Cell Journal (Yakhteh)

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.

222-228

Authors: Jun Li; Yafei Bo; Bo Ding; Lei Wang

Objective: Gastric cancer is the fifth most common neoplasm and the fourth reason for mortality globally. Incidence rates are highly variable and dependent on risk factors, epidemiologic and carcinogenesis patterns. Previous studies reported that Helicobacter pylori (H. pylori) infection is one the strongest known risk factor for gastric cancer. USP32 is a deubiquitinating enzyme identified as a potential factor associated with tumor progression and a key player in cancer development. On the other hand, SHMT2 is involved in serine-glycine metabolism to support cancer cell proliferation. Both USP32 and SHMT2 are reported to be upregulated in many cancer types, including gastric cancer, but its complete mechanism is not fully explored yet. The present study explored possible mechanism of action of USP32 and SHMT2 in the progression of gastric cancer. Materials and Methods: In this experimental study, Capsaicin (0.3 g/kg/day) and H. pylori infection combination was used to successfully initiate gastric cancer conditions in mice. It was followed by 40 and 70 days of treatment to establish initial and advanced conditions of gastric cancer. Results: Histopathology confirmed formation of signet ring cell and initiation of cellular proliferation in the initial gastric cancer. More proliferative cells were also observed. In addition, tissue hardening was confirmed in the advanced stage of gastric cancer. USP32 and SHMT2 showed progressive upregulated expression, as gastric cancer progress. Immunohistologically, it showed signals in abnormal cells and high-intensity signals in the advanced stage of cancer. In USP32 silenced tissue, expression of SHMT2 was completely blocked and reverted cancer development as evident with less abnormal cell in initial gastric cancer. Reduction of SHMT2 level to one-fourth was observed in the advanced gastric cancer stages of USP32 silenced tissue. Conclusion: USP32 had a direct role in regulating SHMT2 expression, which attracted therapeutic target for future treatment.

238-246

Authors: Nahid Khosronezhad; Vahideh Hassanzadeh; Maryam Hezavehei; Abdolhossein Shahverdi; Maryam Shahhoseini

Objective: Choosing the optimal method for human sperm cryopreservation seems necessary to reduce cryoinjury. The aim of this study is to compare two cryopreservation methods including rapid-freezing and vitrification, in terms of cellular parameters, epigenetic patterns and expression of paternally imprinted genes (PAX8, PEG3 and RTL1) in human sperm which play a role in male fertility. Materials and Methods: In this experimental study, semen samples were collected from 20 normozoospermic men. After washing the sperms, cellular parameters were investigated. DNA methylation and expression of genes were investigated using methylation-specific polymerase chain reaction (PCR) and real-time PCR methods, respectively. Results: The results showed a significant decrease in sperm motility and viability, while a significant increase was observed in DNA fragmentation index of cryopreserved groups in comparison with the fresh group. Moreover, a significant reduction in sperm total motility (TM, P<0.01) and viability (P<0.01) was determined, whereas a significant increase was observed in DNA fragmentation index (P<0.05) of the vitrification group compared to the rapid-freezing group. Our results also showed a significant decrease in expression of PAX8, PEG3 and RTL1 genes in the cryopreserved groups compared to the fresh group. However, expression of PEG3 (P<0.01) and RTL1 (P<0.05) genes were reduced in the vitrification compared to the rapid-freezing group. Moreover, a significant increase in the percentage of PAX8, PEG3 and RTL1 methylation was detected in the rapid-freezing group (P<0.01, P<0.0001 and P<0.001, respectively) and vitrification group (P<0.01, P<0.0001 and P<0.0001, respectively) compared to the fresh group. Additionally, percentage of PEG3 and RTL1 methylation in the vitrification group was significantly increased (P<0.05 and P<0.05, respectively) compared to the rapid-freezing group. Conclusion: Our findings showed that rapid-freezing is more suitable method for maintaining sperm cell quality. In addition, due to the role of these genes in fertility, changes in their expression and epigenetic modification may affect fertility.

255-263

Authors: Mohammad Azimi Lamouty; Niloufar Shayan Asl; Abdollah Safari; Marzieh Ebrahimi; Hamed Daemi

Objective: The biological factors secreted from cells and cell-based products stimulate growth, proliferation, and migration of the cells in their microenvironment, and play vital roles in promoting wound healing. The amniotic membrane extract (AME), which is rich in growth factors (GFs), can be loaded into a cell-laden hydrogel and released to a wound site to promote the healing of the wound. The present study was conducted to optimize the concentration of the loaded AME that induces secretion of GFs and structural collagen protein from cell-laden AME-loaded collagen-based hydrogels, to promote wound healing in vitro. Materials and Methods: In this experimental study, fibroblast-laden collagen-based hydrogel loaded with different concentrations of AME (0.1, 0.5, 1, and 1.5 mg/mL, as test groups) and without AME (as control group), were incubated for 7 days. The total proteins secreted by the cells from the cell-laden hydrogel loaded with different concentrations of AME were collected and the levels of GFs and type I collagen were assessed using ELISA method. Cell proliferation and scratch assay were done to evaluate the function of the construct. Results: The results of ELISA showed that the concentrations of GFs in the conditioned medium (CM) secreted from the cell-laden AME-loaded hydrogel were significantly higher than those secreted by only the fibroblast group. Interestingly, the metabolic activity of fibroblasts and the ability of the cells to migrate in scratch assay significantly increased in the CM3-treated fibroblast culture compared to other groups. The concentrations of the cells and the AME for preparation of CM3 group were 106 cell/mL and 1 mg/mL, respectively. Conclusion: We showed that 1 mg/ml of AME loaded in fibroblast-laden collagen hydrogel significantly enhanced the secretion of EGF, KGF, VEGF, HGF, and type I collagen. The CM3 secreted from the cell-laden AME-loaded hydrogel promoted proliferation and scratch area reduction in vitro.

273-286

Authors: Parisa Esmaeili Tazangi; Faisal Alosaimi; Fatemeh Bakhtiarzadeh; Amir Shojaei; Ali Jahanshahi; Javad Mirnajafi-Zadeh

Objective: The mechanisms behind seizure suppression by deep brain stimulation (DBS) are not fully revealed, and the most optimal stimulus regimens and anatomical targets are yet to be determined. We investigated the modulatory effect of low-frequency DBS (L-DBS) in the ventral tegmental area (VTA) on neuronal activity in downstream and upstream brain areas in chemically kindled mice by assessing c-Fos immunoreactivity. Materials and Methods: In this experimental study, 4-6 weeks old BL/6 male mice underwent stereotaxic implantation of a unilateral stimulating electrode in the VTA followed by pentylenetetrazole (PTZ) administration every other day until they showed stage 4 or 5 seizures following 3 consecutive PTZ injections. Animals were divided into control, sham-implanted, kindled, kindled-implanted, L-DBS, and kindled+L-DBS groups. In the L-DBS and kindled+L-DBS groups, four trains of L-DBS were delivered 5 min after the last PTZ injection. 48 hours after the last L-DBS, mice were transcardially perfused, and the brain was processed to evaluate c-Fos expression by immunohistochemistry. Results: L-DBS in the VTA significantly decreased the c-Fos expressing cell numbers in several brain areas including the hippocampus, entorhinal cortex, VTA, substantia nigra pars compacta, and dorsal raphe nucleus but not in the amygdala and CA3 area of the ventral hippocampus compared to the sham group. Conclusion: These data suggest that the possible anticonvulsant mechanism of DBS in VTA can be through restoring the seizure-induced cellular hyperactivity to normal.

158-164

Authors: Reza Chavooshi; Mohammad Reza Ranjkesh; Behnam Hashemi; Leila Roshangar

The goal of tissue engineering is to repair and regenerate diseased and damaged tissues and organs with functional and biocompatible materials that mimic native and original tissues which leads to maintaining and improvement of tissue function. Lignin and cellulose are the most abundant polymers in nature and have many applications in industry. Moreover, recently the physicochemical behaviors of lignin and cellulose, including biocompatibility, biodegradability, and mechanical properties, have been used in diverse biological applications ranging from drug delivery to tissue engineering. To assess these aims, this review gives an overview and comprehensive knowledge and highlights the origin and applications of lignin and cellulose-derived scaffolds in different tissue engineering and other biological applications. Finally, the challenges for future development using lignin and cellulose are also included. Plant-based tissue engineering is a promising technology for progressing areas in biomedicine, regenerative medicine, and nanomedicine, with much research focused on the development of newer material scaffolds with individual specific features to make functional and biocompatible tissues and organs for medical applications.

176-183

Authors: Nazli Servatian; Saeid Abroun; Seyed Abolhassan Shahzadeh Fazeli; Masoud Soleimani

Objective: Beta-thalassemia is a group of inherited hematologic. The most HBB gene variant among Iranian beta-thalassemia patients is related to two mutations of IVSII-1 (G>A) and IVSI-5 (G>C). Therefore, our aim of this study is to use the knock in capability of CRISPR Cas9 system to investigate the correction of IVSII-1 (G>A) variant in Iran. Materials and Methods: In this experimental study, following bioinformatics studies, the vector containing Puromycin resistant gene (PX459) was cloned individually by designed RNA-guided nucleases (gRNAs), and cloning was confirmed by sequencing. Proliferation of TLS-12 was done. Then, the transfect was set up by the vector with GFP marker (PX458). The PX459 vectors carrying the designed gRNAs together with Single-stranded oligodeoxynucleotides (ssODNs) as healthy DNA pattern were transfected into TLS-12 cells. After taking the single cell clones, molecular evaluations were performed on single clones. Sanger sequencing was then performed to investigate homology directed repair (HDR). Results: The sequencing results confirmed that all three gRNAs were successfully cloned into PX459 vector. In the transfection phase, The TLS-12 containing PX459-gRNA/ssODN was selected. Molecular evaluations showed that the HBB gene was cleaved by the CRISPR/Cas9 system, that indicates that the performance of non-homologous end joining (NHEJ) repair system. Sequencing in some clones cleaved by the T7E1 enzyme showed that HDR was not confirmed in these clones. Conclusion: IVS-II-1 (G> A) mutation, which is the most common thalassemia mutation especially in Iran, the CRISPR/ Cas9 system was able to specifically target the HBB gene sequence. This could even lead to a correction in the mutation and efficiency of the HDR repair system in future research.

194-202

Authors: Zahra Keramati; Gholamreza Motalleb; Abbas Rahdar; Mohammad Amin Kerachian

Objective: We investigated whether co-incubation of 5-FU and gum-based cerium oxide nanoparticles (CeO2 NPs) would improve half-maximal inhibitory concentration (IC50) and apoptosis in the Caco-2 cancer cell line Materials and Methods: In this experimental study, we synthesized Ceo-2-XG by the nano perception method. X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and vibrating sample magnetometer (VSM) techniques were employed to characterize the synthesized nanoparticles. The Caco-2 cancer cells were cultured and treated with Ceo-2- XG and 5-FU. Cytotoxicity analysis was carried out using MTT assay on Caco-2 cancer cells. CXCR1, CXCR2, CXCL8, BAX, BCL-2, P53, CASPASE-3, CASPASE-8 and CASPASE-9 gene expression changes were assessed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The Caco-2 cancer cell mortality mechanism was analyzed using Annexin V-FITC/PI flow cytometry. Using the inverted microscope morphology changes of the Caco-2 cancer cells was observed. Results: With a sample size of roughly 11 nm, TEM analysis revealed spherical structures. Interestingly, after 72 hours, 400 μg/ml nanoparticles significantly lowered the IC50 of 5-FU from 101 to 71 μg/ml (P<000.1). Furthermore, qRT-PCR analysis showed that BCL-2, CXCR1, CXCR2 and CXCR8 expressions were significantly decreased in the 5-FU and Ceo-2-XG nanoparticles co-incubated group, compared to the 5-FU alone (P<0.001). Notably, gene expressions of BAX, P53, CASPASE-3, CASPASE-8 and CASPASE-9 were significantly higher in the 5-FU and Ceo- 2-XG nanoparticles co-incubated group, compared to the 5-FU alone (P<0.001). The findings revealed that dead cells owing to apoptosis were more than two times higher in 5-FU and Ceo-2-XG nanoparticles cancer cells than in 5-FU alone treated cancer cells. Conclusion: Co-incubation of 5-FU and Ceo-2-XG nanoparticles significantly increased apoptosis in the Caco-2 cancer cells. The antiproliferative activity of co-incubated 5-FU and Ceo-2-XG nanoparticles on Caco-2 cancer cells was substantially higher than that of 5-FU alone.

212-214

Authors: Mohammad Amin Shahrbaf; Leila Ataei Fashtami; Massoud Vosough

Vitiligo is an autoimmune skin disorder that can significantly affect the quality of life in patients. However, current treatment strategies such as phototherapy, topical glucocorticosteroids, or systemic immunosuppressants can be helpful in vitiligo management, and treatments for disease stability which is the main challenge in vitiligo management. Novel therapeutic modalities have made promising advances in this regard. Molecular targeted therapy to target Janus-activated kinase (JAK) such as Tofacitinib and Ruxolitinib in addition to the cell-based treatments are innovative therapeutic options, which are recently used for vitiligo treatment. Transplantation of non-cultured melanocytes-keratinocytes have been studied in Iran and phase one data published in 2010 on 10 patients that continued on 300 patients as phase three in 2018. Cell Tech Pharmed™ Co. registered and applied this cell-based product, ReColorCell®, as a GMP certified by Iranian Food and Drug Administration (IR-FDA). On 11th December 2021, ReColorCell® officially registered as the first cell-based product in the Iranian drug list (IDL). Currently, the post-market study of this product is ongoing.