCC cells.

427-436

Authors: Jamileh Jahanbakhsh; Maryam Farid Mojtahedi; Nariman Moradi; Reza Fadaei; Afsaneh Tehranian; Rahim Rostami; Ladan Kashani; Ashraf Moeini; Shahin Alizadeh-Fanalou; Sepideh Barzin Tond; Mahshad Khodarahmian; Soudabeh Fallah

Objective: An association between microRNAs (miRNAs) and adhesion proteins expression with repeated implantation failure (RIF) has been recently reported; however, these findings are controversial. This study aims to evaluate the endometrial and circulating expressions of miR-145, miR-155-5p, and miR-224 in addition to the endometrial expressions of membrane protein palmitoylated-5 (MPP-5) and endothelial cell adhesion molecule-1 (PECAM-1) in patients with RIF compared to control subjects. Materials and Methods: This case-control study was carried out between June 2021-July 2022. Subjects included 17 patients with RIF and 17 control subjects, who had previous spontaneous term pregnancy with a live birth, who referred to the Medical Centre of Arash Hospital, Tehran, Iran. Endometrial tissue samples were obtained via hysteroscopy and Pipelle catheter in the RIF and control subjects, respectively. Plasma samples were collected after ovulation in all subjects. The expression levels of MPP5, PECAM-1, miR-224, miR-145, and miR-155-5p were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The student’s t test, chi-square, Mann-Whitney U, and analysis of covariance (ANCOVA) were used for data analyses. Results: RIF patients had less endometrial miR-155-5p expression, and higher endometrial and circulating expressions of miR-145 and miR-224 compared to control subjects. Endometrial PECAM-1 and MPP5 expression significantly decreased in patients with RIF compared to the control group. There was a positive correlation between circulating miR-224 and endometrial miR-155-5p, and between circulating miR-155-5p and endometrial PECAM-1 expression levels in patients with RIF. Conclusion: The present study suggests that circulating miR-224, endometrial miR-145, and PECAM-1 can be reliable, novel biomarkers for diagnosis of RIF.

291-299

Authors: Wenning Cui; Dengfeng Yang; Xiaoyan Chen; Hong Yu

Objective: Osteosarcoma (OS) is an uncommon sarcoma with osteoid formation in conjunction with malignant mesenchymal cells on histological examination. SP-8356 has been reported to exhibit anti-cancer properties in human cancers. However the impact of SP-8356 on OS is largely unknown. The metabolic pathways are coordinated by AMPactivated protein kinase (AMPK), which maintains a balance between the supply and demand of nutrients and energy. This study aimed to investigate effect of SP-8356 on proliferation and apoptosis of OS cells and tumor growth in mice. Furthermore, involvement of PGC-1α/TFAM and AMPK-activation was studied. Materials and Methods: In the experimental study, Saos-2 and MG63 cells were cultured with SP-8356 for 24 hours and analysed for cellular proliferation using MTT assay. DNA fragmentation was studied using ELISA based kit. Furthermore, transwell chambers assay was used to determine cell migration and cell invasion. Targeted protein expression levels were assessed using western blotting. For in vivo studies, mice (5-6 weeks old) were implanted with either Saos-2 or MG63 cells on dorsal surface subcutaneously and they were administered with SP-8356 (10 mg/kg) for two weeks prior to bone tumor induction. Results: We found that SP-8356 exerted anti-proliferative effects on Saos-2 and MG63 cells. Furthermore, SP-8356 treatment significantly restricted migration and invasion of Saos-2 and MG63 cells. Compared to the control, SP-8356 significantly reduced apoptotic cell death, while it increased PGC-1α and TFAM expressions. Without affecting body weight, SP-8356 significantly reduced tumor development in mice, as compared to the control group. Conclusion: SP-8356 was found to inhibit proliferation, suppressed cells migration and invasion and decreased OS tumor growth. Furthermore, SP-8356 was found to act through PGC-1α/TFAM and AMPK activations. SP-8356 can be therefore used as therapeutic agent for OS treatment.

307-316

Authors: Neda Kasiri; Seyed Mostafa Ghannadian; Reza Hosseini; Leila Ahmadi; Mahshid Rahmati; Fereshteh Ashtari; Abbasali Pourazar; Nahid Eskandari

Objective: In spite of the advances in therapeutic modalities, morbidity, due to multiple sclerosis (MS), still remains high. Therefore, a large body of research is endeavouring to discover or develop novel therapies with improved efficacy for treating MS patients. In the present study, we examined the immunomodulatory effects of apigenin (Api) on peripheral blood mononuclear cells (PBMCs) isolated from MS patients. We also developed an acetylated form of Api (apigenin- 3-acetate) to improve In its blood-brain barrier (BBB) permeability. Additionally, we compared its anti-inflammatory properties to original Api and methyl-prednisolone-acetate (a standard therapy), as a potential option in treating MS patients. Materials and Methods: The current study was an experimental-interventional research. The half maximal inhibitory concentration (IC50) values for apigenin-3-acetate, apigenin, and methyl-prednisolone-acetate were determined in healthy volunteers’ PBMCs (n=3). Gene expressions of T-box transcription factor (TBX21 or T-bet) and IFN-γ, as well as proliferation of T cells isolated from MS patients’ PBMCs (n=5), were examined in co-cultures of apigenin-3-acetate, Api and methyl-prednisolone-acetate after 48 hours of treatment, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: Our findings showed that apigenin-3-acetate, apigenin, and methyl-prednisolone-acetate at concentrations of 80, 80, and 2.5 M could inhibit Th1 cell proliferation after 48 hours (P=0.001, P=0.036, and P=0.047, respectively); they also inhibited T-bet (P=0.015, P=0.019, and P=0.022) and interferon-γ (IFN-γ) gene expressions (P=0.0001). Conclusion: Our findings suggested that Api may have anti-inflammatory properties, possibly by inhibiting proliferation of IFN-producing Th1 cells. Moreover, comparative immunomodulatory effects were found for the acetylated version of apigenin-3-acetate versus Api and methyl-prednisolone-acetate.

327-337

Authors: Atiyeh Eghbali; Yasaman Anvarinia; Mostafa Soltan Sanjari; Farzad Pakdel; Maryam Seyedsadr; Fatemehsadat Hosseini Mazinani; Leila Zare; Masoumeh Zarei-Kheirabadi; Leila Satarian

Objective: Traumatic optic neuropathy (TON) causes partial or complete blindness because death of irreplaceable retinal ganglion cells (RGCs). Neuroprotective functions of erythropoietin (EPO) in the nervous system have been considered by many studies investigating effectiveness of this cytokine in various retinal disease models. It has been found that changes in retinal neurons under conditions of glial cells are effective in vision loss, therefore, the present study hypothesized that EPO neuroprotective effect could be mediated through glial cells in TON model. Materials and Methods: In this experiment study, 72 rats were assessed in the following groups: intact and optic nerve crush which received either the 4000 IU EPO or saline. Visual evoked potential and optomotor response and RGC number were assessed and regenerated axons evaluated by anterograde test. Cytokines gene expression changes were compared by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Density of astrocytes cells, assessed by fluorescence intensity, in addition, possible cytotoxic effect of EPO was measured on mouse astrocyte culture in vitro. Results: In vitro data showed that EPO was not toxic for mouse astrocytes. Intravenous injection of EPO improved vision, in terms of visual behavioral tests. RGCs protection was more than two times in EPO, compared to the vehicle group. More regenerated axons were determined by anterograde tracing in the EPO group compared to the vehicle. Moreover, GFAP immunostaining showed while the intensity of reactive astrocytes was increased in injured retina, systemic EPO decreased it. In the treatment group, expression of GFAP was down-regulated, while CNTF was upregulated as assessed by qRT-PCR in the 60th day post-crush. Conclusion: Our study showed that systemic administration of EPO can protect degenerating RGCs. Indeed, exogenous EPO exerted neuroprotective and neurotrophic functions by reducing reactive astrocytic gliosis. Therefore, reduction of gliosis by EPO may be considered as therapeutic targets for TON.

347-353

Authors: Niloofar Rabiei; Ali Reza Soltanian; Maryam Farhadian; Fatemeh Bahreini

Objective: In microarray datasets, hundreds and thousands of genes are measured in a small number of samples, and sometimes due to problems that occur during the experiment, the expression value of some genes is recorded as missing. It is a difficult task to determine the genes that cause disease or cancer from a large number of genes. This study aimed to find effective genes in pancreatic cancer (PC). First, the K-nearest neighbor (KNN) imputation method was used to solve the problem of missing values (MVs) of gene expression. Then, the random forest algorithm was used to identify the genes associated with PC. Materials and Methods: In this retrospective study, 24 samples from the GSE14245 dataset were examined. Twelve samples were from patients with PC, and 12 samples were from healthy control. After preprocessing and applying the fold-change technique, 29482 genes were used. We used the KNN imputation method to impute when a particular gene had MVs. Then, the genes most strongly associated with PC were selected using the random forest algorithm. We classified the dataset using support vector machine (SVM) and naïve bayes (NB) classifiers, and F-score and Jaccard indices were reported. Results: Out of the 29482 genes, 1185 genes with fold-changes greater than 3 were selected. After selecting the most associated genes, 21 genes with the most important value were identified. S100P and GPX3 had the highest and lowest importance values, respectively. The F-score and Jaccard value of the SVM and NB classifiers were 95.5, 93, 92, and 92 percent, respectively. Conclusion: This study is based on the application of the fold change technique, imputation method, and random forest algorithm and could find the most associated genes that were not identified in many studies. We therefore suggest researchers use the random forest algorithm to detect the related genes within the disease of interest.