Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)—induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T₃), thyroxine (T₄), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P₄₅₀ side chain cleavage enzyme (P₄₅₀scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T₃, T₄, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P₄₅₀scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.     

Corticosterone induces steroidogenic lesion in cultured adult rat Leydig cells by reducing the expression of star protein and steroidogenic enzymes

The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34 degrees C in a CO(2) incubator under 95% air and 5% CO(2) using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34 degrees C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P(450) side chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P(450) aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P(450) side chain cleavage enzyme, 3beta- and 17beta-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P(450) aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200-800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein.     

Peroxisomal participation in the cellular response to the oxidative stress of endotoxin

Exposure to a sublethal dose of endotoxin offers protection against subsequent oxidative stresses. The cellular mechanisms involved in generating this effect are not well understood. We evaluated the effect of endotoxin on antioxidant enzymes in liver peroxisomes. Peroxisomes have recently been shown to contain superoxide dismutase (SOD) and glutathione peroxidase (GPX) in addition to catalase. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment increased the specific activity of SOD and GPX in peroxisomes to 208% and 175% of control activity, respectively. These findings correlated with increases in peroxisomal SOD and GPX proteins observed by immunoblot. Although the quantity of catalase protein was increased when assessed by immunoblot analysis, the specific activity of catalase was decreased to 68% of control activity. Activation of catalase with ethanol only restored catalase activity to control levels suggesting that catalase had undergone irreversible inactivation. The observed increase in GPX activity may represent a compensatory mechanism triggered by accumulating H₂O₂. The data presented here suggest for the first time that mammalian peroxisomal antioxidant enzymes are altered during the oxidative injury of endotoxin treatment.     

Intermittent hypobaric hypoxia-induced oxidative stress in rat erythrocytes: protective effects of vitamin E, vitamin C, and carnitine

This study was aimed at determining the effect of vitamin E, vitamin C, and carnitine on intermittent hypobaric-hypoxia-induced oxidative stress (OS) in erythrocytes. For this purpose, male Wistar rats of 4 months of age were orally supplemented with one of the antioxidants prior to exposure to altitudes of 5700 m or 6300 m. Hemoglobin (Hb) and OS indices such as osmotic fragility and hemolysis were measured together with lipid peroxidation (LPO) and protein oxidation. The increase in Hb was accompanied by increase in activities of antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) during exposure to both the altitudes without any further elevation by supplements. The extent of reduction in osmotic fragility and hemolysis by vitamin E and carnitine was greater at 6300 m than at 5700 m. Increase in LPO products, for example, malondialdehyde (MDA) and lipofuscin-like autofluorescent substances (AFS) was noticeable at both the altitudes, and vitamin E and carnitine were effective in reducing LPO. While protein oxidation products such as carbonyl content (PrC) and advanced oxidation protein products (AOPP) increased at 6300 m, protein sulphydryl (P-SH) content decreased. P-SH levels were restored on supplementation of antioxidants. Hence, our results indicate that vitamin E, vitamin C, and carnitine may be beneficial in overcoming OS and hemolysis under situations such as intermittent hypobaric hypoxia (IHH) and hypobarotherapy wherein hypoxia is used to correct many pathological situations in humans. Further, this study suggests that supplementation of vitamin E, vitamin C, and L-carnitine alone and not in combination can be beneficial in attenuating the OS associated with IHH compared to the unsupplemented rats exposed to two different altitudes.     

Insulin involved Akt/ERK and Bcl-2/Bax pathways against oxidative damages in C6 glial cells

Insulin, a hypoglycemic hormone, has multiple functions in the brain. The aim of this study to identify the mechanisms of insulin in hydrogen peroxide (H₂O₂)-induced toxicity in the C6 glial cells. Cytotoxicity, lactate dehydrogenase, nitric oxide, reactive oxygen species and calcium ion, lipid peroxidation, protein oxidation and glutathione levels were determined. Signaling pathway molecules were assessed by western blotting and RT-PCR. The results showed that treatment with insulin reduced the cell death and cell membrane damages against H₂O₂-induced toxicity. Furthermore, insulin interfered H₂O₂-induced intracellular generation of reactive oxygen species and calcium-ion transport, apoptosis, including lipid and protein oxidation products. Cells treated with insulin reverted H₂O₂-induced suppression of reduced glutathione levels by blocking oxidized glutathione. Moreover, insulin treatment activates Akt, restores ERK1/2 and Bcl-2 by preventing Bax and Bax/Bcl-2 ratio. Our results suggest that treatment of insulin exerts potential role against 24?h of H₂O₂-induced toxicity in C6 cells.     

The protective effect of melatonin against cypermethrin-induced oxidative stress damage in Spodoptera litura (Lepidoptera: Noctuidae)

Antioxidant enzymes form the first-line defense against free radicals damage in organisms. Their regulation depends mainly on the oxidant and antioxidant status of the cell, given that oxidants are their principal modulators. Therefore, the aim of the present study was to investigate the effect of melatonin on synthetic pyrethroid insecticide-induced antioxidative enzymes activity in Spodoptera litura larvae. In addition, activities of enzymatic antioxidants viz. superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), α, β-esterase, and acetylcholine esterase (AChE) were assessed. There was no significant change in GST levels in the melatonin-treated groups. Melatonin modulates cypermethrin-induced changes in the activities of esterase and AChE, whereas SOD, CAT, and GR activity was significantly increased in melatonin-treated samples when compared to control. In conclusion, the results of the current study revealed that SP toxicity activated oxidant systems in all antioxidant systems in some tissues of insects. Melatonin administration led to a marked increase in antioxidant activity and inhibited GST and AChE in most of the tissues studied.     

Ameliorative effect of an oxovanadium (IV) complex against oxidative stress and nephrotoxicity induced by cisplatin

Objective: The present study was designed to investigate the chemoprotective efficacy of an L-cysteine-based oxovanadium (IV) complex, namely, oxovanadium (IV)-L-cysteine methyl ester complex (VC-IV) against cisplatin (CDDP)-induced renal injury in Swiss albino mice.

Methods: CDDP was administered intraperitoneally (5 mg/kg body weight) and VC-IV was administered orally (1 mg/kg body weight) in concomitant and 7 days pre-treatment schedule.

Results: CDDP-treated mice showed marked kidney damage and renal failure. Administration of VC-IV caused significant attenuation of renal oxidative stress and elevation of antioxidant status. VC-IV also significantly decreased serum levels of creatinine and blood urea nitrogen, and improved histopathological lesions. Western blot analysis of the kidneys showed that VC-IV treatment resulted in nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) through modulation of cytosolic Kelch-like ECH-associated protein 1. Thus, VC-IV stimulated Nrf2-mediated activation of antioxidant response element (ARE) pathway and promoted expression of ARE-driven cytoprotective proteins, heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1, and enhanced activity of antioxidant enzymes. Interestingly, VC-IV did not alter the bioavailability and renal accumulation of CDDP in mice.

Discussion: In this study, VC-IV exhibited strong nephroprotective efficacy by restoring antioxidant defense mechanisms and hence may serve as a promising chemoprotectant in cancer chemotherapy.     

AZT induces oxidative damage to cardiac mitochondria: protective effect of vitamins C and E

AZT (zidovudine) is a potent inhibitor of HIV replication and a major antiretroviral drug used for AIDS treatment. A major limitation in the use of AZT is the occurrence of severe side effects. The aim of this work was to test whether AZT causes oxidative damage to heart mitochondria and whether this can be prevented by supranutritional doses of antioxidant vitamins. An experimental animal model was used in which mice were treated with AZT for 35 days (10 mg/kg/day) in drinking water. Animals treated with antioxidant vitamins were fed the same diet as controls but supplemented with vitamins C (ascorbic acid, 10 g/ kg diet) and E (alpha-dl-tocopherol, 0.6 g/kg diet) for 65 days before sacrifice. This resulted in a daily intake of 1250 mg/kg/day (vitamin C) and 75 mg/kg/day (vitamin E). Cardiac mitochondrial DNA (mtDNA) of mice treated with AZT had over 120% more oxo-dG (8-oxo-7,8-dihydro-2'-deoxyguanosine, which is a biomarker of oxidative damage to DNA) in their mitochondrial DNA than untreated controls. AZT treatment also caused an increase in mitochondrial lipid peroxidation and an oxidation of mitochondrial glutathione. Dietary supplementation with supranutritional doses of the antioxidant vitamins C and E protected against these signs of mitochondrial oxidative stress. The oxidative effects of AZT are probably due to an increase in production of reactive oxygen species by mitochondria of AZT-treated animals, raising the possibility that oxidative stress may play an important role in the cardiotoxicity of AZT.     

Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis

Abstract

Although endothelial progenitor cells (EPCs) have been used to promote revascularization after peripheral or myocardial ischemia, excess amounts of reactive oxygen species (ROS) are often involved in senescence and apoptosis of EPCs, thereby causing defective neovascularization and reduced or failed recovery. Here, we examined the cytoprotective effect of Ecklonia cava-derived antioxidant dieckol (DK) on oxidative stress-induced apoptosis in EPCs to improve EPC bioactivity for vessel repair. Although H₂O₂ (10 ^{? 3} M) increased the intracellular ROS level in EPCs, DK (10ug/ml) pretreatment suppressed the H₂O₂-induced ROS increase and drastically reduced the ratios of apoptotic cells. H₂O₂-induced ROS increased the phosphorylation of p38 MAPK and JNK; this was inhibited by DK pretreatment. H₂O₂ treatment increased the phosphorylation of NF-κB, which was blocked by pretreatment with SB 203580, a p38 MAPK inhibitor, or SP 600125, a JNK inhibitor. H₂O₂ decreased the cellular levels of Bcl-2 and c-IAPs, cellular inhibitors of apoptosis proteins, but increased caspase-3 activation. However, all these effects were inhibited by pretreatment with DK. Injection of DK-mixed EPCs (DK + EPCs) into myocardial ischemic sites in vivo induced cellular proliferation and survival of cells at the ischemic sites and, thereby, enhanced the secretion of angiogenic cytokines at the ischemic sites. These results show that DK + EPC exhibit markedly enhanced anti-apoptotic and antioxidative capabilities, unlike that shown by EPCs alone; thus, they contribute to improved repair of ischemic myocardial injury through cell survival and angiogenic cytokine production.     

Seasonal variation in the activity of the Leydig cells in Egyptian Nubian goat (Zaraibi) bucks

Ten Egyptian Nubian goat bucks were used to evaluate the effect of season on testicular hormonal activity and ultrastructure. Parameters were recorded for 7 consecutive weeks in the middle of the four seasons, with blood samples being collected weekly. At the end of each of these seasons, testicular biopsies were obtained surgically for histological and cytological studies. Season had a significant effect on plasma testosterone concentration, being at its lowest level (P < 0.01) during winter and spring (1.2 and 2.6 ng/ml, respectively), while at its highest during summer (10 ng/ml). The effect of season on plasma LH concentration was higher (P < 0.01) in autumn (2.9 mIU/ml) and less in spring and summer (0.4 mIU/ml). Season of the year influenced the percentage of sectional tissue area occupied by the seminiferous tubules and interstitial tissue. Seminiferous tubules occupied the majority of the testicular tissue during winter (76.6%), with the least being occupied during spring (49.8%). The thickness of the seminiferous tubules was maximal during autumn, followed by summer (53 and 36 μm, respectively). In summer the Leydig cells contained abundant smooth endoplasmic reticulum (sER), while some areas of the cytoplasm were occupied exclusively by tubular sER, arranged in parallel—indicating the highest activity of these cells. A characteristic multivesicular structure with numerous large lipid droplets and vacuoles was recorded in the Leydig cells during spring and winter, denoting low or even arrested activity of the cells. It could be concluded that season influences the activity of the Leydig cells of Egyptian Nubian bucks, and this is reflected by their ultrastructure and secretive activity.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H₂O₂) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H₂O₂-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H₂O₂-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H₂O₂, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca^{2 +}) chelator, overexpression of Homer 1a had no significant effects on H₂O₂-induced oxidative stress. These results suggest that Homer 1a has protective effects against H₂O₂-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca^{2 +} homeostasis.     

Potential antitumour and pro-oxidative effects of (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR)

Context: Characterization of the pro-oxidant activity of QNACR.

Objectives: Reactive oxygen species (ROS) induce cellular damage and represent unique opportunities to kill malignant cells. In this study, we synthesized and evaluated the new compound, (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR) as potential pro-oxidative agent against breast cancer.

Methods: Oxidative stress biomarkers such as ROS, thiobarbuturic acid reactive species (TBARs) and different antioxidant enzyme activities were determined in cell lysates.

Results: QNACR showed cytotoxic and more selective effects to tumour MCF7 cells (IC₅₀ < 25 µM) compared to antitumour controls, inducing ROS and TBARs parallel to inhibitions of catalase (CAT), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Longer exposures to QNACR triggered adaptive effects increasing the overall activities of CAT, glutathione reductase, G6PDH and 6PGDH, but eventually the adaptation changes faded and cells died.

Conclusion: QNACR led to remarkable modifications in the oxidative status of tumour cells, proposing this compound as potential alternative for antitumour therapy.     

Effect of stationary magnetic field strengths of 150 and 200 mT on reactive oxygen species production in soybean

Our previous investigation reported the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean. In this study, soybean seeds treated with static magnetic fields of 150 and 200 mT for 1 h were evaluated for reactive oxygen species (ROS) and activity of antioxidant enzymes. Superoxide and hydroxyl radicals were measured in embryos and hypocotyls of germinating seeds by electron paramagnetic resonance spectroscopy and kinetics of superoxide production; hydrogen peroxide and antioxidant activities were estimated spectrophotometrically. Magnetic field treatment resulted in enhanced production of ROS mediated by cell wall peroxidase while ascorbic acid content, superoxide dismutase and ascorbate peroxidase activity decreased in the hypocotyl of germinating seeds. An increase in the cytosolic peroxidase activity indicated that this antioxidant enzyme had a vital role in scavenging the increased H(2)O(2) produced in seedlings from the magnetically treated seeds. Hence, these studies contribute to our first report on the biochemical basis of enhanced germination and seedling growth in magnetically treated seeds of soybean in relation to increased production of ROS.     

Zearalenone induces ROS‐mediated mitochondrial damage in porcine IPEC‐J2 cells

In this study, the mitochondrial damage effect and mechanism of zearalenone (ZEA) in swine small intestine IPEC‐J2 cells in vitro were comprehensively characterized. The analyses revealed that ZEA at high doses (8 and 7 μg/mL) can significantly increase P < 0.05 the malondialdehyde levels and decrease antioxidant enzymes activities after 48 h of exposure. Meanwhile, the reactive oxygen species (ROS) accumulation increased in high dose ZEA‐treated groups after 2 h treatment, but decreased due to the ROS‐induced mitochondrial damage and the caused cell apoptosis after 48 h of high does ZEA treatment. Moreover, the decreasing of mitochondrial membrane potential (MMP; ΔΨ) in high dose ZEA exposure was observed in line with the increasing ROS production in mitochondria. Results suggest that ZEA exposure can induce mitochondrial damage by reducing antioxidant enzyme activities, accumulation of ROS, and decreasing MMP. The mitochondrial damage had a dramatic concentration–effects relationship with ZEA.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

ROS与造血干细胞损伤研究进展

辐射可以通过引起造血干细胞（hematopoietic stem cell,HSC）内活性氧（reactive oxygen species,ROS）系统水平升高导致HSC损伤。HSC损伤患者出现难治性血液系统疾病,严重影响患者生存质量,甚至威胁患者生命。ROS可以通过多种机制引起组织、器官和细胞损伤。ROS的来源包括：线粒体、NOX（NADPH oxidases）、细胞色素P450酶、黄嘌呤氧化酶、非偶联NO合酶。已证实HSC内ROS来源于NOX。ROS升高后影响HSC在成骨细胞微环境定位,导致HSC与微环境相互作用减弱,从而影响HSC功能。此外,ROS升高后通过激活P38MAPK-P16Ink4途径,损伤HSC自我更新能力,并且使HSC定向分化产生更多的髓系克隆而不是红细胞系克隆;P13K—Akt-mTOR途径可能也是ROS诱导HSC损伤途径。ROS对细胞周期影响为：促使HSC离开G0期进入细胞周期,导致干细胞池的耗尽。基于NOX在氧化还原信号传递过程中的重要作用,证实辐射通过NOX产生的ROS以及鉴定产生ROS的NOX亚型,这一工作会为临床靶向治疗辐射诱发的血液系统疾病提供重要的价值。     

Protective role of Cytoglobin and Neuroglobin against the Lipopolysaccharide (LPS)-induced inflammation in Leydig cells ex vivo

Leydig cells are responsible for testosterone production in male testis upon stimulation by luteinizing hormone. Inflammation and oxidative stress related Leydig cell dysfunction is one of the major causes of male infertility. Cytoglobin (CYGB) and Neuroglobin (NGB) are two globin family member proteins which protect cells against oxidative stress.In the current study, we established a Lipopolysaccharide (LPS)-induced inflammation model in TM3 Leydig cell culture to study the function of CYGB and NGB proteins under inflammatory conditions. CYGB and NGB were downregulated using siRNA and shRNA based experimental strategies. Overexpression was conducted using lentiviral pLenti-III-CYGB-2A-GFP, and pLenti-III-NGB-2A-GFP vector systems. As testicular macrophages regulate immune function upon inflammation and steroidogenesis of Leydig cells, we generated direct/indirect co-culture systems of TM3 and mouse macrophage (RAW264.7) cells ex vivo.Downregulation of CYGB and NGB induced nitride oxide (NO) release, blocked cell cycle progression, reduced testosterone production and increased inflammatory and apoptotic pathway gene expression in the presence and absence of LPS. On the other hand, CYGB and NGB overexpression reduced TNFα and COX-2 protein expressions and increased the expression of testosterone biogenesis pathway genes upon LPS stimulation. In addition, CYGB and NGB overexpression upregulated testosterone production. The present study successfully established an inflammatory interaction model of TM3 and RAW264.7 cells. Suppression of CYGB and NGB in TM3 cells changed macrophage morphology, enhanced macrophage cell number and NO release in co-culture experiments upon LPS exposure.In summary, these results demonstrate that globin family members might control LPS induced inflammation by regulating apoptotic mechanisms and macrophage response.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.