Exercise Improves Diaphragm Dysfunction Of Cigarette Smoke-induced Chronic Obstructive Pulmonary Disease:the Role Of RhoA/ROCK

ObjectiveDiaphragm dysfunction is an importantly extrapulmonary pathological manifestation of chronic obstructive pulmonary disease(COPD)and is closely related to COPD respiratory symptoms.Cigarette smoking,as a risk factor for diaphragm dysfunction in COPD,can directly damage extrapulmonary tissues.Exercise is an important component of pulmonary rehabilitation,and plays an essential role in improving COPD diaphragm dysfunction via regulating inflammation.Exercise can improve the mobility of the diaphragm,respiratory muscle strength,and reduce the inflammation level of lung and whole body in COPD.Our previous study found that chemerin/CMKLR1 may be a vital way for exercise to regulate inflammation and improve diaphragm dysfunction in COPD.It’s downstream Rho A/ROCK can act as a molecular switch by combining with GTP or GDP,playing an important role in the inflammatory response of COPD.However,whether Rho A/ROCK signaling pathway plays a key role in exercise improving COPD diaphragm dysfunction remains unknown.Therefore,this study intends to further reveal the specific mechanism that exercise prevents and treats COPD diaphragm dysfunction from the perspective of exercise regulating inflammation.Methods(1)Eight-week-old male C57BL/6 mice were randomly divided into control group(CG),model control group(MG)and model exercise group(MEG),with 8 mice in each group.MG and MEG mice received incremental cigarette smoke exposure for 25 weeks to establish the COPD model;then MEG mice received moderate-intensity aerobic exercise for 9 weeks,during which MG mice were routinely fed without any intervention.CG mice were routinely housed throughout the study without any intervention.After the exercise intervention,the lung function and the isolated diaphragm muscle function of mice in all groups were detected;then the lung tissue and diaphragm muscle tissue were collected.The structure of the lung and diaphragm muscle tissue was observed by HE staining,protein expressions of Rho A,ROCK1,IL-1β,TNF-α,Atrogin-1,Mu RF-1,Myo D1,and myf5 in the diaphragm muscle were detected by Western blot.(2)C2C12 myoblasts were randomly divided into 3 groups: control group(CG),LPS group(LG),and LPS stretch group(LSG),which were plated onto Bio Flex flexiblebottomed 6-well plates.After culturing at 37°C for 24 hours,LG and LSG were added with an appropriate concentration of LPS and incubated at 37°C for 24 hours.Then,the LSG cells were mechanically stretched with appropriate stretch parameters.CG cells were routinely cultured without any intervention during the whole experimental process.24 hours after the end of stretching,the cell supernatant and cells were collected.The expression of IL-1β and TNF-α in the cell supernatant were detected by ELISA,the protein expressions of Rho A,ROCK1,Atrogin-1,Mu RF-1,Myo D and Myf5 were detected by Western blot.(3)C2C12 myoblasts were seeded onto Bio Flex flexible-bottomed 6-well plates and randomly divided into 6 groups: control group(CG),LPS group(LG),LPS agonist group(UG),LPS agonist stretch group(USG),LPS inhibitor group(YG),LPS inhibitor stretch group(YSG).CG cells did not receive any intervention.After culturing at 37°C for 24 hours,LG,UG,USG,YG,and YSG cells were added with LPS and then cultured for another 24 hours.Subsequently,UG and USG cells were added with an appropriate concentration of U46619,and YG and YSG cells were added with an appropriate concentration of Y27632.After 30 minutes,USG and YSG cells underwent mechanical stretch intervention.24 hours after the end of stretching,the cell supernatant and cells were collected.The expression of IL-1β and TNF-α in the cell supernatant were detected by ELISA,the protein expressions of Rho A,ROCK1,Atrogin-1,Mu RF-1,Myo D and Myf5 were detected by Western blot.Results(1)Compared with CG,MG mice had significantly increased cross-sectional area of alveolar,decreased lung function(p < 0.05),and structural and functional impairment of diaphragm;the protein expression of ROCK1,TNF-α and Mu RF-1 was elevated,and the protein expression of Myf5 was significantly reduced(p < 0.05).Compared with MG,MEG mice had a significant decrease in the cross-sectional area of alveolar,improved lung function(p < 0.05),and a significant rise in the cross-sectional area and isometric muscle contractions force of the diaphragm(p < 0.05).The force-frequency curve of diaphragm shifted significantly up;the protein expression of Rho A,ROCK1 reduced significantly(p < 0.05);the expression level of inflammation declined,but the difference between groups was not statistically significant;the expression of Mu RF1 was significant decreased(p < 0.05).(2)Compared with CG,the expression of Rho A protein in LG cells was significantly increased(p = 0.024);the expression levels of IL-1β and TNF-α were significantly elevated(p < 0.05);the expression of Atrogin-1 and Mu RF-1 protein increased relatively,but did not reach statistical significance.Compared with LG,the expression of Rho A protein in LSG cells was significantly decreased(p = 0.012);the levels of inflammation and protein degradation were reduced,and the level of muscle regeneration was increased but did not reach statistical significance.(3)Compared with CG,the proliferation level of YSG cells increased significantly,and the proliferation of cells in other groups decreased significantly(p < 0.01);the protein levels of Rho A and ROCK1 in LG and UG cells were significantly increased,but did not reach statistical significance;inflammatory levels in LG and UG cells were significantly increased(p < 0.01);Atrogin-1 protein expression in LG and UG cells was significantly increased(p < 0.01).Compared with LG,the proliferation levels of YG and YSG cells increased(p < 0.01);the inflammation levels of UG and USG elevated,and the inflammation levels of YG and YSG decreased(p < 0.05);the protein degradation levels of USG,YG,and YSG reduced(p < 0.01).Compared with UG,the expression of ROCK1 protein in YSG cells was significantly lower(p = 0.042);the levels of IL-1β in USG,YG,and YSG cells were significantly decreased,and the levels of TNF-α in YG and YSG cells were significantly reduced(p < 0.001);Atrogin-1 protein expression in USG,YG,YSG cells was significantly decreased(p < 0.01).Compared with YG,TNF-α levels in YSG cells were significantly declined(p = 0.003).Conclusion(1)Moderate-intensity aerobic exercise can improve COPD mice diaphragmatic dysfunction,inhibit Rho A/ROCK signaling pathway and pro-inflammatory factors expression,regulate protein degradation and muscle regeneration.(2)Mechanical stretch can improve the proliferation of C2C12 myoblasts cultured with LPS,and to some extent inhibit the Rho A/ROCK signaling and promote the expression levels of inflammatory factors,regulate ubiquitin ligases and myogenetic regulating factors.(3)Rho A/ROCK signaling pathway can affect the proliferation of C2C12 myoblasts cultured with LPS;mechanical stretch can alleviate the reduced cell proliferation and the pro-inflammatory effect caused by agonists;enhance the increased cell proliferation caused by inhibitors and inhibit the inflammation and protein degradation.