| Purpose Chronic obstructive pulmonary disease(COPD)is a common chronic systemic disease with high incidence.In addition to local lung damage,COPD can also cause a variety of extrapulmonary injuries,among which diaphragmatic dysfunction is an important factor causing respiratory function decline.Systemic inflammation is the key link of COPD diaphragm dysfunction.Exercise training is currently an effective means of rehabilitation treatment for diaphragmatic dysfunction of COPD,and the regulation of exercise on inflammatory response has also become one of the ways to improve diaphragmatic dysfunction.However,the specific mechanism of this effect has not yet been clarified.Recently,the biological effects of adipokines have attracted a lot of attention,among which the close relationship between chemerin and COPD has been gradually confirmed,and chemerin is an indispensable part of the inflammatory response of COPD.The regulation effect of exercise on the chemerin/CMKLR1 signal axis has been confirmed in other studies,but there is still no clear conclusion on the regulation of the upstream signal of the chemerin/CMKLR1 axis by exercise.Studies have shown that in obesity and diabetes models,PPAR? has been proved to be an upstream signal of exercise regulation of chemerin/CMKLR1,which is involved in exercise improvement of glucose and lipid metabolism,and the pharmacologic ligand of PPAR? has also been used in clinical management of COPD patients.However,for COPD diaphragm dysfunction,whether PPAR? is the upstream signal of exercise regulation of chemerin/CMKLR1 to improve COPD diaphragm dysfunction is still unclear,and it is worth exploring.MethodsPart one: Effects of exercise on diaphragm dysfunction and PPAR?-chemerin/CMKLR1 axis and its downstream inflammatory factors in COPD rats.Thirty-two two-month-old male SD rats were randomly divided into control group(CG),model group(MG),aerobic exercise group(AEG)and resistance exercise group(REG),with 8 rats in each group.COPD rat model was established by cigarette smoke exposure in MG,AEG and REG.After the model was established,AEG and REG received either aerobic or resistance exercise training for 9 weeks.Aerobic exercise was carried out by swimming,while resistant exercise was carried out by climbing ladder.MG was free to move during the exercise intervention,while CG did not receive any intervention throughout the study.The body weight of the rats was measured monthly during the experiment.After exercise training,lung function and diaphragm function of the rats were detected.HE staining of lung and diaphragm was used to observe the changes of tissue structure.The protein expressions of PPAR?,chemerin,CMKLR1,IL-1?,IL-6,IL-8,TNF-?,atrogin-1,Mu RF1 and My OD1 in the diaphragmatic muscle of rats were detected by Western blot.Part two: Effects of mechanical stretching on L6 cell proliferation,PPAR?-chemerin/CMKLR1 axis and downstream factors under LPS-induced inflammation.1.Screening the concentration of LPS that could induce low proliferative activity of L6 myoblast cellsL6 cells were inoculated in a 96-well plate and cultured in an incubator at 37? for 24 h.LPS culture wells with different concentrations were set separately,and 6compound wells were set at each concentration.Ten microliters LPS solution was added to each well and cultured in an incubator at 37? for 24 h.CCK-8 kit was used to detect cell proliferation.2.Effect of mechanical stretching on the proliferation level of L6 cells in LPS-induced inflammatory environment.L6 cells were randomly divided into control group(CG),LPS group(LG),and LPS stretching group(LSG).LSG was subjected to periodic mechanical stretching with a tensile strength of 15%,a frequency of 0.5Hz,and a duration of 6 h in the Flexcell stretching device.CG and LG were cultured under the same conditions without cell pull stimulation.CCK-8 kit was used to detect the proliferation activity of cells 24 h after the end of stretching.Cells were collected and the protein expressions of PPAR?,chemerin,CMKLR1,IL-1?,IL-6,IL-8,TNF-?,atrogin-1,Mu RF1 and My OD1 in each group were detected by Western blot.Part three: The role of PPAR? in mechanical stretching regulating chemerin/CMKLR1 signal to promote L6 cell proliferation in inflammatory environment.1.The appropriate concentration of GW9662 and rosiglitazone solution to affect the proliferation level of L6 myoblast cells in the inflammatory environment was screened.L6 myoblast cells were inoculated in 96-well plates and added with LPS solution after affixed to the wall.After cultured for 24 h,the cells were randomly divided into 10 groups,including LPS group,DMSO group and 4 groups of rosiglitazone and GW9662 with 4 different concentration gradients,respectively.The proliferation activity of each group of cells was detected by CCK-8 kit after 24 h of drug culture.2.Effects of PPAR? protein expression on mechanical stretching induced activation of L6 myoblasts and PPAR?-chemerin/CMKLR1 signal.L6 cells were randomly divided into control group(CG),LPS group(LG),rosiglitazone group(RG),rosiglitazone + stretching group(RSG),GW9662 group(GWG),and GW9662 + stretching group(GSG).The two stretching groups were treated with mechanical stretching intervention in the Flexcell stretching device.All the control groups were cultured in the same environment without stretching stimulation.Indicators were detected 24 h after the end of stretching,and the indicators were the same as in part two.Results1.Effects of exercise on diaphragmatic dysfunction and PPAR?-chemerin/CMKLR1 pathway and its downstream inflammatory factors in COPD rats.(1)Compared with MG,lung function and diaphragmatic function of AEG rats were significantly increased(p < 0.05),and the diaphragmatic tissue structure was ameliorated.The lung function of REG rats was also improved(p < 0.05),but the diaphragmatic function was not significantly changed.(2)Compared with MG,the expression of PPAR? protein in diaphragm of AEG and REG rats were significantly up-regulated(p < 0.05),and the chemerin/CMKLR1 signal in diaphragm of AEG rats was inhibited(p < 0.05).However,there was no significant variation in chemerin /CMKLR1 signal in the diaphragm of REG rats.(3)Compared with MG,the expression of IL-8 in the diaphragm of AEG rats was significantly inhibited(p < 0.05),and the expression of IL-1? and IL-6 tended to be inhibited.There was no significant change of inflammatory factors in diaphragm of REG rats.(4)The expression of Myo D1 in the diaphragmatic muscle of AEG rats was increased compared with that of MG(p < 0.05),and the expression of atrogin-1 and Mu RF1 had a downward trend.The expression of Mu RF1 in the diaphragm of REG rats was also restrained(p < 0.05).2.Effects of mechanical stretching on L6 cell proliferation,PPAR?-chemerin/CMKLR1 and downstream factors in LPS-induced inflammatory environment.2.1 Appropriate LPS concentration induced low proliferative activity of L6 myoblast cells.The concentration of 2 mg/m L,5 mg/m L and 10 mg/m L LPS solution inhibited the level of cell proliferation.After screening,the concentration of 2 mg/m L LPS was used as the stimulation concentration of LPS in the subsequent part of this study.2.2 Influence of mechanical stretching on the proliferation level of L6 cells in inflammatory environment.(1)The proliferation level of LG cells was decreased compared with CG cells(p <0.05),and the proliferation activity of LSG cells was significantly increased compared with LG cells(p < 0.05).(2)Compared with LG and CG,the expression of PPAR? protein in LSG cells was significantly increased(p < 0.05),and the expression of CMKLR1 protein in LSG cells tended to be inhibited.(3)The protein levels of IL-1? and TNF-? in LG cells were significantly increased compared with CG cells(p < 0.05),while the levels of these inflammatory cytokines in LSG cells were significantly decreased after mechanical traction(p < 0.05).(4)The expression level of atrogin-1 in LG cells was significantly higher than that in CG cells,and showed a tendency to be inhibited decreased after stretching,but without statistically significance.The expression of Mu RF1 in each group also showed a similar trend.However,the expression of Myo D1 showed a downward trend in LG and an upward trend in LSG.3.Role of PPAR? in mechanical strertching regulating chemerin/CMKLR1 signal to promote L6 cells proliferation in inflammatory environment.3.1 Appropriate concentration of GW9662 and rosiglitazone solution affected the L6 myoblast proliferation level in the inflammatory environment.GW9662 at 50 ?M tended to inhibit the proliferation activity of L6 cells in the inflammatory environment,while rosiglitazone at 10 ?M and 50 ?M tended to enhance the proliferation activity of L6 cells.3.2 Effects of mechanical stretching combined with rosiglazone or GW9662 on L6 myoblast activity and PPAR?-chemerin/CMKLR1 signal in LPS-induced inflammatory culture environment.(1)Rosiglitazone/GW9662 alone promoted/inhibited the proliferation activity of L6 cells.Compared with GWG,the proliferation level of L6 cells was increased(p <0.05).Compared with RG,the proliferation level of L6 cells was further elevated(p <0.05).(2)PPAR? protein was activated in RG cells,which was significantly higher than that in GWG and GSG(p < 0.05);The expression level of RSG protein was the highest,which was significantly different from CG,LG,GWG and GSG(p < 0.05).The expression of CMKLR1 in RG and RSG was significantly lower than that in LG(p <0.05).(3)IL-1? decreased significantly in RSG cells compared with GWG cells(p < 0.05).Compared with LG,IL-6 in GWG,RG and RSG cells was significantly decreased(p< 0.05).TNF-? in LG,GWG,GSG and RG cells was significantly increased compared with CG cells(p < 0.05),and TNF-? protein expression in RSG cells was significantly decreased compared with LG and GWG cells(p < 0.05).(4)The expressions of atrogin-1 and Mu RF1 in RG cells were significantly inhibited(p < 0.05).The expression of Mu RF1 in RSG cells was significantly inhibited(p <0.05).The expression of Myo D1 in GSG,RG and RSG cells tended to elevate but without statistically significance.Conclusion1.COPD rats showed diaphragmatic dysfunction.Aerobic exercise had more effective rehabilitation effect on COPD lung function and diaphragmatic function.Exercise up-regulated the expression of PPAR? protein in the diaphragm,inhibited chemerin/CMKLR1 signal,decreased the levels of IL-8 and TNF-? in the diaphragm,and regulated the imbalance of protein degradation/growth in the diaphragm.2.Appropriate concentration of LPS induced low proliferative activity of L6 cells.Mechanical stretching enhanced the proliferation activity of L6 cells cultured in LPS environment,and activated PPAR? protein,to a certain extent,affecting the expression of chemerin/CMKLR1 signal and inhibiting the protein levels of IL-1? and TNF-?,and affect the expression of E3 ligase and its substrate.3.Exogenous enhancement of PPAR? signal combined with mechanical stretching enhanced the activity of L6 cells,inhibited the chemerin/CMKLR1 signal and the expression of IL-6 and TNF-?,and further restrained the expression of atrogin-1 and Mu RF1,which degraded myocyte protein.In contrast,by inhibiting PPAR? signaling,these effects were inhibited.In conclusion,this study confirmed the role of PPAR? in the improvement of COPD diaphragm dysfunction by exercise regulation of chemerin/CMKLR1 signal in vivo and in vitro. |
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