Resveratrol relieves HFD-induced insulin resistance in skeletal muscle tissue through antioxidant capacity enhancement and the Nrf2-Keap1 signaling pathway.

BACKGROUND: Resveratrol has received much attention due to its beneficial effects including antioxidant activity. The purpose of this study was to investigate the therapeutic effects of resveratrol treatment on oxidative stress and insulin resistance in the skeletal muscle of high-fat diet (HFD)-fed animals. METHODS AND RESULTS: A total of 30 six-week-old C57BL/6J mice were randomly allocated to three groups (10 animals in each group): The control group in which mice were fed a normal chow diet (NCD); the HFD group in which mice were fed an HFD for 26 weeks; and the HFD-resveratrol group in which HFD was replaced by a resveratrol supplemented-HFD (400 mg/kg diet) after 10 weeks of HFD feeding. At the end of this period, gastrocnemius muscle samples were examined to determine insulin resistance and the oxidative status in the presence of HFD and resveratrol. Resveratrol supplementation in HFD-fed mice reduced body and adipose tissue weight, improved insulin sensitivity, and decreased oxidative stress as indicated by lower malonaldehyde (MDA) levels and higher total antioxidant capacity. The supplement also increased the expression and activity of antioxidative enzymes in gastrocnemius muscle and modulated Nrf2 and Keap1 expression levels. CONCLUSIONS: These results suggest that resveratrol is effective in improving the antioxidant defense system of the skeletal muscle in HFD-fed mice, indicating its therapeutic potential to combat diseases associated with insulin resistance and oxidative stress.

Design of novel solar-light-induced KBi6O9I/Ag-AgVO3 nanophotocatalyst with Ag-bridged Z-scheme charge carriers separation and boosted photo-elimination of hospital effluents.

In this research, a novel solar-light-induced KBi6O9I/Ag-AgVO3 nanophotocatalyst with an Ag-bridged Z-scheme structure has been designed and synthesized through a sonochemical method to photo-degrade antibiotic hospital contaminants under simulated solar-light irradiation. Synthesized nanophotocatalysts with varying KBi6O9I to Ag-AgVO3 weight ratios underwent N2 Adsorption-Desorption, XRD, TEM, UV-Vis DRS, FESEM and PL analyses. The Ag-bridged Z-scheme-structured KBi6O9I/Ag-AgVO3 (1:1) nanophotocatalyst, demonstrated broad light absorption within the solar-light spectrum and showcased effective photocatalytic efficacy in degrading tetracycline antibiotic (88.3% and 83.5% removal for 25 and 50 mg/L, respectively, after 120 min). This performance outperformed other composited photocatalysts, as well as pure Ag-AgVO3 and KBi6O9I photocatalysts. The enhanced degradation efficiency of the KBi6O9I/Ag-AgVO3 (1:1) composite can be ascribed to the synergistic interaction of various elements. These include the surface plasmon resonance impact of silver nanoparticles, their pronounced sensitivity to solar irradiation, and the Z-scheme heterojunction configuration. Collectively, these factors work together to minimize the recombination rate of photoinduced electron-hole pairs, thereby amplifying the efficacy of photodegradation. Furthermore, the KBi6O9I/Ag-AgVO3 (1:1) composite photocatalyst displayed sustained pollutants elimination performance even after undergoing four consecutive cycles.

Microglia/macrophage polarization regulates spontaneous remyelination in intermittent cuprizone model of demyelination.

Central nervous system (CNS) lesions can repeatedly be de-and remyelinated during demyelinating diseases such as multiple sclerosis (MS). Here, we designed an intermittent demyelination model by 0.3 % Cuprizone feeding in C57/BL6 mice followed by two weeks recovery. Histochemical staining of luxol fast blue (LFB) was used for study of remyelination, detection of glial and endothelial cells was performed by immunohistochemistry staining for the following antibodies: anti Olig2 for oligodendrocyte progenitor cells, anti APC for mature oligodendrocytes, anti GFAP for astrocytes, and anti Iba-1 for microglia/macrophages, anti iNOS for M1 microglia/macrophage phenotype, anti TREM-2 for M2 microglia/macrophage phenotype and anti CD31 for endothelial cells. Also, real-time polymerase chain reaction was performed for assessment of the expression of the targeted genes. LFB staining results showed enhanced remyelination in the intermittent cuprizone (INTRCPZ) group, which was accompanied by improved motor function, increased mature oligodendrocyte cells, and reduction of astrogliosis and microgliosis. Moreover, switching from M1 to M2 polarity increased in the INTRCPZ group that was in association with downregulation of pro-inflammatory and upregulation of anti-inflammatory genes. Finally, evaluation of microvascular changes revealed a remarkable decrease in the endothelial cells in the cuprizone (CPZ) group which recovered in the INTERCPZ group. The outcomes demonstrate enhanced myelin content during recovery in the intermittent demyelination model which is in association with reshaping macrophage polarity and modification of glial and endothelial cells.

Effect of Nigella sativa Consumption on Lipid Profile and Glycemic Index in Patients with Metabolic Syndrome: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Metabolic syndrome is a multifactorial disorder and genetics, lifestyle, and aging play important roles in its prevalence. Nigella sativa has several pharmacological benefits, including anti-inflammatory, antitumor, anti-diabetic, antioxidant, and hypolipidemic effects. This meta-analysis of randomized controlled trials assesses the effect of N. sativa consumption on lipid profile and glycemic indices in patients with metabolic syndrome. METHODS: We systematically researched Cochrane Library, PubMed, Scopus, and Web of Science databases. The literature research identified 171 studies with duplication. Of those, 73 articles were screened for titles and abstracts, and 7 studies were finally selected for the meta-analysis. Because of the high degree of heterogeneity, we performed subgroup analyses based on the dose of N. sativa (<=500 mg/day or >500 mg/day). RESULTS: The results revealed that N. sativa intake significantly decreased total cholesterol (SMD: -0.71; 95% CI, -1.44 to -0.38; P = 0.00), LDL-C (SMD: -1.06; 95% CI, -1.45 to -0.66; P = 0.00) and HDL-C (SMD: -0.31; 95% CI, 0.09 to 0.53; P = 0.01) concentrations. In addition, N. sativa significantly decreased FBS (SMD: -0.8; 95% CI, -1.21 to -0.39; P = 0.00) and HbA1c (SMD: -0.37; 95% CI, -0.66 to -0.09; P = 0.01) concentrations. No publication bias was observed, and sensitivity analysis showed stable results. CONCLUSION: The current systematic review and meta-analysis indicates that N. sativa could improve lipid profile and glycemic index in patients with metabolic syndrome.

Crosstalk between autophagy and insulin resistance: evidence from different tissues.

Insulin is a critical hormone that promotes energy storage in various tissues, as well as anabolic functions. Insulin resistance significantly reduces these responses, resulting in pathological conditions, such as obesity and type 2 diabetes mellitus (T2DM). The management of insulin resistance requires better knowledge of its pathophysiological mechanisms to prevent secondary complications, such as cardiovascular diseases (CVDs). Recent evidence regarding the etiological mechanisms behind insulin resistance emphasizes the role of energy imbalance and neurohormonal dysregulation, both of which are closely regulated by autophagy. Autophagy is a conserved process that maintains homeostasis in cells. Accordingly, autophagy abnormalities have been linked to a variety of metabolic disorders, including insulin resistance, T2DM, obesity, and CVDs. Thus, there may be a link between autophagy and insulin resistance. Therefore, the interaction between autophagy and insulin function will be examined in this review, particularly in insulin-responsive tissues, such as adipose tissue, liver, and skeletal muscle.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

Potato-on-rod like of Z-scheme plasmon Ag2CrO4-Ag2Mo2O7 heterojunction nanophotocatalyst with high stability and accelerated photo-degradation evolution of organic contaminants.

The shocking increase of resistant dye pollutants in the environment and their harmful effects has become a potential threat to the ecosystem. In the current work, the novel and highly efficient potato-on-rod-like Z-scheme plasmon Ag2CrO4-Ag2Mo2O7 heterojunction nano-photocatalyst was synthesized by precipitation method to photodegrade different organic dyes under artificial sunlight. The required analysises were carried out to characterize nanophotocatalysts. FESEM and TEM results showed the placement way of potato-like Ag2CrO4 between/on rod-like Ag2Mo2O7 which was leading to suitable structure and surface morphology. Besides, the morphology observations released the meso-/macroporous potato-on-rod like architecture self-assembled by nanoparticles. DRS analysis also confirmed two band gap energies of 2.55 and 1.72 eV in Ag2CrO4-Ag2Mo2O7 (3:1) resulting from forming a heterojunction structure and the plasmon Ag. Ag2CrO4-Ag2Mo2O7 (3:1) nanophotocatalyst exhibited the most remarkable activity in the photodegradation of 10 mg/L 2-naphthol orange (97.8%), 10 mg/L rhodamine B (99.7%), 10 mg/L crystal violet (98.9%), and 10 mg/L methyl orange (56.1%) with a catalyst dosage of 0.1 gr for about 90 min. The appropriate energy band gap, the formation of the heterostructure, the presence of meso (0.0038 cm3/g) and macro (0.0044 cm3/g) holes, and pore diameter at about 17.2 nm based on BET-BJH analysis that facilitated the penetration of pollutant molecules, increased pollutant adsorption and demonstrated stunning capability of efficient light harvesting, the reason was electron-hole pairs recombination rate reduction. Moreover, the fabricated samples showed tremendous catalyst constancy and reusability even after the fourth run. Results have shown the remarkable photocatalytic activity under visible light and provide an environment-friendly and green strategy to overcome the challenges of organic pollutants present in aqueous solutions.

Higher daily physical activity levels may facilitate pre-diabetes regression to normoglycemia: A longitudinal study among an Iranian population.

The possible association of habitual physical activity (PA) and the risk of pre-diabetes (Pre-DM) progression to type 2 diabetes (T2D) or the chance of returning to normoglycemia was investigated. This cohort study included 1167 Pre-DM individuals (mean age of 53.5 years, and 45.3% men) who participated in the third phase of the Tehran Lipid and Glucose Study (2006-2008) and followed up to a median of 9 years. PA, including leisure time and job activities, was measured using a reliable and validated Iranian version of the Modifiable Activity Questionnaire and reported as metabolic equivalent (MET)-minutes per week. The odds ratios (ORs) and 95% confidence intervals (CIs) of incident T2D and returning to normoglycemia were estimated in relation to PA levels (i.e., per every 500 MET-minutes/week, or across categories of PA levels < 600 as a reference, 600-1500 and > 1500 MET-minutes/week). During the study follow-up, 39.0 % progressed to T2D, and 37.8% returned to normoglycemia. Compared to subjects with a PA < 600 MET-minutes/week, the chance of regression to normoglycemia increased by 58% [OR = 1.58, 95% CI = 1.03-2.40 âˆ¼ relative risk (RR) = 1.32, 95% CI = 1.02-1.63] among the participants who had a PA > 1500 MET-minutes/week. We further noted that each 500 MET-min/week activity corresponded to an elevated chance of returning to normoglycemia by 5% (OR = 1.05, 95% CI = 1.01-1.11). The study's findings provided evidence that higher daily PA levels may facilitate Pre-DM regression to normoglycemia. The beneficial effect of PA in Pre-DM subjects needs to exceed the recommended levels (i.e., 600 MET-minutes/week).

Development of a comprehensive assessment tool to measure the quality of care for individuals with traumatic spinal cord injuries.

OBJECTIVE: To develop a comprehensive assessment tool to evaluate the Quality of Care (QoC) in managing individuals with traumatic spinal cord injuries (TSCI). METHOD: At first, the concepts of QoC for TSCI were identified by conducting a qualitative interview along with re-evaluation of the results of a published scoping review (conceptualization). After operationalization of indicators, they were valued by using the expert panel method. Afterward, the content validity index (CVI) and content validity ratio (CVR) were calculated and served as cut-offs for indicator selection. Then specific questions were developed for each indicator and classified into three categories: pre-hospital, in-hospital, and post-hospital. Data availability of the National Spinal Cord Injury Registry of Iran (NSCIR-IR) was subsequently used to design questions that represent indicators in an assessment tool format. The comprehensiveness of the tool was evaluated using a 4-item Likert scale by the expert panel. RESULT: Twelve experts participated in conceptualization and 11 experts participated in operationalization phase. Overall, 94 concepts for QoC were identified from published scoping review (87 items) and qualitative interviews (7 items). The process of operationalization and indicator selection led to the development of 27 indicators with acceptable content validity. Finally, the assessment tool contained three pre-hospital, twelve in-hospital, nine post-hospital, and three mixed indicators. Ninety-one percent of experts evaluated the entire tool as comprehensive. CONCLUSION: Our study presents a health-related QoC tool that contains a comprehensive set of indicators to assess the QoC for individuals with TSCI. However, this tool should be used in various situations to establish construct validity further.

In vitro and Molecular Docking Analysis of Quercetin as an Anti-inflammatory and Antioxidant.

INTRODUCTION: Quercetin (3,3',4',5,7-pentahydroxyflavone) is a dietary flavonoid with good antioxidant and anti-inflammatory properties. AIMS: The present study aims to determine these effects in peripheral blood mononuclear cells (PBMCs) evoked by lipopolysaccharides (LPS). METHODS: The mRNA expression and protein secretion of inflammatory mediators were evaluated by enzyme- linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (PCR), respectively. Western blotting was utilized for assessing p65-NF-κB phosphorylation. Ransod kits evaluated the glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in the cell lysates. Ultimately, the molecular docking approach was performed to investigate the biological activity of Quercetin against NF-κB pathway proteins and antioxidant enzymes. RESULTS: The findings revealed that quercetin significantly attenuated the expression and secretion of inflammatory mediators and p65-NF-κB phosphorylation in LPS-induced PBMCs. Additionally, quercetin dose-dependently improved the activities of SOD and GPx enzymes and decreased LPS-mediated oxidative stress in PBMCs. Moreover, quercetin has a considerable binding affinity to IκKb, the core element of the NF-κB pathway and the antioxidant enzyme SOD. CONCLUSION: The data show that quercetin plays a vital role in ameliorating inflammation and oxidative stress caused by LPS in PBMCs.

Alterations of SOCS1 and SOCS3 transcript levels, but not promoter methylation levels in subcutaneous adipose tissues in obese women.

BACKGROUND: Animal model studies suggest that change in the members of the suppressor of the cytokine signaling (SOCS) family (mainly SOCS1 and SOCS3) is linked to the pathogenesis of obesity-related metabolic disorders. Moreover, epigenetic modification is involved in the transcriptional regulation of the SOCS gene family. Here, we aimed to evaluate the mRNA expression as well as gene promoter methylation of SOCS1 and SOCS3 in subcutaneous adipose tissue (SAT) from obese women compared to normal-weight subjects. We also intend to identify the possible association of SOCS1 and SOCS3 transcript levels with metabolic parameters in the context of obesity. METHODS: This study was conducted on women with obesity (n = 24) [body mass index (BMI) ≥ 30 kg/m 2] and women with normal-weight (n = 22) (BMI < 25 kg/m 2). Transcript levels of SOCS1 and SOCS3 were evaluated by real-time PCR in SAT from all participants. After bisulfite treatment of DNA, methylation-specific PCR was used to assess the putative methylation of 10 CpG sites in the promoter of SOCS1 and 13 CpG sites in SOCS3 in SAT from women with obesity and normal weight. RESULTS: It was found that unlike SOCS3, which disclosed an elevating expression pattern, the expression level of SOCS1 was lower in the women with obesity as compared with their non-obese counterparts (P-value = 0.03 for SOCS1 transcript level and P-value = 0.011 for SOCS3 transcript level). As for the analysis of promoter methylation, it was found that SOCS1 and SOCS3 methylation were not significantly different between the individuals with obesity and normal weight (P-value = 0.45 and P-value = 0.89). Correlation analysis indicated that the transcript level of SOCS1 mRNA expression had an inverse correlation with BMI, hs-CRP levels, HOMA-IR, and insulin levels. However, the SOCS3 transcript level showed a positive correlation with BMI, waist-to-height ratio, waist circumference, hip circumference, hs-CRP, HOMA-IR, insulin, fasting blood glucose, and total cholesterol. Interestingly, HOMA-IR is the predictor of the transcript level of SOCS1 (ß = - 0.448, P-value = 0.003) and SOCS3 (ß = 0.465, P-value = 0.002) in SAT of all participants. CONCLUSIONS: Our findings point to alterations of SOCS1 and SOCS3 transcript levels, but not promoter methylation levels in subcutaneous adipose tissues from women with obesity. Moreover, mRNA expression of SOCS1 and SOCS3 in SAT was associated with known obesity indices, insulin resistance, and hs-CRP, suggesting the contribution of SOCS1 and SOCS3 in the pathogenesis of obesity-related metabolic abnormalities. However, further studies are required to establish this concept.

Amelioration of obesity-induced white adipose tissue inflammation by Bacillus coagulans T4 in a high-fat diet-induced obese murine model.

AIM: Fresh evidence suggests that B. coagulans can be regarded as a promising therapeutic alternative for metabolic disorders. However, the possible effects of this probiotic on obesity-induced adipose tissue inflammation are unknown. METHODS: C57BL/6j male mice were assigned to a normal-chow diet (NCD) or a high-fat diet (HFD) for 10 weeks. After this period, HFD-fed mice were randomly divided into two groups; HFD control group and HFD plus B. coagulans T4 (IBRC-N10791) for another 8 weeks. B. coagulans T4 was administrated daily by oral intragastric gavage (1 × 109 colony-forming units). KEY FINDINGS: Here, we found that B. coagulans successfully mitigated obesity and related metabolic disorder, as indicated by reduced body weight gain, decreased adiposity, and improved glucose tolerance. B. coagulans T4 administration also inhibited HFD-induced macrophage accumulation in white adipose tissue and switched M1 to M2 macrophages. In parallel, B. coagulans T4 treatment attenuated HFD-induced alteration in mRNA expression of pro/anti-inflammatory cytokines and Tlr4 in white adipose tissue. Moreover, B. coagulans T4 supplementation reduced the Firmicutes/Bacteriodetes ratio and increased the number of Lactobacillus and Faecalibacterium compared to the HFD group. Additionally, a significant increase in propionate and acetate levels in the HFD group was seen following B. coagulans T4 administration. SIGNIFICANCE: Taken together, the present study provides evidence that B. coagulans T4 supplementation exerts anti-obesity effects in part through attenuating inflammation in adipose tissue. The present study will have significant implications for obesity management.

Evaluation of Apoptosis-related Genes and Hormone Secretion Profiles Using Three Dimensional Culture System of Human Testicular Organoids.

BACKGROUND: In reproductive biology, testicular organoids can be used to treat infertility and to study testicular development and spermatogonial stem cells (SSCs) differentiation. Generating organoid from primary cells is challenging. In this study, testicular organoids were created using human primary testicular cells and evaluated the apoptotic gene expression and hormone secretion profiles of the organoids. MATERIALS AND METHODS: Primary human testicular cells were isolated using 2-step enzymatic digestion from three brain-dead donors. Immunocytochemistry and flow cytometry analyses were performed to confirm human SSCs. Isolated cells were cultured in three experimental groups: control group (2 dimensional (2D)), group 1 (organoid culture after 2D culture), and group 2 (organoid culture immediately after enzymatic digestion). Testicular organoids were cultured in DMEM/F-12 media supplemented with follicle-stimulating hormone (FSH) and fetal bovine serum (FBS) for four weeks. After 24 hours and four weeks of culture, reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the relative expression of apoptotic genes (caspase 3, 9, Bax, and Bcl-2). At 24 hours, two weeks, and four weeks after culture, enzyme-linked immunoassay (ELISA) was used to determine the testosterone and inhibin B concentrations. Light microscopy and toluidine blue staining were also used for morphological analysis. RESULTS: RT-qPCR results revealed that pro-apoptotic (caspase 3, 9, Bax) gene expression levels were highest in group 2 after 24 h and four weeks of culture. In contrast, the expression level of Bcl-2 (anti-apoptotic) was lower in group 2 compared to other groups. The hormone secretion levels decreased in a time-dependent manner during the cultivation. According to morphological evaluations, testicular organoids are compact, spherical structures with two to three elongated cells organized along their border. CONCLUSION: Our findings revealed that the testicular organoid culture system maintained hormonal secretory abilities, demonstrating the function of Sertoli and Leydig cells in the absence of testis-specific environments.

Glial Response to Intranasal Mesenchymal Stem Cells in Intermittent Cuprizone Model of Demyelination.

Intranasal mesenchymal stem cells (MSCs) delivery is a non-invasive method that has received interests for treatment of neurodegenerative diseases, such as multiple sclerosis (MS). The impact of intranasal MSCs on intermittent cuprizone model of demyelination was a focus of this study. C57/BL6 mice were fed with 0.3% cuprizone in an intermittent or single ways. Luxol fast blue (LFB), Rotarod test, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot (WB) were used for interpretation of outcomes. MSCs effectively homed to the corpus callosum area, were able to improve motor coordination and to promote myelin recovery in the intermittent cuprizone (INTRCPZ/MSCs). Astrogliosis (GFAP+ cells) and microgliosis (Iba-1+ cells) were hampered, and more mature oligodendrocyte cells (APC+ cells) were identified in mice receiving INTRCPZ/MSCs. Such treatment also considerably reduced markers related to the macrophage type 1 (M1) cells, namely iNOS and CD86, but it recovered the M2 markers MRC-1 and TREM-2. In addition, a remarkable decrease in the expressions of pro-inflammatory IL-1ß and TNFα but an increase in the rate of anti-inflammatory TGF-ß and IL-10 were identified in mice that underwent INTRCPZ/MSCs therapy. Finally, microvascular changes were evaluated, and a noticeable increase in the expression of the endothelial cell marker CD31 was found in the INTRCPZ/MSCs-treated mice (p < 0.05 for all). The outcomes are representative of the efficacy of intranasal MSCs delivery in intermittent cuprizone model of MS for reshaping macrophage polarity along with modification of glial, inflammatory, and angiogenic markers in favor of therapy.

Preconditioning exercise reduces hippocampal neuronal damage via increasing Klotho expression in ischemic rats.

Considerable amounts of oxidants are produced in cerebral ischemia, where oxidative stress plays a key role in neuronal damage after ischemia. Klotho, an anti-aging protein, alleviates oxidative stress by activating the transcription of an important antioxidant enzyme, manganese superoxide dismutase (MnSOD), in the nervous system. Thus, increased Klotho expression level could lead to a reduction in neuronal damages after brain ischemia via lowering oxidative stress. It is known that physical activity increases Klotho expressions. In this study, we assessed neuroprotective effects of preconditioning exercise in rats (treadmill running at a speed of 20 m/min,30 min/day, six days/week, for3 weeks) on hippocampal Klotho and MnSOD expression in the brain using an animal model of stroke, middle cerebral artery occlusion (MCAO). Our study revealed a reduction in hippocampal Klotho and MnSOD expression as well as CA1 neuronal activity in MCAO compared to the sham group. Exercise prevented the ischemia-induced decline in Klotho and MnSOD expression levels as well as CA1 neuronal activity in Exercise + MCAO compared to the MCAO group. Also, exercise significantly improved the neurological scores and reduced brain infarction area in Exercise + MCAO in comparison to MCAO group. There was a post-ischemia deficit in the working memory, as measured by spontaneous alternation percentage using Y-maze test, in MCAO compared to the sham group. The latter effect was not observed in the Exercise + MCAO group, which could be related to an increase in the antioxidant capacity as exhibited by Klotho and MnSOD up-regulation. The results were confirmed with a positive correlation between Klotho expression and MnSOD expression which allows proposing Klotho as a potential neuroprotective protein in ischemic stroke with respect to antioxidant defense. In general, the present study suggested that preconditioning exercise induced upregulation of Klotho and MnSOD, as well as attenuated the post-ischemic injuries. The upregulation of Klotho might be an underlying mechanism by which preconditioning exercise plays as a neuroprotective factor against post-ischemic neural injuries in ischemic rats.

Melatonin in cryopreservation media improves transplantation efficiency of frozen-thawed spermatogonial stem cells into testes of azoospermic mice.

BACKGROUND: Cryostorage of spermatogonial stem cells (SSCs) is an appropriate procedure for long-term storage of SSCs for fertility preservation. However, it causes damage to cellular structures through overproduction of ROS and oxidative stress. In this study, we examined the protective effect of melatonin as a potent antioxidant in the basic freezing medium to establish an optimal cryopreservation method for SSCs. METHODS: SSCs were obtained from the testes of neonatal male mice aged 3-6 days. Then, 100 µM melatonin was added to the basic freezing medium containing DMSO for cryopreservation of SSCs. Viability, apoptosis-related markers (BAX and BCL2), and intracellular ROS generation level were measured in frozen-thawed SSCs before transplantation using the MTT assay, immunocytochemistry, and flow cytometry, respectively. In addition, Western blotting and immunofluorescence were used to evaluate the expression of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins in frozen-thawed SSCs after transplantation into recipient testes. RESULTS: The data showed that adding melatonin to the cryopreservation medium markedly increased the viability and reduced intracellular ROS generation and apoptosis (by decreasing BAX and increasing BCL2) in the frozen-thawed SSCs (p < 0.05). The expression levels of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins and resumption of spermatogenesis from frozen-thawed SSCs followed the same pattern after transplantation. CONCLUSIONS: The results of this study revealed that adding melatonin as an antioxidant to the cryopreservation medium containing DMSO could be a promising strategy for cryopreservation of SSCs to maintain fertility in prepubertal male children who suffer from cancer.

Evidence for the effect of soluble uric acid in augmenting endoplasmic reticulum stress markers in human peripheral blood mononuclear cells.

There is evidence regarding the association of hyperuricemia with inflammatory disorders. Hence, it has been of particular interest to dissect the exact role of alteration in uric acid (UA) levels in the context of inflammation. Recently, the endoplasmic reticulum (ER) stress pathway has come into the forefront as a possible mechanism linking hyperuricemia to inflammation. Here, we intended to examine the role of UA in the presence or absence of a second stimulus, LPS, in human peripheral blood mononuclear cells (PBMCs), and analyzed ROS production as well as expression of ER stress markers: GRP78 and CHOP, and inflammatory cytokines.PBMCs were isolated using Ficoll gradient centrifugation from healthy volunteers. Cell viability was measured by MTT assay. PBMCs were treated with an increasing concentration of soluble UA (0, 5, 12, and 20 mg/dl) for 20 h, followed by the addition of 100 ng/mL of LPS or vehicle for another 4 h. Real time-PCR was performed to investigate the mRNA expression of GRP78, CHOP, TNF-α, IL-1ß, and IL-6, and western blot was used to investigate the protein levels of GRP78 and CHOP. Moreover, ELISA was used to evaluate the protein levels of TNF-α, IL-1ß, and IL-6. Finally, intracellular ROS production was determined using fluorescent probes (DCFH-DA).High concentrations of UA either alone or combined with LPS increased the protein levels of GRP78 and CHOP. On the other hand, LPS alone increased the protein levels of GRP78 and CHOP. However, there was no significant difference between the mRNA expression of GRP78, CHOP, TNF-α, IL-1ß, and IL-6 when PBMCs were treated with UA. High concentrations of UA augmented LPS-stimulated IL-1ß transcript and protein levels as well as TNF-α protein levels in PBMC culture. Moreover, high concentrations of UA along with LPS significantly increased intracellular ROS production.It seems that a high concentration of UA not only induces the protein levels of ER stress markers in PBMCs but also augments the impact of LPS on the levels of pro-inflammatory markers and ROS production.

Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model.

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion-a mouse model for long-term infertility-was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C-Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6-integrin, and ß1-integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.

Metformin Therapy Attenuates Pro-inflammatory Microglia by Inhibiting NF-κB in Cuprizone Demyelinating Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS.