Research On The Mechanism And Prevention Approach Of Rat Neutrophils Peroxidation Damage Induced By Overtraining

Objective: The aim of this study is to investigate the impact of reactive oxygen species (ROS) activated by overtraining on neutrophil peroxidation damage,which lead to exercise-related immunosuppression. The first series is to investigate the impact of high temperature, H₂O₂ concentration, low oxgen and acidity changes caused by lactic acid on the phagocytes cell respiratory burst and the phagocytic function in peripheral whole blood. The second series is to probe into the mechanism of neutrophils peroxidation damage inducd by overtraining. The last series focused on the role of ROS, which mediated production by NADPH oxidase, in the overtraining induced oxidative stress, the inhibitor of NADPH oxidase diphenyleneiodonium(DPI) and Glutamine were employed ated by and the combined effect of supplementation and on the function of neutrophils in vitro after overtraining.Methods:Series 1: Peripheral whole blood from healthy male divided into Ctr, high tempreature,low oxygen?H2O2 and lactic acid groups,which combined DPI treatment respectively, after the treatment of high tempreature,low oxygen?H2O2 and lactic acid respectively, respiratory burst and phagocytosis of neutrophils were detected by flow cytometery. Also, the activity of NADPH oxidase was detected in isolated neutrophile granulocyte, and then regression equation between respiratory burst and the activity of NADPH oxidase was established. The lactic acid, which was selected in the point of highest phagocytize, was added before neutrophile granulocyte isolation, the co-localization of the gp91^phox and p47^phox of NADPH-oxidase was checked by immunocytochemistry and confocal technology.Series 2: Fifty male Wistar rats were randomly divided into three groups: a negative control group (C, n = 10), an overtraining group (E, n = 10) and an overtraining + DPI intervention group (D, n =10). Groups E and D were trained on a standard treadmill with progressive load for 11 weeks. After 36-40 h from the last training, eight rats were randomly selected from each group, and blood was sampled from the orbital vein. ELISAs were used to measure serum cytokine levels and lipid peroxidation in blood plasma. Flow cytometry with Annexin V / PI double staining was used to measure neutrophil apoptosis and necrosis. DNA damage in lymphocytes was tested using single cell gel electrophoresis (SCGE).Series 3. Based on a rat overtraining protocol, fifty male Wistar rats were randomly divided into five groups:a control group(C), an overtraining group (E), an overtraining and DPI administration group (D), an overtraining and glutamine supplementation group (G), and an overtraining combined DPI administration and glutamine supplementation group(D+G). Rats were trained on a standard treadmill for 11 weeks. After 36-40 h from the last training, eight rats were randomly selected from each group, and blood was sampled from the orbital vein. ELISAs were used to measure serum cytokine levels and lipid peroxidation in blood plasma; the co-localization between gp91phox and p47phox of the NADPH-oxidase subunit using immunocytochemistry and confocal microscopy was examined.The activity of NADPH oxidase was assessed by chemiluminescence.Neutrophils respiratory burst and phagocytosis function was measured by FCM.Results:1. Low concentration (less than 4.81mmol/L) of lactic acid can be inhibited the function of phagocytosis and respiratory burst of phagocytic cells, moderate concentration (4.81mmol/L 8.14mmol/L) of lactic acid can lead to the increase respiratory burst of phagocytic cells. The curve of respiratory burst can be declined sharply while above this concentration lactic acid and the curve of sharp decline of the function of phagocytosis was early than the curve of respiratory burst.2. There is co-localization between the subunit p47^phox and the gp91^phox in NADPH-oxidase on the plasma membrane in neutrophils when the lactic acid concentration in blood is 7.69mmol/L. It is a high degree of correlation between the activity NADPH oxidase and the respiratory burst in phagocytic cells.3. Compared with group C, the concentrations of IL-1?, IL-8, and TNF-?were significantly increased and MCP-1, and CINC were significantly decreased in blood plasma from group E (P < 0.01 and P < 0.05, respectively). Concentrations of IL-1?and MCP-1 were decreased (P < 0.05), and IL-8 and TNF-?were significantly increased (P <0.05) in blood plasma from group D. MDA and MPO were elevated in plasma from groups E and D (P < 0.01 and P < 0.05, respectively).4. Compared with group C, the percentage of neutrophils apoptosis were significantly elevated (P < 0.01) in both groups E and D, and the percentage of cell death was raised in group E (P < 0.05). No significant change was observed in group D;Compared with group C, the number of comet cells, an indicator of DNA damage, was significantly increased (P < 0.01), and the width and tail length of comet cells were notably increased in group E, while no significant increase was observed in group D.5. The co-localization between gp91^phox and p47^phox of the NADPH-oxidase subunit was induced by overtraining. There is high positive correlation between NADPH oxidase activity and lipid peroxidation, DNA damage, apoptosis and the activation of neutrophils.but it is negative correlation between NADPH oxidase activity and leukomonocyte proliferation.6. Compared with group C, the plasma concentrations of NO increased in group G, andthe NO, CINC concentrations in group DG increased significantly. It is no significant changes in the other groups. 7. Serum cytokine levels and lipid peroxidation in blood plasmaand the activity of NADPH oxidase and ROS in neutrophils were markedly increased, but the respiratory burst and phagocytosis function of neutrophils were decreased in group E when compared to group C.The index in group D and group G was decreased, but it was increased in group DG, though this was not statistically significant.Furthermore, the respiratory burst and phagocytosis function in group DG were increased when compared to group E. 8. After overtraining ,the expression gp91^phox and p47^phox was up regulated in group E.It is no significant changes in the other groups except group DG,in which the expression gp91^phox is down regulated. Compared with group E, the expression gp91^phox and p47^phox was up regulated in group D,groupG and groupDG.Conclusion:1. The acidity induced by lactic acid in peripheral blood can regulate the activity NADPH oxidase and lead to the change of the phagocytosis function and respiratory burst in phagocytic cells.2. The lactic acid 69 mmol/L is a seemly concentration which can induce the respiratory burst of neutrophile granulocyte, which is useful to evaluate the role of ROS mediated generation by NADPHA oxidase.3. Excessive exercise led to an increased secretion of inflammatory cytokines and chemokines in peripheral blood, activated peripheral blood neutrophils NADPH oxidase to generate more ROS, led to oxidative stress, which is a main source of ROS induced by Excessive exercise.4. The activation of NADPH oxidase is responsible for the production of superoxide anions, which leads to excessive oxidative stress in the blood after overtraining. This oxidative stress may be related to the decrease in neutrophil function after over training.5. Over training activated NADPH oxidase, and ROS generated by NADPHA oxidase is the main source of oxidative damage, which is the mechanism of exercise-related immunosuppression.6. The DPI treatment combined glutamine supplementation can reverse the decrease neutrophils function after overtraining in vitro. DPI by the way of depress the ROS generation, as well as glutamine regulate the energy balance.