Research on DNA Damage of Lymphocytes Induced by ROS Mediated by NADPH-Oxidase in Neutrophils and Intervention after Overtraining

Objective:To investigate the effects of reactive oxygen species(ROS) mediated by NADPH-oxidase in neutrophils and intervention of biphenyl iodide(DPI) on DNA damage of lymphocytes induced by over training.Methods:30 male vistar rats were randomly divided into three groups:blank control group(C,n=10),overtraining group(E,n=10) and DPI intervention group(G,n =10).Group E and G were training in standard treadmill with an increasing load for 11 weeks,within 36h-40h after the last training,eight rats were randomly pick out from each group and the orbital venous blood were obtained.ELISA was used to detect the level of cytokine and blood lipid peroxidation in blood plasma.Utilizing flow cytometer and AnnexinV / PI double staining to measure the apoptosis and necrosis of neutrophil,using immunocytochemistry and confocal microscopy to indentify the co-localization both gp91phox and p47phox,which is the key subunits of the NADPH-oxidase and single cell gel electrophoresis(SCGE) was used to detect DNA damage of lymphocytes.Results:1.Compared with group C,the concentration of IL-1b,IL-8,TNF-α were significantly increased and the MCP-1,CINC were significantly decreased in rat blood plasma of group E(respectively,P<0.01 and P<0.05);the concentration of IL-1b,MCP-1 were decreased significantly(P<0.05),and IL-8,TNF-α were significantly increased(P<0.05) in rat blood plasma of group D;the level of MDA,MPO were increased in plasma of group E and D(respectively P<0.01 and P<0.05).2.Compared with group C,the percentage of apoptosis of neutrophils significantly increased(P<0.01) in group E and D,and the percentage of cell death increased in groups E(P<0.05) while no significant change in group D.3.Compared with group C,the number of comet cells,which indicate the DNA damage significantly increased(P<0.01) and the width and the tail length of comet cells were significant increased in group E while no increase in group D.4.the co-location confocal of gp91phox and p47phoxhe,a key subunit of NADPH oxidase was localized display in group E while no location in group C.Conclusion:1.Excessive exercise can lead to the upgrade of inflammatory cytokines and chemokines in peripheral blood,which may induce tissue inflammation and regulate the differentiation of T lymphocytes.2.Overtraining can activate NADPH oxidase of peripheral blood neutrophils which can mediate to produce too much ROS and lead to the lipid peroxidation in blood.3.The production of ROS from these pathway and upgrade of the levels of peroxide can cause apoptosis or death of neutrophils and DNA damage of lymph cell.4 Too much ROS generated by this pathway in the phagocytic cells,the upgrade of inflammatory cytokines,immune regulatory factors and the peroxidation injury of lymph cell that all induced by overtraining can regulate cellular immune function,which may be the factors caused by exercise-induced immunosuppression.