N-Acetyl-L-cysteine prevents exercise-induced intestinal lymphocyte apoptosis by maintaining intracellular glutathione levels and reducing mitochondrial membrane depolarization

Intense exercise leads to post-exercise lymphocytopenia and immunosuppression, possibly by triggering lymphocyte apoptosis. To test the role of oxidative stress on exercise-induced lymphocyte apoptosis, we administered the antioxidant N-acetyl--cysteine (NAC) and measured apoptosis in intestinal lymphocytes (IL) from exhaustively exercised animals. Eighty-seven female C57BL/6 mice were randomly assigned to receive NAC (1 g/kg) or saline 30 min prior to treadmill exercise for 90 min at 2degrees slope (30 min at 22 m min(-1), 30 min at 25 m min(-1), and 30 min at 28 m min(-1)) and sacrificed immediately (Imm) or 24 hours (24 h) after cessation of exercise. Control mice (nonexercised) were exposed to treadmill noise and vibration without running. Exercise increased IL phosphatidylserine externalization (p<0.001), mitochondrial membrane depolarization (p<0.05), and decreased intracellular glutathione concentrations (p<0.05) immediately following exercise in saline relative to nonexercised mice. At 24 h post-exercise, saline injected mice had fewer total (p<0.001) and CD3+ (p<0.005) IL compared to nonexercised animals. NAC injection in mice maintained intracellular glutathione levels, prevented phosphatidylserine externalization, mitochondrial membrane depolarization, and loss of IL immediately and 24 h after exercise. These data suggest that lymphocyte apoptosis precedes post-exercise lymphocytopenia and may be due to oxidative stress.     

Adrenalectomy in mice does not prevent loss of intestinal lymphocytes after exercise.

Exhaustive exercise is associated with an increase in circulating glucocorticoids (GCs), lymphocyte apoptosis, and a reduction in intestinal lymphocyte number. The present study examined the role of GCs on the numerical changes seen in intestinal lymphocytes after exercise. Female C57BL/6 mice were bilaterally adrenalectomized (ADX; n = 18) or given sham surgery (Sham; n = 18) and assigned to one of three exercise conditions: treadmill running (28 m/min, 90 min, 2 degrees slope) and killed immediately or after 24 h recovery, or not exercised and killed immediately after 90-min exposure to the treadmill environment. Lymphocytes were isolated from the intestines with CD45(+) cells collected by positive selection using magnetic bead separation columns, and lymphocyte subpopulations were analyzed by flow cytometry for CD45(+), CD3alphabeta(+), CD3gammadelta(+), CD8beta(+), CD8alpha(+), CD4(+), and NK(+) phenotypic markers. ADX mice had significantly more intestinal CD45(+) leukocytes (P < 0.05) and CD3alphabeta(+) (P < 0.05), CD3gammadelta(+) (P < 0.01), CD8alpha(+) (P < 0.001), and NK(+) (P < 0.05) intestinal lymphocytes than Sham mice. There was a significant effect of exercise condition on total intestinal CD45(+) leukocytes (P < 0.01) and CD3alphabeta(+) (P < 0.05), CD8alpha(+) (P < 0.001), and CD4(+) (P < 0.05) intestinal lymphocytes, with fewer cells at 24 h postexercise compared with the other treatment conditions. There were no surgical x exercise interaction effects on the CD3 and CD8 phenotype numbers. Plasma corticosterone was virtually nil in ADX mice regardless of exercise condition but was significantly elevated in Sham mice immediately postexercise (P < 0.001). The data indicate that ADX does not prevent the loss of lymphocytes from the intestinal mucosa 24 h after strenuous exercise and GCs are not directly causal in the leukopenia of exercise.     

Endogenous glucocorticoids play a positive regulatory role in the anti-keyhole limpet hemocyanin in vivo antibody response

Glucocorticoids (GCs) are commonly reported to be immunosuppressive. Studies that support this involve the administration of synthetic GCs such as dexamethasone at high pharmacological doses and using in vitro assay systems that may have limited relevance to the role of GCs during normal in vivo immune responses. Therefore, the following experiments tested the conclusion that GCs are generally immunosuppressive. Adult male Sprague Dawley rats received adrenalectomy (ADX) or sham surgery. ADX rats were given either basal corticosterone (CORT) replacement in their drinking water (25 microg/ml) or no CORT. Rats were immunized with keyhole limpet hemocyanin (KLH), and blood samples were taken. ADX rats with no CORT replacement had reduced anti-KLH IgM and IgG responses compared with sham-operated controls. ADX rats that received basal CORT replacement had partially restored anti-KLH IgM, but still had suppressed anti-KLH IgG. Administration of GC receptor type I (RU28318) and type II (RU40555) receptor antagonists also reduced the anti-KLH IgM and IgG responses. ADX rats that received both basal CORT replacement and low dose injections of CORT on days 5 and 7 after KLH had anti-KLH IgG levels equal to those of sham-operated controls. Finally, the GC elevation 4-7 days after immunization may play a role in stimulating the IgM to IgG2a switch. GC receptor blockade reduced the anti-KLH IgG2a and splenic IFN-gamma, but not the anti-KLH IgG1, response. Given that IFN-gamma is an important regulator of the IgM to IgG2a switch, it is possible that the small rise in GC found 4-7 days after KLH facilitates IgG2a isotype switching.     

A combination of an iron chelator with an antioxidant exerts greater efficacy on cardioprotection than monotherapy in iron-overload thalassemic mice

Many recent studies have shown that antioxidant compounds decrease cardiac oxidative stress, decrease cardiac iron deposition, and improve cardiac dysfunction in iron-overload induced cardiomyopathy in animal models. Interestingly, a therapy including the combination of the iron chelator deferiprone (DFP) plus the antioxidant N-acetylcysteine (NAC) has been shown to significantly decrease oxidative stress and restore heart and brain function in iron-overloaded rats. However, the cardioprotective effects of this combined DFP and NAC treatment in thalassemic mice have not been investigated. We hypothesised that the combination of DFP and NAC exerts better cardioprotection than monotherapy via decreasing cardiac iron accumulation, oxidative stress, and apoptosis in thalassemic mice. The iron-overload condition was induced in heterozygous β^KO HT and wild-type mice by instigating high iron diet consumption (FE) for three months. Then, iron chelator DFP (75?mg/kg/day twice a day), antioxidant NAC (100?mg/kg/day once a day), and combined DFP plus NAC were fed via oral gavage for one month with continuous iron feeding. Left ventricular (LV) function, heart rate variability (HRV), apoptosis, and cardiac iron accumulation were determined. Chronic iron-overload in mice led to increased cardiac iron deposition, oxidative stress, apoptosis, and impaired LV function and HRV. Although DFP and NAC showed similar cardioprotective efficacy, combined DFP plus NAC exerted greater efficacy in reducing both cardiac iron deposition and cellular apoptosis than monotherapy. In conclusion, combined iron chelator and NAC treatment exert the greatest cardioprotective efficacy when compared with either of the monotherapies in iron-overload thalassemic mice.     

N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset

Background

We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC), a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells.

Methodology/Principal Findings

Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220⁺CD43⁺) and pre-B (B220⁺CD43⁻) cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21–38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle.

Conclusions/Significance

The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.     

Estrogen administration, postexercise tissue oxidative stress and vitamin C status in male rats

Estrogen can putatively act as an antioxidant and protect tissues from exercise-induced oxidative stress. To test the in vivo efficacy of estrogen, the effects of 2 weeks of daily estrogen (40 microg x kg(-1) body weight beta-estradiol 3-benzoate) injection on indices of immediate postexercise oxidative stress and antioxidant status were determined in adult male rats, with and without 8 weeks of prior dietary vitamin E deprivation. The treadmill running protocol (60 min at 21 m x min(-1), 12% grade) induced significant oxidative stress as indicated by muscle glutathione status. Estrogen administration had little effect on postexercise tissue glutathione status, superoxide dismutase and glutathione peroxidase activity, and vitamin E levels. Estrogen administration induced significant reductions in muscle, liver, and heart vitamin C concentrations following exercise, as well as in unexercised male rats. Tissue vitamin C loss was not directly mediated through liver glycogen or glutathione status. Thus, estrogen administration generally did not appear to influence postexercise tissue indices of oxidative stress or antioxidant status and may have contributed to a decline in overall antioxidant protection by inducing losses in tissue vitamin C content.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

The HIV-1 transactivator factor (Tat) induces enterocyte apoptosis through a redox-mediated mechanism

The intestinal mucosa is an important target of human immunodeficiency virus (HIV) infection. HIV virus induces CD4+ T cell loss and epithelial damage which results in increased intestinal permeability. The mechanisms involved in nutrient malabsorption and alterations of intestinal mucosal architecture are unknown. We previously demonstrated that HIV-1 transactivator factor (Tat) induces an enterotoxic effect on intestinal epithelial cells that could be responsible for HIV-associated diarrhea. Since oxidative stress is implicated in the pathogenesis and morbidity of HIV infection, we evaluated whether Tat induces apoptosis of human enterocytes through oxidative stress, and whether the antioxidant N-acetylcysteine (NAC) could prevent it. Caco-2 and HT29 cells or human intestinal mucosa specimens were exposed to Tat alone or combined with NAC. In an in-vitro cell model, Tat increased the generation of reactive oxygen species and decreased antioxidant defenses as judged by a reduction in catalase activity and a reduced (GSH)/oxidized (GSSG) glutathione ratio. Tat also induced cytochrome c release from mitochondria to cytosol, and caspase-3 activation. Rectal dialysis samples from HIV-infected patients were positive for the oxidative stress marker 8-hydroxy-2'-deoxyguanosine. GSH/GSSG imbalance and apoptosis occurred in jejunal specimens from HIV-positive patients at baseline and from HIV-negative specimens exposed to Tat. Experiments with neutralizing anti-Tat antibodies showed that these effects were direct and specific. Pre-treatment with NAC prevented Tat-induced apoptosis and restored the glutathione balance in both the in-vitro and the ex-vivo model. These findings indicate that oxidative stress is one of the mechanism involved in HIV-intestinal disease.     

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [3H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.     

Long-duration freewheel running and submandibular lymphocyte response to forced exercise in older mice

Submandibular lymph nodes (SLN) are crucial for immune surveillance of the anterior ocular chamber and upper respiratory tract; little is known about how training and exercise affect SLN lymphocytes. The intent of this study was to describe the impact of long term freewheel running followed by acute strenuous exercise on SLN lymphocytes in mice. Female C57BL/6 mice were assigned to running wheels or remained sedentary for 8 months, and further randomized to treadmill exercise and sacrifice immediately, treadmill exercise and sacrifice 24 h after exercise cessation, or no treadmill exposure. SLN lymphocytes were isolated and analyzed for CD3, CD4, CD8, and CD19 cell surface markers, phosphatidylserine externalization as a marker of apoptosis, and intracellular glutathione as a marker of oxidative stress. Compared with running wheel mice, older sedentary mice had a lower percent of T cells and higher percent of B cells (p < 0.05). Although intracellular glutathione did not differ between groups, running mice had a lower percent of Annexin V(+) SLN lymphocytes 24 h after treadmill exercise. Further research will be needed to determine if voluntary exercise translates into improved anterior ocular and upper respiratory tract health.     

Training does not protect against exhaustive exercise-induced lactate transport capacity alterations

The effects of endurance training on lactate transport capacity remain controversial. This study examined whether endurance training 1) alters lactate transport capacity, 2) can protect against exhaustive exercise-induced lactate transport alteration, and 3) can modify heart and oxidative muscle monocarboxylate transporter 1 (MCT1) content. Forty male Wistar rats were divided into control (C), trained (T), exhaustively exercised (E), and trained and exercised (TE) groups. Rats in the T and TE groups ran on a treadmill (1 h/day, 5 days/wk at 25 m/min, 10% incline) for 5 wk; C and E were familiarized with the exercise task for 5 min/day. Before being killed, E and TE rats underwent exhaustive exercise (25 m/min, 10% grade), which lasted 80 and 204 min, respectively (P < 0.05). Although lactate transport measurements (zero-trans) did not differ between groups C and T, both E and TE groups presented an apparent loss of protein saturation properties. In the trained groups, MCT1 content increased in soleus (+28% for T and +26% for TE; P < 0.05) and heart muscle (+36% for T and +33% for TE; P < 0.05). Moreover, despite the metabolic adaptations typically observed after endurance training, we also noted increased lipid peroxidation byproducts after exhaustive exercise. We concluded that 1) endurance training does not alter lactate transport capacity, 2) exhaustive exercise-induced lactate transport alteration is not prevented by training despite increased MCT1 content, and 3) exercise-induced oxidative stress may enhance the passive diffusion responsible for the apparent loss of saturation properties, possibly masking lactate transport regulation.     

Effect of corticosterone on the prolactin response to psychological and physical stress in rats

Both corticosterone and prolactin (PRL) levels increase in response to stress. In these studies we examined the effect of corticosterone on the PRL response to both physical (footshock) and psychological (novel environment) stress. Three groups of rats were used: sham adrenalectomized (SHAM), adrenalectomized (ADX), and adrenalectomized with corticosterone replacement (ADX+CORT). The corticosterone-treated animals received 80 ug corticosterone/ml drinking water. Blood samples were drawn via an indwelling cannula and PRL values determined using radioimmunoassay. ADX rats showed a consistently greater PRL response to being placed on a platform above water (novel environment) or when receiving intermittant footshock than did ADX+CORT rats. The PRL response of the latter group was similar to that of the SHAM animals. These findings indicate that corticosterone levels of an animal can significantly attenuate the magnitude of the PRL response to both physical and psychological stress. These findings further emphasize that the PRL response to stress is dependent not only upon the immediate action of the stressor, but also the prior stress history of the animal.     

Mixed interleukins and thymosin fraction V synergistically induce T lymphocyte development in hydrocortisone-treated aged mice.

Analysis of the role of interleukins in T cell ontogeny in vitro indicates that the regulation of T cell development involves interleukins (ILs) as well as thymic hormones (THs). In order to assess their respective roles in T lymphocyte development in vivo, chemically thymectomized mice were treated with ILs and THs. After 2 days of hydrocortisone treatment, aged mice showed acute thymic involution (weight was less than 30% of control) and reduced spleen size (less than 80% of control) with progressive recovery to 8 days. After 2 days of hydrocortisone treatment, adult mice were injected for 5 days with mixed buffy coat interleukins (BC-IL; 50 units IL2 equivalence), purified IL2 (50 units), rIL1 beta (4 ng), and thymosin fraction V (TF5; 100 micrograms). The animals were sacrificed and spleens and thymuses were analyzed for weight, cellularity, T cell number, subsets, and function as determined by proliferative responses to concanavalin A and ILs. BC-IL treatment increased the recovery of spleen and thymus weights and cellularity with corresponding augmentation of number and function of T lymphocytes; neither IL1 or IL2 or their combination had this effect. TF5 had no effect alone but strongly potentiated the effect of BC-IL on T lymphocyte function. These data indicate that BC-IL in combination with thymic peptides potently promotes T lymphocyte development. The combination may be therapeutically relevant for immunorestoration.     

N-Acetyl Cysteine Depletes Reactive Oxygen Species and Prevents Dental Monomer-Induced Intrinsic Mitochondrial Apoptosis In Vitro in Human Dental Pulp Cells

Purpose

To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC) on this process.

Methods

Human dental pulp cells (hDPCs) were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.

Results

Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS) and depletion of glutathione (GSH), differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI) staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.

Conclusions

Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis.     

NAC Attenuates LPS-Induced Toxicity in Aspirin-Sensitized Mouse Macrophages via Suppression of Oxidative Stress and Mitochondrial Dysfunction

Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.     

Effects of unaccustomed downhill running on muscle damage,oxidative stress,and leukocyte apoptosis

[Purpose]

The purpose of this study was to investigate the effect of unaccustomed downhill running on muscle damage, oxidative stress, and leukocyte apoptosis.

[Methods]

Thirteen moderately trained male subjects performed three 40 min treadmill runs at ~70% VO_2max on separate days: a level run (L) followed by two downhill runs (DH1 and DH2). Blood samples were taken at rest (PRE) and immediately (POST), 2 h, 24 h, and 48 h after each run. Data were analyzed using 2-way repeated measures ANOVA with post hoc Tukey tests.

[Results]

Creatine kinase (CK) activity and oxidative stress level were significantly elevated at 24 h and 48 h following DH1 (P < 0.05). The level of oxidative stress at the POST measurement following DH1 and DH2 was greater than PRE. The rate of leukocyte apoptosis was significantly increased at the POST measurement following all three runs, and remained elevated for up to 48 h following DH1 (P < 0.01).

[Conclusion]

CK activity and oxidative stress were elevated following an acute bout of moderate intensity downhill running, resulting in a greater apoptotic response at 24 h and 48 h post-exercise in comparison with level grade running or a second downhill run. These elevations were blunted following DH2. Although the link between exercise-induced muscle damage and leukocyte apoptosis is currently unknown, the differential response to DH1 vs. L and DH2 indicates that it may be mediated by the elevation of oxidative stress.     

Exercise-induced activation of cardiac sympathetic nerve triggers cardioprotection via redox-sensitive activation of eNOS and upregulation of iNOS

We investigated the mechanism of exercise-induced late cardioprotection against ischemia-reperfusion (I/R) injury. C57BL/6 mice received treadmill exercise (60 min/day) for 7 days at a work rate of 60-70% maximal oxygen uptake. Exercise transiently increased oxidative stress and activated endothelial isoform of nitric oxide synthase (eNOS) during exercise and increased expression of inducible isoform of NOS (iNOS) in the heart after 7 days of exercise. The mice were subjected to regional ischemia by 30 min of occlusion of the left coronary artery, followed by 2 h of reperfusion. Infarct size was significantly smaller in the exercised mice. Ablation of cardiac sympathetic nerve by topical application of phenol abolished oxidative stress, activation of eNOS, upregulation of iNOS, and cardioprotection mediated by exercise. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine during exercise also inhibited activation of eNOS, upregulation of iNOS, and cardioprotection. In eNOS(-/-) mice, exercise-induced oxidative stress was conserved, but upregulation of iNOS and cardioprotection was lost. Exercise did not confer cardioprotection when the iNOS selective inhibitor 1400W was administered just before coronary artery occlusion or when iNOS(-/-) mice were employed. These results suggest that exercise stimulates cardiac sympathetic nerves that provoke redox-sensitive activation of eNOS, leading to upregulation of iNOS, which acts as a mediator of late cardioprotection against I/R injury.     

N‐acetylcysteine counteracts oxidative stress and prevents hCG‐induced apoptosis in rat Leydig cells through down regulation of caspase‐8 and JNK

We have earlier reported that following persistent stimulation with hCG, oxidative stress‐induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N‐acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione‐S‐transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase‐8, up‐regulated following repeated hCG exposure, were significantly down‐regulated following NAC co‐incubation. While Bcl‐2 expression was fully restored, Bax and caspase‐9 remained unchanged. NAC treatment induced down‐regulation of upstream JNK/pJNK and down‐stream caspase‐3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis. Mol. Reprod. Dev. 77:900–909, 2010. © 2010 Wiley‐Liss, Inc.