| Objective(1) To evaluate the effects of overtraining on phagocytosis, reactive oxygenspecies (ROS) generation and inflammatory cytokines production of peritonealmacrophages (MФs), especially the mechanisms involved. It will provide experimentevidence for strengthen our understanding of overtraining.(2) To determine the effects of supplement with glutamine on the function ofMФs (i.e., phagocytosis, ROS generation and inflammatory cytokines production)from the overtraining group, and to explore a way to ameliorate the situation inducedby overtraining.(3) To investigate the effects of IGF-1and MGF peptide on phagocytosis, ROSgeneration and inflammatory cytokines production of MФs in vitro, and attempt todiscover the mechanisms of the inhibiton of MФs induced by overtraining from theview of IGF-1and MGF.Methods(1) In vivo.40male wistar rats (8-wk-old) were randomly divided into5groups:sedentary group(C, n=8), overtraining group (E), overtraining supplement with Glngroup (EG). E and EG group were respectively divided into two groups whichsacrificed at36h (E1, EG1, n=8) and7days (E2, EG2, n=8) after the last training. Allgroups except C were training in standard treadmill with an increasing load for11weeks. The criteria to evaluate the condition of the rats and judge whether theprotocol was successful were as follows: locomotory capacity, mental state, weightchange, and concentrations of hemoglobin and testosterone in blood. Peritonealmacrophages were isolated and purified after all rats were sacrificed by decapitation.The phagocytosis and the ROS generation of MФs were measured by the uptake ofneutral red and the flow cytometry respectively; The producitin of TNF-α and IL-1βwere tested by ELISA; The NADPH-oxidase activity was tested by colorimetry;Real-time PCR was used to test the expression of the following genes:NADPH-oxidase subunits (gp91phox, p22phox, p47phox, p40phox, p67phox), PKC-δ,G6PD, iNOS, IGF-1and MGF.(2) In vitro. MФs were exposed to diffrent concentrations (1,10,50,100,200ng/ml) of IGF-1or MGF peptide for2h. The phagocytosis, the ROS generationand the inflammatory cytokines production of MФs were measured by the uptake ofneutral red, the flow cytometry and ELISA respectively. Results(1) Index of overtraining. The rats had to be assisted by hand to complete thejob and their mental state worsened in the later stage of exercise. Overtrainingsignificantly decreased the body weight (19.3%, P<0.01), the hemoglobin (13.5%,P<0.01) and the testosterone (55.3%, P<0.01) in blood.(2) Phagocytosis. The phagocytosis of MФs from group E1was significantlylower than group C (decreased by27%, P<0.05). There was no significant differencein the phagocytosis of MФs between group E2and group C. Although thephagocytosis of MФs from group EG1was still lower than group C, no differencewas observed between them. There was no significant difference in the phagocytosisof MФs between group EG2, group E2and group C.(3) ROS. The ROS generation of MФs from group E1was significantly lowerthan group C (decreased by35%, P<0.01). The ROS generation of MФs from groupE2was significantly higher than group E1(P<0.01), and no difference was observedbetween group E2and group C. The ROS generation of MФs from group EG1did notchange as compared with group E1, it still significantly lower than group C (P<0.01).The ROS generation of MФs from group EG2was significantly higher than groupEG1(P<0.01), and there was no difference as compared with group E2or group C.(4) Response ability of inflammatory cytokines. The production of TNF-α andIL-1β of MФs from group E1was significantly lower than group C as LPSstimulation. The response ability of inflammatory cytokines of MФs from group E2was significantly higher than group E1(P<0.05), and no difference was observedbetween group E2and group C. The response ability of inflammatory cytokines ofMФs from group EG1was significantly higher than group E1, and no difference wasobserved between group EG1and group C. There was no difference between groupEG2, group E2and group C.(5) NADPH-oxidase activity. The NADPH-oxidase activity of MФs from groupE1was significantly lower than group C. The NADPH-oxidase activity of MФs fromgroup E2was significantly higher than group E1(P<0.05), and no difference wasobserved between group E2and group C. The NADPH-oxidase activity of MФs fromgroup EG1and EG2did not change as compared with group E1and E2, respectively.The correlation analysis showed that NADPH-oxidase activity and ROS generationwere positive correlation and the pearson correlation was0.62(P<0.01)(data from5groups).(6) NADPH-oxidase subunits. The NADPH-oxidase subunits (gp91phox,p22phox, p40phox, p67phox) mRNA levels of MФs from group E1did not change ascompared with group C, however, the expression of p47phox of MФs from group E1was significantly higher than group C (P<0.05). No difference was observed betweengroup E2, group E1and group C in all subunits of NADPH-oxidase. There was no significant difference in the NADPH-oxidase subunits (gp91phox, p22phox, p40phox,p67phox) mRNA levels of MФs between group EG1and group E1, however, theexpression of p47phox of MФs from group EG1was significantly lower than groupE1(P<0.05). All subunits of NADPH-oxidase of MФs from group EG2did not changeas compared with group E2, group C and group EG1.(7) PKC-δ. The PKC-δ mRNA level of MФs from group E1was significantlylower than group C (P<0.05). The expression of PKC-δ of MФs from group E2wassignificantly higher than group E1(P<0.05), and no difference was observed betweengroup E2and group C. The PKC-δ mRNA level of MФs from group EG1and EG2did not change as compared with group E1and E2, respectively. The correlationanalysis showed that PKC-δ mRNA and NADPH-oxidase activity were positivecorrelation and the pearson correlation was0.51(P<0.01), PKC-δ mRNA and ROSgeneration were positive correlation and the pearson correlation was0.69(P<0.01)(data from5groups).(8) There was no difference between group C, group E1, group E2, group EG1and group EG2in the expression of G6PD of MФs.(9) There was no difference between group C, group E1, group E2, group EG1and group EG2in the expression of iNOS of MФs.(10) IGF-1anf MGF. IGF-1and MGF mRNA levels in MФs from E1groupincreased significantly compared with the control group (21-fold and92-fold,respectively; p<0.01). IGF-1and MGF mRNA levels in MФs from E2group wassignificantly lower than group E1(p<0.01), and no difference was observed betweengroup E2and group C. IGF-1and MGF mRNA levels in MФs from EG1group wassignificantly lower than group E1(p<0.01), however, they were still higher thangroup C (p<0.01). IGF-1and MGF mRNA levels in MФs from EG2group wassignificantly lower than group EG1(p<0.01), and no difference was observed ascompared with group C and group E2.(11) In vitro experiments showed that different concentrations of IGF-1(1,10,50,100,200ng/ml) had no significant effect on the phagocytosis and the ROSgeneration of MФs. IGF-1(100ng/ml) significantly increased the secretion of TNF-αof MФs as LPS stimulation. IGF-1(100,200ng/ml) significantly increased theproduction of IL-1β of MФs as LPS stimulation.(12) In vitro experiments showed that MGF peptide impaired the phagocytosis ofMФs in dose-independent manner. Additonally, MGF peptide of some concentrations(i.e.,1,10,50,100ng/ml) significantly inhibited the ROS generation of MФs.However, different concentrations of MGF (1,10,50,100,200ng/ml) had nosignificant effect on the production of TNF-α and IL-1β of MФs as LPS stimulation. Conclusion(1) Overtraining inhibits the phagocytosis, the ROS generation and theinflammatory cytokines production of MФs, it may be a mechanism of whyovertraining cause immunesuppression.(2) Supplement with glutamine ameliorates the decline of the phagocytosis andthe inflammatory cytokines production of MФs induced by overtraining, however, ithas no significant effect on the ROS generation of MФs.(3) The mechanisms of overtraining inhibit ROS production of MФs: Thedecline of ROS production in overtraining group did not cause by suppressing theNADPH-oxidase subunits or the pathway of NADPH-oxidase production; The declineof NADPH-oxidase activity mediated by PKC-δ may play important roles in theinhibiton of ROS production; MGF produced by macrophages may play a key role inovertraining inhibit ROS production.(4) The mechanisms of overtraining inhibit phagocytosis of MФs: MGFproduced by macrophages may play a key role in overtraining inhibit phagocytosis ofMФs.(5) The mechanisms of overtraining inhibit inflammatory cytokines productionof MФs: IGF-1and MGF produced by macrophages do not involved, there must beother mechanisms involved in this process. |
PDF Full Text Request