Cell Journal (Yakhteh)

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.

85-91

Authors: Sepideh Shahkarami; Samareh Younesian; Shahrbano Rostami; Farzad Kompani; Davood Bashash; Seyed Asadollah Mousavi; Seyed H. Ghaffari

Objective: Minimal residual disease (MRD) is considered the greatest prognostic factor in acute lymphoblastic leukemia (ALL). MRD is a valuable tool for anticipating impending relapse and treatment response assessment. The objective of the present study was to investigate whether the detection of IgH gene rearrangement using polymerase chain reaction (PCR)-based GeneScan analysis could be a complementary method to monitor MRD along with the quantitative realtime PCR (qPCR). Materials and Methods: In this cross-sectional study, we valued the MRD levels, based on the GeneScanning analysis (GSA), and then compared the data with quantitative real-time polymerase chain reaction at different time points in peripheral blood (PB) samples of adult B-lineage ALL patients (n=35). The specific polymerase chain reaction (PCR) primers for IGH gene FR-1 and fluorescence-labeled J-primer were used and analyzed by capillary gel electrophoresis on a sequencer. The results of this study were compared with the previously reported MRD results obtained by the IGH rearrangements allele-specific oligonucleotide (ASO) -qPCR methods. Results: The total concordance rate was 86.7%, with a P<0.001. MRD results obtained by GSA and ASO-qPCR methods were concordant in all diagnostic samples and samples on the 14th and 28th days of induction therapy. The results of these 2.5 years’ follow-ups demonstrated a significant correlation between the two techniques (r=0.892, P<0.001). Conclusion: It seems that the PCR-based GeneScan analysis of IGH gene rearrangement detection may be a valuable molecular technique to distinguish monoclonality from polyclonality. And also, it may be a precise tool to detect the residual leukemic DNA in the PB follow-up samples of patients.

102-109

Authors: Behdokht Jamali; Malihe Entezari; Nahid Babaei; Mehrdad Hashemi

Objective: Parkinson’s disease (PD) is a severely debilitating disease for which no suitable treatment has been found so far. In recent years, nanoparticles (NPs) have shown therapeutic potential in PD. Thus, in the current research, for the first time, we investigated the effects of vitamin E and TiO2 nanoparticles (TiO2-NPs) on a rat model of PD. Materials and Methods: In this experimental study, after preparation and induction of PD, rats were administrated with vitamin E and TiO2-NPs. One day after receiving the last dose of treatments, rats were killed and substantia nigra was extracted from the brain and its cell suspension was prepared. In each group, female rats were mated, and after confirmation that the female rats were pregnant by vaginal smear test, the fetus was removed. Cell viability was studied by the MTT method and apoptosis, and necrosis were studied by the flow cytometry technique. Also, after RNA extraction and cDNA synthesis, the expression of Bcl-2 and circRNA 0001518 genes were studied using the real time polymerase chain reaction (RT-PCR) technique. For analyzing the data, two-way ANOVA was used. Results: The results showed a sharp decrease in cell viability in female PD rats and fetuses resulting from PD female rats. Vitamin E treatment showed the greatest effect on increasing cell viability. Decreased expression of the Bcl-2 gene and increased expression of circRNA 0001518 were observed in PD conditions. Conclusion: Administration of vitamin E may be a good option for reducing PD-induced cell death.

118-125

Authors: Junhao Fu; Min Yu; Wenxia Xu; Shian Yu

Objective: Chemotherapeutic drug resistance is the main obstacle that affects the efficacy of current therapies of hepatocellular carcinoma (HCC), which needs to be addressed urgently. High expression of histone methyltransferase G9a was reported to play a pivotal role in the progression of HCC. Regulatory mechanism of aberrant activation of G9a in HCC and the association with subsequent cisplatin (DDP) resistance still remains ambiguous. This study strived to investigate mechanism of G9a overexpression and its impact on cisplatin resistance in HCC cells. Materials and Methods: In this experimental study, we investigated effects of different concentrations of cisplatin in combination with BIX-01294 or PR-619 on viability and apoptosis of HuH7 and SNU387 cells via CCK-8 kit and flow cytometric analysis, respectively. Colony formation capacity was applied to evaluate effect of cisplatin with or without BIX-01294 on cell proliferation, and western blotting was used to verify expression level of the related proteins. Global mRNA expression profile analysis was adopted to identify differentially expressed genes associated with overexpression of G9a. Results: We observed that overexpression of G9a admittedly promoted cisplatin resistance in HCC cells. Global mRNA expression profile analysis after G9a inhibition showed that DNA repair and cell cycle progression were downregulated. Moreover, we identified that deubiquitination enzymes (DUBs) stabilized high expression of G9a in HCC through deubiquitination. Additionally, cisplatin could significantly inhibit proliferation of DUBs-deficient HCC cells, while promoting their apoptosis. Conclusion: Collectively, our data indicated that DUBs stabilize G9a through deubiquitination, thereby participating in the cisplatin resistance of HCC cells. The elucidation of this mechanism contributes to propose a potential alternative intervention strategy for the treatment of HCC patients harboring high G9a levels.

135-142

Authors: Somayeh Karimi Majd; Mandana Gholami; Behzad Bazgir

Objective: Satellite cells play an important role in muscle regeneration, which this process can be affected by different genes including PAX7 and MyoD. Exercise training known as an important strategy for mediating the satellite cell’s function. Therefore, the main purpose of the present study is to investigate the changes in PAX7 and MyoD protein expression in response to eccentric and concentric resistance exercise in healthy young men. Materials and Methods: In this semi-experimental and cross-sectional study, 10 healthy men (age range 18-30 years old) participated. They were randomly divided into two equal groups (n=5) to perform one of two high-intensity eccentric or concentric knee extensions muscle contraction protocols. The contractions included a maximum of 12 sets of 10 repetitions, with a 30 second rest time interval between sets. PAX7 and MyoD protein expression was assessed using Immunohistochemistry analysis from the Vastus Lateralis muscle needle biopsy samples that have been taken 24 hours before and 3 to 4 hours after the end of the exercise protocol. Results: We observed that the PAX7 protein expression level increased significantly after eccentric (47.75%) and concentric (39.21%) (P=0.01) intervention. While, the MyoD protein expression level reduced (38.14%) significantly following acute eccentric resistance exercise (P=0.01). Conclusion: It seems that eccentric or concentric muscular contraction modulates the expression of PAX7 and MyoD protein expression in the skeletal muscle, with further effects observed in eccentric resistance exercise.

822-828

Authors: Mansoureh Sabzalizadeh; Mohammad Reza Afarinesh; Ali Derakhshani; Vahid Sheibani

Objective: Stem cells (SCs) can improve the functional defects of brain injury. Rodents use their whiskers to get tactile information from their surroundings. The aim of this study was to investigate whether the transplantation of SCs into the lesioned barrel cortex can help neuronal function in the contralateral cortex. Materials and Methods: Sixteen male Wistar rats (200-230 g) were used in this experimental study. We induced a mechanical lesion in the right barrel cortex area of rats by removing this area by a 3 mm skin punch. Four groups containing one intact group of rats: group 1: control, and three lesion groups, group 2: lesion+un-differentiated dental pulp SCs (U-DPSCs), group 3: lesion+differentiated dental pulp SCs (D-DPSCs), and group 4: cell medium (vehicle) that were injected in the lesion area. Three weeks after transplantation of SCs or cell medium, the rats’ responses of left barrel cortical neurons to controlled deflections of right whiskers were recorded by using the extracellular single-unit recordings technique. Results: The results showed that the neural spontaneous activity and response magnitude of intact barrel cortex neurons in the lesion group decreased significantly (P<0.05) compared to the control group while ON and OFF responses were improved in the D-DPSCs (P<0.001) group compared to the vehicle group three weeks after transplantation. Conclusion: Transplantation of dental pulp mesenchymal SCs significantly improved the neural responses of the left barrel cortex that was depressed in the vehicle group.

839-846

Authors: Lei Yang; Liyi Hu

Objective: Non-small cell lung cancer (NSCLC) stands as a prominent contributor to cancer-related fatalities on a global scale, necessitating the search for novel therapeutic agents. SP-8356, a derivative of (1S)-(–)-verbenone, has shown promise as an anticancer agent in preclinical studies. However, specific mechanisms underlying its effects in NSCLC remain to be elucidated. The aim of this research was to explore the in vitro anti-NSCLC effects of SP-8356, elucidate its mechanisms of action, and assess its efficacy in inhibiting tumor formation in a murine model. Materials and Methods: In this experimental study, NSCLC cell lines were treated with various concentrations of SP- 8356. Cell viability and proliferation were assessed using MTT and colony formation assays, respectively. Cell cycle distribution was analyzed by flow cytometry, and apoptosis was evaluated by determining apoptotic protein expression. Western blot analysis was conducted to assess protein expression levels of the both p53 and MDM2. Additionally, we evaluated efficacy of the SP-8356 in inhibiting tumor formation of the nude mouse model. Results: SP-8356 demonstrated a concentration-dependent inhibition of cell proliferation in the NSCLC cell lines. Flow cytometric analysis showed that SP-8356 led to cell cycle arrest at the G2/M phase, indicating its potential influence on regulating the cell cycle. SP-8356 treatment was associated with the downregulation of CDK1 and Cyclin B1. Additionally, SP-8356 significantly enhanced apoptosis in NSCLC cells. SP-8356 treatment was associated with the downregulation of Bcl-2, while Bax expression was upregulated. Mechanistically, SP-8356 led to accumulation of the p53 protein levels within the NSCLC cells. This accumulation was mediated through inhibition of its negative regulator, MDM2. Using a nude mouse model demonstrated that SP-8356 effectively inhibited tumor formation in vivo. Conclusion: Our findings shed light on the molecular mechanisms underlying anticancer activity of SP-8356 and highlight its potential as a promising therapeutic candidate for NSCLC treatment.

854-862

Authors: Mahnaz Babaahmadi; Nima Makvand Gholipour; Behnoosh Tayebi; Jed Pheneger; Ensiyeh Hajizadeh-Saffar; Mohamadreza Baghaban Eslaminejad; Seyedeh-Nafiseh Hassani

Objective: The collagen-induced arthritis (CIA) model is the most commonly studied autoimmune model of rheumatoid arthritis (RA). In this study, we investigated the usefulness of collagen type II emulsified in Freund's incomplete adjuvant (CII/IFA) as a suitable method for establishing RA in Lewis rats. The aim of the present study was to present a straightforward and effective method for inducing CIA in rats. Materials and Methods: In this experimental study, animals were divided into two equal groups (n=5); control and CIA. Five rats were injected intradermally at the base of the tail with a 0.2 ml CII/IFA emulsion. On the seventh day, a 0.1 ml CII/IFA emulsion booster was injected. Arthritis symptoms that arose were evaluated at clinical, histological, radiological, and at protein expression levels to find out if the disease had been induced successfully. Results: Our finding showed a decreasing trend in the body weight during the RA induction period, while the arthritis score and paw thickness were increased during this period. The results of the enzyme-linked immunosorbent assay (ELISA) for serum samples revealed that the levels of proinflammatory cytokines, interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor (TNF)-α and anti-CII IgG were significantly increased in CIA rats compared to the control group. After CIA induction, the level of anti-inflammatory protein IL-10 was decreased significantly. Radiographic examination of the hind paws showed soft tissue swelling, bone erosion, and osteophyte formation in CIA rats. Additionally, based on histological evaluations, the hind paws of the CIA group showed pannus formation, synovial hyperplasia, and bone and cartilage destruction. Conclusion: It seems that CII/IFA treatment can be an appropriate and effective method to induce RA disease in Lewis rats. This well-established and well-characterized CIA model in female Lewis rats could be considered to study aspects of RA and develop novel anti-arthritic agents.

874-882

Authors: Mansoureh Farhangniya; Farzaneh Mohamadi Farsani; Najmeh Salehi; Ali Samadikuchaksaraei

Objective: Wound healing is a complex process involving the coordinated interaction of various genes and molecular pathways. The study aimed to uncover novel therapeutic targets, biomarkers and candidate genes for drug development to improve successful wound repair interventions. Materials and Methods: This study is a network-meta analysis study. Nine wound healing microarray datasets obtained from the Gene Expression Omnibus (GEO) database were used for this study. Differentially expressed genes (DEGs) were described using the Limma package and shared genes were used as input for weighted gene co-expression network analysis. The Gene Ontology analysis was performed using the EnrichR web server, and construction of a protein-protein interaction (PPI) network was achieved by the STRING and Cytoscape. Results: A total of 424 DEGs were determined. A co-expression network was constructed using 7692 shared genes between nine data sets, resulting in the identification of seven modules. Among these modules, those with the top 20 genes of up and down-regulation were selected. The top down-regulated genes, including TJP1, SEC61A1, PLEK, ATP5B, PDIA6, PIK3R1, SRGN, SDC2, and RBBP7, and the top up-regulated genes including RPS27A, EEF1A1, HNRNPA1, CTNNB1, POLR2A, CFL1, CSNk1E, HSPD1, FN1, and AURKB, which can potentially serve as therapeutic targets were identified. The KEGG pathway analysis found that the majority of the genes are enriched in the "Wnt signaling pathway". Conclusion: In our study of nine wound healing microarray datasets, we identified DEGs and co-expressed modules using WGCNA. These genes are involved in important cellular processes such as transcription, translation, and posttranslational modifications. We found nine down-regulated genes and ten up-regulated genes, which could serve as potential therapeutic targets for further experimental validation. Targeting pathways related to protein synthesis and cell adhesion and migration may enhance wound healing, but additional experimental validation is needed to confirm the effectiveness and safety of targeted interventions.