NADPH Oxidase: a Target for the Modulation of the Excessive Oxidase Damage Induced by Overtraining in Rat Neutrophils

Objective: The purpose of this study is to demonstrate that NADPH oxidase mediating the ROS production is the major pathway for ROS generation in neutrophils during exercise. NADPH oxidase, as a target can modulate oxidative damage induced by overtraining, which can be value to the prevention of exercise-induced immunosuppression. Methods: Thirty 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). The co-localization between gp91(phox) and p47(phox) of the NADPH-oxidase was detected using immunocytochemistry and confocal microscopy. Results: 1) Compared with group C, the concentrations of IL-1 beta, IL-8, and TNF-alpha 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 beta and MCP-1 were decreased (P < 0.05), and IL-8 and TNF-alpha 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). 2) 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. 3) 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. 4) The p47(phox) protein translocated to the cell membrane and co-localized with the gp91(phox) subunit of NADPH oxidase in neutrophils activated by overtraining. Conclusion: 1) Excessive exercise led to an increased secretion of inflammatory cytokines and chemokines in peripheral blood, and it may have induced tissue inflammation 2) Overtraining can activate the NADPH oxidase-mediated overproduction of ROS, leading to increased lipid peroxidation. 3) NADPHoxidase in neutrophils as a target, was responsible for ROS, oxidative damage to phagocytes and lymphocytes and changes to inflammatory cytokines and immune regulatory factors all affect cellular immune functions and may be causative factors for exercise-induced immunosuppression.