Upregulation of apoptotic genes and downregulation of target genes of Sonic Hedgehog signaling pathway in DAOY medulloblastoma cell line treated with arsenic trioxide.

Sonic hedgehog (SHH) medulloblastoma etiology is associated with the SHH molecular pathway activation at different levels. We investigated the effect of arsenic trioxide as a downstream-level inhibitor of the SHH signaling pathway on morphology, cytotoxicity, migration, and SHH-related and apoptotic gene expression of DAOY cells. Cells were treated at various arsenic trioxide (ATO)concentrations (1, 2, 3, 5, and 10 µM) for different times (24 and 48 hr). Following treatments, the morphology of the cells was investigated at ×20 and ×40 magnification by an inverted microscope. Then, cytotoxicity was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. Cell migration was analyzed through the wound-healing assay. Furthermore, the expression of SHH-related (GLI1, GLI2, SMO, and MYCN) and apoptotic genes (BAX, BCL2, and TP53) was assessed by real-time quantitative polymerase chain reaction (qPCR). Finally, GLI1, SMO, and MYCN markers were analyzed through immunocytochemistry. Data were analyzed by SPSS (version 16) and P≤0.05 was considered significant. Morphological changes were seen at 3 and 2 µM in 24 and 48 hr of treatment, respectively. The MTT assay showed a dose-dependent cytotoxicity indicating an IC50 value of 3.39±0.35 and 2.05±0.64 µM in 24 and 48hr treatment, respectively. In addition, the trypan blue assay showed higher IC50 values of 4.29±0.25 and 3.92±0.22 µM in 24 and 48 hr treatment, respectively. The wound-healing assay indicated a dose-dependent reduction of cell migration speed showing a 50% reduction at 2.89±0.26 µM. Significant downregulation of GLI1 and GLI2, as well as the upregulation of BAX, BAX/BCL2 ratio, and TP53 were evident. Significant increases in GLI1 and MYCN markers were also evident in immunocytochemistry. ATO, as a downstream effective inhibitor of the SHH pathway, substantially leads to cell death, cell migration inhibition, apoptosis upregulation, and downregulation of SHH target genes in DAOY medulloblastoma. Since ATO is a toxic chemotherapeutic agent, it must be used at low concentrations (2 µM) in order not to damage healthy cells.

Evaluation of the prevalence of bovine leukemia virus DNA in peripheral blood mononuclear cells of multiple sclerosis patients.

Multiple sclerosis (MS) is an immune system-mediated neurodegenerative disease. Recent studies suggest that viral agents, especially the Epstein Barr virus (EBV), are etiological agents for MS. The roles of other viruses in MS have been investigated. Studies have shown an increase in the level of antibodies against bovine leukemia virus (BLV) in patients with MS. In this regard, our study aimed to examine the presence of BLV DNA in peripheral blood mononuclear cells (PBMCs) of MS patients in Iran. In this cross-sectional study, the presence of BLV in 109 Iranian MS patients and 60 healthy controls was evaluated. The isolated PBMCs were used for DNA extraction and PCR, using specific primers for two distinct genes. The mean age of the participants was 39 ± 9.5 years, and 27 (24.77%) of them were male. Clinical evaluation of these patients showed the most frequent MS type to be relapsing-remitting MS (RRMS) (71; 65.14%). BLV evaluation did not show any BLV DNA presence in the PBMCs of individuals in either the MS or healthy control groups. Therefore, our study showed no evidence of BLV infection in Iranian MS patients.

Differentiation of bone marrow derived mesenchymal stem cells into male germ-like cells in co-culture with testicular cells.

OBJECTIVE: Stem cell therapy, specifically, pre-induction of mesenchymal stem cells toward male germ-like cells may be useful in patients with azoospermia. The aim of this study was to evaluate in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) into male germ-like cells by indirect co-culture with testicular cells in the presence of bone morphogenetic protein 4 (BMP4). METHODS: Experimental groups included: control (mouse BMSCs), treatment group-1 (BMSCs treated with BMP4), treatment group-2 (indirect co-culture of BMSCs with mouse testicular cells in the presence of BMP4) and treatment group-3 (indirect co-culture of BMSCs with testicular cells). BMSCs-derived male germ-like cells were evaluated by the expression of Dazl, and Stra8 using RT-qPCR. RESULTS: Stra8 gene expression was significantly increased in the treatment group-2 and Dazl gene was significantly increased in the treatment group-1 compared to other groups. In conclusion, indirect co-culturing of BMSCs with testicular cells and BMP4 leads to the differentiation of BMSCs into male germ-like cells which express specific male germ-like genes. Testicular cells released factors that contributed to the differentiation of BMSCs into male germ progenitor cells. CONCLUSION: This study suggests that mesenchymal stem cells may be differentiated into male germ-like cells and therefore, may be a novel treatment option for men with azoospermia.

The effect of hydroethanolic extract of Matricaria chamomilla on the reproductive system of male rats exposed to formaldehyde.

Formaldehyde is a common agent in our surrounding environment and can adversely affect the male reproductive system. In this study, the effectiveness of Matricaria chamomilla (MC) extract as an antioxidant was investigated in rats treated with formaldehyde. Thirty-two male Wistar rats were randomly divided into six groups: F (10 mg/kg formaldehyde), M200 (200 mg/kg MC extract), M500 (500 mg/kg MC extract), FM200 (10 mg/kg formaldehyde and 200 mg/kg MC extract), FM500 (10 mg/kg formaldehyde and 500 mg/kg MC extract) and control group (0.9% normal saline). Formaldehyde and MC extract were administered daily for 30 consecutive days via intraperitoneal injection. Hormonal status, sperm parameters, testis tissue histology, germinal cells apoptosis and stereological analyses of testis tissue were investigated. Testosterone and LH levels were significantly increased in FM200, FM500, F200 and F500 groups compared to F group (p ≤ 0.05). Sperm count, motility and viability were significantly enhanced in FM200, FM500, F200 and F500 groups compared to F group (p ≤ 0.05). A decrease in the number of apoptotic germ cells in FM200, FM500, M200 and M500 groups (p ≤ 0.05) was evident. In particular, the MC extract in dose 500 mg/kg is seen to reduce the adverse effects of formaldehyde on the reproductive system of male rats.

The effect of lithium chloride on BDNF, NT3, and their receptor mRNA levels in the spinal contusion rat models.

OBJECTIVE: Nowadays, there seems to be no decisive way for treatment of spinal cord injury (SCI).Extensive cell death (apoptosis and necrosis) occurring in SCI can cause considerable progressive sensorimotor disabilities. Preventing cell death by improving endogenous regenerative capability could an effective strategy for the treatment of SCI. This study was designed to evaluate the effects of lithium chloride (LiCl) on the cell survival through overexpression of BDNF and NT3 mRNA level and their receptors in the contusion rat models. METHODS: Rats were randomly divided into four experimental groups (eight rats/group) including: contused animals (the non-treatment group); contused animals (the control group) which received laminectomy; contused animals received normal saline (vehicle)and contused animals received intraperitoneal injection of 20 mg/kg LiCl three days after surgery. Injection continued for 14 days as treatment. Basso, Beattie, Bresnahan (BBB) rating scale was used to assess the motor function of the rats. To evaluate the histopathological and gene expression analysis, rats were sacrificed 28 days after surgery. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to obtain the relative levels of mRNA for BDNF, NT3 and their receptors. RESULTS: The results showed LiCl ameliorates BBB scores via up-regulation of BDNF and TrkB receptors. Also, histological analysis showed that the numerical density per area of TUNEL- positive cells and the percentage of cavity significantly decreased in the LiCl-treated group. CONCLUSION: Our findings suggest that LiCl protects neural cells and effectively enhances locomotor function, which was done through up-regulation of endogenous BDNF expression in rats with SCI. ABBREVIATIONS: SCI: spinal cord injury; LiCl: lithium chloride; BDNF: Brain-derived neurotrophic factor; NT3: Neurotrophin-3; BBB: Basso, Beattie, Bresnahan; TrkB: Tropomyosin receptor kinase B; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Indirect Co-Culture of Testicular Cells with Bone Marrow Mesenchymal Stem Cells Leads to Male Germ Cell-Specific Gene Expressions.

OBJECTIVE: Non-obstructive azoospermia is mostly irreversible. Efforts to cure this type of infertility have led to the application of stem cells in the reproduction field. In the present study, testicular cell-mediated differentiation of male germ-like cells from bone marrow-derived mesenchymal stem cells (BM-MSCs) in an in vitro indirect co-culture system is investigated. MATERIALS AND METHODS: In this experimental study, mouse BM-MSCs were isolated and cultured up to passage three. Identification of the cells was evaluated using specific surface markers by flow-cytometry technique. Four experimental groups were investigated: control, treatment with retinoic acid (RA), indirect co-culture with testicular cells, and combination of RA and indirect co-culture with testicular cells. Finally, following differentiation, the quantitative expression of germ cell-specific markers including Dazl, Piwil2 and Stra8 were evaluated by real-time polymerase chain reaction (PCR). RESULTS: Molecular analysis revealed a significant increase in Dazl expression in the indirect co-culture with testicular cells group in comparison to the control group. Quantitative expression level of Piwil2 was not significantly changed in comparison to the control group. Stra8 expression was significantly higher in RA group in comparison to other groups. CONCLUSION: Indirect co-culture of BM-MSCs in the presence of testicular cells leads to expression of male germ cell-specific gene, Dazl, in the induced cells. Combination of co-culture with testicular cells and RA did not show any positive effect on the specific gene expressions.

Neuroprotective effects of selegiline on rat neural stem cells treated with hydrogen peroxide.

Oxidative stress and reactive oxygen species generation have been implicated in the pathogenesis of several neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and multiple sclerosis. In the present study, the neuroprotective effects of selegiline against hydrogen peroxide-induced oxidative stress in hippocampus-derived neural stem cells (NSCs) were evaluated. NSCs isolated from neonatal Wistar rats were pretreated with different doses of selegiline for 48 h and then exposed to 125 µM H2O2 for 30 min. Using MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, acridine orange/ethidium bromide staining and reverse transcription-quantitative polymerase chain reaction, the effects of selegiline on cell survival, apoptosis and the expression of B-cell lymphoma 2 (Bcl-2) and heat shock protein 4 (Hspa4) in pretreated stem cells were assessed compared with a control group lacking pretreatment. The results indicated that the viability of cells pretreated with 20 µM selegiline was significantly increased compared with the control group (P<0.05). Additionally, 20 µM selegiline increased the mRNA expression of Bcl-2 and Hspa4 (P<0.05 vs. control) and suppressed oxidative stress-induced cell death (apoptosis and necrosis; P<0.05 vs. control and 10 µM groups). From these findings, it was concluded that selegiline may be a therapeutic candidate for the treatment of neurological diseases mediated by oxidative stress.