Effects Of Cigarette Smoke On Diaphragm Inflammation And Chemerin-RhoA/ROCK In Mice

ObjectiveCigarette smoke exposure is the most important and common risk factor for chronic obstructive pulmonary disease(COPD),and diaphragmatic dysfunction is an important extrapulmonary pathological effect of COPD.However,the direct damaging effect of cigarette smoke exposure on the diaphragmatic tissue remains to be further studied,and the pathogenesis of diaphragmatic dysfunction in COPD also remains to be further explored.Inflammatory response is an important factor leading to diaphragm dysfunction.Our previous studies have found that chemerin is involved in the regulation of COPD inflammation level,but the specific mechanism is unclear.Some studies have shown that the chemerin/CMKLR1 chemotaxis response of inflammatory cells depends on the activity of Rho A/ROCK signaling pathway.Therefore,this study focused on the perspective of inflammation and explored the inflammatory factors,chemerin-Rho A/ROCK signaling pathway in the diaphragm tissue,and the expression of proteins related to diaphragm protein degradation and muscle regeneration through progressive cigarette smoke exposure program,so as to explore the effects of cigarette smoke on the diaphragm.This study provides a reference basis for further exploring the mechanism of COPD diaphragm dysfunction,and lays a good foundation for improving the treatment of COPD respiratory muscle dysfunction with chemerin-Rho A/ROCK as the target.MethodsTwelve 8-week-old male SPF C57BL/6 mice were randomly divided into blank group and smoked group,including 6 mice in blank group and 6 mice in smoked group.Mice in the blank group were routinely fed in the conventional air without any other intervention.Mice in the smoking group were smoked with a 25-week progressive and incremental whole-body cigarette smoke exposure program.Samples were collected from the diaphragmatic tissue of mice after the smoking cycle.Hematoxylin-eosin(HE)staining was used to observe the pathological changes and the changes of diaphragm fiber cross-sectional area(CSA)in the two groups.The expression of chemerin,CMKLR1,Rho A,ROCK1,ROCK2,IL-1β,IL-6,TNF-α,Atrogin-1,Mu RF-1,Myostatin,Myo D1 and Myf5 in the diaphragm of the two groups of mice was detected by Western blotting(WB).Results(1)The observation and analysis under HE staining microscope showed that the fibers of the diaphragm were loosely arranged,the interstitial fibers were increased,the muscle nucleus was disordered,and the CSA was significantly decreased in the smoking group(p<0.05).In the blank group,the diaphragm fibers were tightly arranged and regular,uniform in size,full in muscle fibers,and no interstitial hyperplasia was observed.(2)Compared with blank group,the expression level of chemerin protein in the diaphragm of smoked group was significantly increased by WB detection(p<0.01).Compared with blank group,ROCK1 content of smoked group was significantly increased(p<0.05).Compared with blank group,the protein expressions of CMKLR1,Rho A and ROCK2 in the smoked group were increased,but did not reach statistical significance.(3)Compared with blank group,the expression level of TNF-α protein in the diaphragm of smoked group was significantly increased by WB detection(p<0.05).Compared with blank group,the expression levels of IL-1β and IL-6 protein in the diaphragm of smoked group were increased,but did not reach statistical significance.(4)WB analysis showed that compared with blank group,Atrogin-1,Mu RF-1and Myostatin expression levels were increased in smoked group,and Atrogin-1 and Myostatin protein expression levels were significantly increased(p<0.01).Mu RF-1protein expression was significantly increased(p<0.05).Compared with blank group,the expression levels of Myo D1 and Myf5 protein in the diaphragm of smoked group were decreased,but there was no statistical significance.Conclusions(1)Cigarette smoke exposure affects extrapulmonary tissue and causes significant structural damage to the diaphragm in mice.(2)Cigarette smoke exposure increases the expression levels of inflammatory cytokines IL-1β,IL-6 and TNF-α in the diaphragm of mice,increases the expression levels of chemerin-Rho A/ROCK signaling pathway,and affects the degradation of diaphragm protein and muscle regeneration,which may be one of the mechanisms of diaphragm dysfunction in COPD.