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In the present work, we examined the beneficial efficacy of chrysin as a natural flavonoid on testicular torsion/detorsion damages among adult male rats. Forty-eight male Wistar rats were randomly allocated into six groups: sham, torsion/detorsion (TD) group, TDC30 group in which TD process was applied and treated with chrysin (30 mg/kg), TDC50 group in which TD process was applied and treated with chrysin (50 mg/kg), HC30 group in which animals were treated with chrysin (30 mg/kg), and HC50 group in which rats were treated with chrysin (50 mg/kg). Serum samples were tested for testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels. The sperm parameters, histopathological analysis, and expressions of apoptosis-related genes were examined among different groups. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels in the testicles and serum were measured. Serum levels of SOD, GPx, FSH, LH, and “Bax” expression significantly increased in the TD group compared to the sham group ( P < 0.05 ). Sperm parameters, serum testosterone level, Johnson’s scores, seminiferous tubule diameter, the height of the germinal epithelium (HE) measurements, and expression of “Bcl2” meaningfully decreased ( P < 0.05 ). Administration of chrysin impeded ischemia/reperfusion damages in testis tissue and improved sperm quality. The result of our study indicates that treatment with chrysin can protect testis tissue from ischemia/reperfusion damages induced by TD procedure.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 μM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p

Can sublethal stress induced by nitric oxide on fresh human spermatozoa protect the functional properties of post-thaw human spermatozoa?Semen samples were obtained from 46 donors. Twenty semen samples were used to determine toxicity level of nitric oxide by incubation of semen with different concentrations of nitric oxide (0.01 to 400 μM). Then, 26 semen samples were cryopreserved with optimized ranges of nitric oxide: control (NO-0.00), 0.01 μM nitric oxide (NO-0.01), 0.1 μM nitric oxide (NO-0.1), 1 μM nitric oxide (NO-1), 10 μM nitric oxide (NO-10), 100 μM nitric oxide (NO-100). Frozen-thawed spermatozoa were assessed for motion characteristics, viability, morphology, apoptosis-like changes, caspase 3 activity, DNA fragmentation and intracellular reactive oxygen species levels. Fertilization potential was investigated by heterologous piezo-intracytoplasmic sperm injection (piezo-ICSI) of human spermatozoa into mouse oocytes.In fresh spermatozoa, nitric oxide did not induce a negative effect, except a significant reduction in motility and viability at 200 µM and 400 µM (P0.05). Cryopreservation significantly reduced sperm motility and increased reactive oxygen species, apoptosis-like changes, caspase 3 activity, and DNA damage (P0.05). NO-0.01 significantly increased total and progressive motility versus the other groups (P0.05). The lowest percentage of caspase 3 activity was in the NO-0.01 and NO-0.1 compared with the other freezing groups. In the fertilization trial, the rate of two-cell embryo formation after heterologous piezo-ICSI was higher (P0.05) in NO-0.01 (69%) versus controls (42%).Sublethal oxidative stress induced by nitric oxide might improve human sperm function after cryopreservation.

Telomeres through maintaining chromosomal integrity have key roles in the cell life span. The autophagy is typically a pro-survival process and important for maintaining cellular homeostasis. Conversely, in some conditions, autophagy acts as caspase-independent cell death program. Beclin1 gene plays a principal role in the initiation of autophagy.The aim of this study was to evaluate the effect of autophagy induction via recombinant Beclin1 on telomerase activity and programmed cell death (apoptosis) in MCDK cells.The recombinant Beclin1-pcDNA3.1(-) was transfected into MDCK cells. Next, the autophagy information was detected by LC3II staining as autophagy marker using flow cytometry. The telomerase activity was measured by telomeric repeat amplification protocol method in MDCK cells. To detection of the cell death in MDCK cells, apoptosis assay was done through Annexin V staining method.The results of flow cytometry analysis indicated that following overexpression of Beclin1 gene, the percentage of the LC3II was 16.08% compared with control group (0.48%). Following induction of autophagy, telomerase activity reduced 10 folds in comparison with the control group. The rate of apoptosis in transfected MDCK cells increased up to 12.74%.Crosstalk between telomerase, autophagy, and apoptosis may determine the fate of the cancer cell aging. Hence, manipulation of autophagy may create a novel area to design new compounds and combination therapy to shorten the cancer cell survival.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 μM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated that the abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.

The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.

Autophagy is a cellular homeostatic process whereby damaged proteins and organelles are encapsulated into double membrane vesicles, called autophagosomes, for lysosomal digestion. Beclin1 plays a key role in the initial steps of autophagosome formation. In this study, we evaluated the effect of Beclin 1 overexpression in induction of autophagy and the relationship between autophagy induction and telomerase activity in HeLa cells. We found that overexpression of Beclin 1 in HeLa cells leads to autophagosome formation as shown by intracellular autophagosomal marker LC3-II staining. Expression of Beclin1 reduced telomerase activity for about 100 fold compared with the control while it did not affect TERT expression level. The results of cell cycle analysis indicated that the cell cycle and proliferation progressed normally up to 48h post-transfection. Understanding the role of autophagy induction and telomerase in the pathophysiology of aging and human cancer reveal new strategies that hold much promise for intervention and therapeutic uses.

Neuronal apoptosis and impaired hippocampal neurogenesis are major players in cognitive/memory dysfunctions including Alzheimer's disease (AD). Interferon beta (IFNβ) is a cytokine with anti-apoptotic and neuroprotective properties on the central nervous system (CNS) cells which specifically affects neural progenitor cells (NPCs) even in the adult brain. In this study, we examined the effect of IFNβ on memory impairment as well as hippocampal neurogenesis and apoptosis in a rat model of AD. AD model was induced by lentiviral-mediated overexpression of mutant APP in the hippocampus of adult rats. Intranasal (IN) administration of IFNβ (0.5 μg/kg and 1 μg/kg doses) was started from day 23 after virus injection and continued every other day to the final day of experiments. The expression levels of APP, neurogenesis (Nestin, Ki67, DCX, and Reelin) and apoptosis (Bax/Bcl-2 ratio, cleaved-caspase-3 and seladin-1) markers were evaluated by immunohistochemistry, real-time PCR, immunofluorescence and western blotting. Moreover, thioflavin T and Nissl stainings were used to assess Aβ plaque levels and neuronal degeneration in the hippocampus, respectively. Our results showed that IFNβ treatment reduced APP expression and Aβ plaque formation, and concomitantly ameliorated spatial learning and memory deficits examined in Y-maze and Morris water maze tests. Moreover, in parallel with reducing apoptosis and neural loss in the hippocampal subfields, IFNβ decreased ectopic neurogenesis in the CA1 and CA3 regions of the AD rat hippocampus. However, IFNβ increased neurogenesis in the dentate gyrus neurogenic niche. Our findings suggest that IFNβ exerts neuroprotective effects at least partly by inhibition of apoptosis and modulation of neurogenesis. Taken together, IFNβ can be a promising therapeutic approach to improve cognitive performance in AD-like neurodegenerative context.

Purpose: Most cancer cells exhibit a defect in their capacity to mature into nonreplicating adult cells and existing in a highly proliferating state. Differentiation therapy by agents such as 1,25-dihydroxyvitamin D3(1,25-(OH)2 VD3) represents a useful approach for the treatment of cancer including acute myeloid leukemia. Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-(OH)2 VD3. However, usage of these findings in the clinical trials is limited by calcemic effects of 1,25-(OH)2 VD3. Attempts to overcome this problem have focused on a combination of low concentrations 1,25-(OH)2 VD3 with other compounds to induce differentiation of HL-60 cells. In this study, the effect of honey bee venom (BV) and 1,25-(OH)2 VD3, individually and in combination, on proliferation and differentiation of human myeloid leukemia HL-60 cells were assayed. Materials and Methods: In this in vitro study, toxic and nontoxic concentrations of BV and 1,25-(OH)2 VD3 were tested using Trypan blue stained cell counting and (3[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In addition, differentiation of cells was assayed using a Wright-Giemsa staining and nitroblue tetrazolium reduction test. Data were analyzed by a one-way analysis of the variance test using SPSS software. Results: Our findings showed that both the BV and 1,25-(OH)2 VD3, in a dose and time-dependent manner, caused cell death at high concentrations and inhibited cell proliferation at lower concentrations. About 5 nM of 1,25-(OH)2 VD3 induced differentiation of HL-60 cells to monocytes after 72 h. 2.5 μg/ml of BV suppressed proliferation of HL-60 cells but had not any effects on their differentiation, whereas in combination with 5 nM of 1,25-(OH)2 VD3, it enhanced antiproliferative and differentiation potency of 1,25-(OH)2 VD3. Conclusions: These results indicate that BV potentiates the 1,25-(OH)2 VD3-induced HL-60 cell differentiation into monocytes

Background: Telomeres through maintaining chromosomal integrity have key roles in the cell life span. The autophagy is typically a pro-survival process and important for maintaining cellular homeostasis. Conversely, in some conditions, autophagy acts as caspase-independent cell death program. Beclin1 gene plays a principal role in the initiation of autophagy. Objective: The aim of this study was to evaluate the effect of autophagy induction via recombinant Beclin1 on telomerase activity and programmed cell death (apoptosis) in MCDK cells. Materials and Methods: The recombinant Beclin1-pcDNA3.1(-) was transfected into MDCK cells. Next, the autophagy information was detected by LC3II staining as autophagy marker using flow cytometry. The telomerase activity was measured by telomeric repeat amplification protocol method in MDCK cells. To detection of the cell death in MDCK cells, apoptosis assay was done through Annexin V staining method. Results: The results of flow cytometry analysis indicated that following overexpression of Beclin1 gene, the percentage of the LC3II was 16.08% compared with control group (0.48%). Following induction of autophagy, telomerase activity reduced 10 folds in comparison with the control group. The rate of apoptosis in transfected MDCK cells increased up to 12.74%. Conclusion: Crosstalk between telomerase, autophagy, and apoptosis may determine the fate of the cancer cell aging. Hence, manipulation of autophagy may create a novel area to design new compounds and combination therapy to shorten the cancer cell survival.

Electrophysiological measurements are indispensable tool for investigating the functional integrity of peripheral nerves in both clinical and laboratory environments. Nerve conduction study is a property of both the axons and myelin sheath. The nerve conduction velocity is highly dependent on rapid signal propagation enabled by myelination. The compound motor action potential (CMAP) which correlates with the number of functional axons is an indicator for axonal damage when significantly reduced. Because nerve conduction velocity (NCV) is sensitive to both myelin sheath and axonal changes, it is an excellent measure of the function of the peripheral nervous system. Nerve conduction studies may be diagnostically helpful in patients suspected of having almost any peripheral nervous system disorder including disorders of nerve roots, peripheral nerves, muscle and neuromuscular junction. Cranial nerves and spinal cord function may also be assessed. Here, we mentioned the components and properties of nerve conduction study.

Background Curcumin has immunomodulatory anti-inflammatory, antioxidant, and neuroprotective properties. The goal of this study was to determine the effects of curcumin and biodegradable membrane on nerve healing in rat sciatic nerve transected injuries. Methods Rats were divided into groups: (1) control group (Ctrl), (2) curcumin group (Cur), (3) membrane group (Mem), and (4) membrane and curcumin group (Mem + Cur). Functional recovery was evaluated at 2, 4, 6, and 8 weeks after surgery. At the end of the eighth week after surgery, histological assessments were done. Results At the end of 8th week after surgery, functional assessments (sciatic nerve index, withdrawal reflex latency, and electromyography) in the Mem + Cur group improved compared with other groups (P < 0.05). Histological results (number of nerve fibers, diameter of nerve fibers, and myelin thickness) improved in the Mem + Cur group compared with the control, Cur, and Mem groups (P < 0.05). Conclusion The present study showed the positive effects of Mem + Cur on nerve regeneration of transected sciatic nerve in rat model. © 2018 Wolters Kluwer Health, Inc. All rights reserved.

MicroRNAs have a critical role in oligodendrocyte development including cell proliferation, differentiation, and myelin formation. MicroRNA 338 (miR-338) is necessary to promote oligodendrocyte differentiation by repressing negative regulators of oligodendrocyte differentiation. Oligodendrocytes (OLs) are responsible for myelin sheath synthesis around nerve fibers in the central nervous system (CNS) that form the myelin sheath of axons. Human endometrial-derived stromal cells (hEnSCs) are a new source of mesenchymal-like stem cells for cell replacement therapy. The hEnSCs, after treating with fibroblast growth factor 2/epidermal growth factor (20 ng/mL) and platelet-derived growth factor (PDGF)-AA (10 ng/mL) for 12 days, were divided in two groups: in the first group, the cells were treated by triiodothyronine (T3), and in the second group, the cells were infected by miR-338-green fluorescent protein-expressing lentiviruses. Six days after infection, the cells were collected and analyzed for the expression of stage-specific markers Nestin, microtubule-associated protein 2, neurofilament-L, oligodendrocyte lineage transcription factor, SRY-box containing gene 10, PDGF receptor alpha, 2',3'-cyclic nucleotide 3' phosphodiesterase, A2B5, O4, and myelin basic protein by immunocytochemistry and quantitative reverse transcription PCR. Result showed that in the infected cells, the expression of pre-oligodendrocyte markers was higher than that of T3-treated cells. The EnSCs can differentiate to oligodendrocyte cells by the overexpression of miR-338, and these cells can be used as a unique source for cell therapy of neurodegenerative disease.

Background Polycystic ovary syndrome (PCOS) is a common but complex endocrine disorder and is the major cause of anovulation and consequent subfertility. In this study the effect of grape seed extract (GSE) on triglyceride (TG), total cholesterol (TC), highdensity lipoprotein-cholestrol (HDL-C), low-density lipoprotein-cholestrol (LDL-C) and interleukin-6 (IL-6) in PCOS Wistar rats were assessed. Materials and Methods In this experimental study, 84 adult female Wistar rats were divided into 7 groups (n=12) including control (intact), Sham (estradiol valerate solvent injection), control PCOS and 4 experimental PCOS groups. To induce the syndrome, a single subcutaneous injection of 2 mg estradiol valerate was applied. In experimental groups, PCOS rats were treated with different doses of 50, 75, 100 and 200 mg/kg body weight (BW) GSE by intraperitoneal injection for 10 consecutive days. After harvesting blood serum, TG was measured by Glycerol-3-phosphate Oxidase-Peoxidase (GPO- PAP), TC by Cholesterol Oxidase-Peroxidase (CHOD-PAP), and HDL-C by sedimentation method, LDL-C by Friedwald calculation and IL-6 by ELISA method. The serum values of each parameter were analyzed using one-way ANOVA at P≤0.05. Results In all experimental groups significant decrease of visceral fat was obvious as compared with control PCOS group. LDL-C, TC and IL-6 levels in experimental groups, particularly at dose of 50 mg/kg of GSE, were significantly decreased as compared with PCOS group. However, HDL-C levels were not significantly changed. Conclusion : According to the findings of this study, it can be concluded that GSE with its effects on serum TC, LDL-C and IL-6 could reduce the effects of dyslipidemia and inflammation in PCOS rats and improve systemic symptoms of PCOS.

In recent years the increasing use of nanoparticles has led researchers to study their effects on biological systems. The most important effects of nanoparticles on cells are their ability to induce or suppress production of reactive oxygen species (ROS). Changes in reactive oxygen species play an important role in various developmental processes, including proliferation and differentiation in several diseases such as Parkinson. The aim of this study was to investigate the effect of iron oxide nanoparticle with dimensions of less than 20 nanometers on the viability and neuronal differentiation of mouse embryonic stem cell (Royan B1).To assess the effects of Fe2O3 nanoparticles on neuronal differentiation of Royan B1 cells, embryoid bodies were divided into eight groups receiving different amounts of nanoparticle (10, 20, 30 μg/mL) for 12 hours, retinoic acid (1 μM), and both. Differentiation was examined under phase contrast microscope and using immunocytochemistry.Data analysis showed that cell death was increased by a time and concentration manner and there was a direct relevance between iron oxide amount and H2O2 level in cells. Statistical analysis of embryoid bodies showed that neural differentiation of mouse embryonic stem cells in groups that received nanoparticles were significantly lower than other groups and their viability were considerably reduced.According to the findings of this study it can be concluded that iron oxide nanoparticles reduce retinoic acid-neuronal differentiation in mouse embryonic stem cells and it seems that the main mechanism involved in the reduction of viability and neural differentiation was enhanced levels of ROS within the cells.

Objective Methotrexate (MTX) is an antimetabolite drug commonly prescribed for the various cancers and autoimmune diseases. Despite its considerable therapeutic effects, nephrotoxicity is the most important side-effect of treatment with MTX. Aquaporin1 (AQP1) is a water channel proteins which is present in mammalian kidney. Raspberry fruit with antioxidant properties is able to protect biological systems from the harmful effects of free radicals. The purpose of this study was to investigate the effect of raspberry extract on expression of AQP1 and the MTX-induced nephrotoxicity in rats. Materials and Methods In this experimental study, 60 adult male Wistar rats were divided into nine groups including control, sham, MTX treated group [single dose of 20 mg/kg of body weight (BW) MTX at the third day], raspberry treated groups [intraperitoneal (I.P) injection of 100, 200, 400 mg/kg of BW raspberry extract for ten consecutive days], MTX and raspberry treated groups. At day 11, rats were sacrificed via chloroform inhalation and kidney tissues were fixed in formalin solution for histological and immunohistochemistry analysis. The serological assays for urea, creatinine, uric acid and interleukin-6 (IL-6) levels were also performed. Results MTX elevated serum level of the urea, creatinine, uric acid, IL-6, renal tissue damage and decreased the AQP1 expression level. Raspberry fruit extract improved the kidney function and reduced side effects of MTX in treated rats. Expression of AQP1, in a dose dependent manner was also ameliorated, as compared to control group. Conclusion According to the findings of this study, it can be concluded that biological activity of compounds presented in raspberry fruit extract especially anthocyanins may have chemo-protective effect on kidney function and AQP1 expression in rats treated by MTX.

Oligodendrocytes are myelinating cells in the central nervous system that form the myelin sheath of axons to support rapid nerve conduction. Human endometrial stromal cells (EnSCs) are the abundant and easy available source for cell replacement therapy. In the present study, the EnSCs were coaxed to oligodendrocyte progenitor programming by induction of neuronal condition media, including bFGF, epidermal growth factor, and platelet-derived growth factor (PDGF)-AA signaling molecules as well as triiodothyronine. Differentiated cells were analyzed for expression of oligodendrocytic markers by quantitative reverse transcription PCR and immunocytochemistry. The results showed the expression of oligodendrocyte lineage markers such as nestin, PDGF receptor alpha (PDGFRα), Sox10, and Olig2 in the level of mRNAs. The expression of nestin and PDGFRα increased after 8 days posttreatment. Interestingly, the expression of nestin and PDGFRα genes at the levels of mRNA and proteins decreased 24 days after induction. The expression of A2B5, O4, and Olig2 proteins in EnSCs was confirmed using immunocytochemistry. The results confirmed that EnSCs could response to the signaling molecules which routinely applied for oligodendrocyte differentiation. Here for the first time, we demonstrated that EnSCs could be programmed into oligodendrocyte progenitor cells and may convince to consider these cells as suitable source for cell therapy of neurodegenerative diseases.

Mouse P19 embryonic carcinoma (EC) cells are pluripotent and can differentiate into a population consisting largely of neurons and glia cells using a concentration of 5x10(-7)M of retinoic acid (RA). Thus, P19 EC cells are a good model system to study events occurring during the critical phases of neuronal differentiation, in vitro. Honey bee venom (BV) consists of mellitin, phospholipase A2, apamin and several other bioactive substances. Previous studies have shown that mellitin and phospholipase A2 - two major components of BV - play an important role in the differentiation of neurons. The purpose of this study was to examine effects of BV and RA on the differentiation of cholinergic neuron in P19 cell line. Preliminary results obtained from morphological examination showed that six days after treatment with 5x10(-7)M RA, P19 cells produced processes, and gradually obtained neuronal phenotype at approximately day-10. All cells then died at day-11. P19 cells treated with 1.3μg/ml BV produced processes on day-6 and neurons appeared in the next four days. They then proceeded to total size until day-10 and produced elongated processes; however, all cells died on day-11. Using BV and RA together had the same effect but more pronounced differentiating results. It can be concluded that applying BV with RA has an additive effect on cell differentiation and proliferation. The presence of acetylcholinesterase (AChE), frequently used as a marker for neuronal differentiation, was also determined and found using DTNB.