The Study Of The Role And Intervention Of RhoA/ROCK1 Signaling Pathway In Sepsis-associated ARDS And Pulmonary Fibrosis

BackgroundAcute respiratory distress syndrome(ARDS)partly caused by sepsis has a high mortality in intensive care unit(ICU).Sepsis-associated ARDS triggers overlapping inflammation and subsequent pulmonary fibrosis.Increasing evidences from experimental and clinical studies suggest the proliferation and differentiation in human lung fbroblast cells have important pathological significance in the development of ARDS.However,the potential molecular mechanism of ARDS-associated lung fibrosis is complex and remains unclear.The morbidity and mortality of ARDS and ARDS-associated lung fibrosis in ICU is still very high.It is vital to understand pathogenesis of pulmonary fibrosis,and provide guidance in choosing suitable treatment to mitigate lung fibrosis.ObjectiveTo determine the espression of proteins of RhoA/ROCK1 pathway in lung tissues of mice with pulmonary fibrosis(PF)induced by Lipopolysaccharide(LPS)and treatmentde by Fasudil.And to investigate the treatment effect of Fasudil on PF and the role of RhoA/ROCK1 pathway in the development of fibrosis.At the cellular level,we wanted to investigate the effect of Lipopolysaccharide(LPS)on the activity of RhoA/ROCKl signaling pathways of human lung fibroblast cells,and examine the role of Fasudil(a specific blocker of Rock)in LPS-induced cells proliferation and differentiation.Then explore the role of RhoA/ROCKl pathway in the proliferation and differentiation of lung fibroblasts,and seek new ideas for the treatment of ARDS.Methods(1)All 90 mice were randomly divided into 3 groups:the control group(n = 30):aerosol inhalation salin(5mg/kg)and abdominal injection was done with salin(20mg/kg/day);LPS group(n = 30):aerosol inhalation LPS(5mg/kg,concentration of LPS was 1mg/mL),and abdominal injection was done with salin(20mg/kg/day);Fasudil group(n = 30):aerosol inhalation LPS(5mg/kg,concentration of LPS was 1mg/mL),and abdominal injection was done with Fasudil(20mg/kg/day).Then each 5 mice were sacrificed at 3th,7th,14th and 28th days respectively,and then the lung tissue was taken from the mice.The left lung was placed in a clean EP tube,frozen in the refrigerator at-80℃,the protein levels of GTP-RhoA,ROCK1,MYPT-1(Myosin phosphatase target subunit),p-MYPT-1(a downstream substrate of ROCK1)and alpha-smooth muscle(a-SMA,a marker of myofibroblast cells differentiation)of left lung at 3th were determined by Western blot;Quantitive real-time polymerase chain reaction was used to determine the level of a-SMA mRNA.The right lung was placed in a clean EP tube with Faure Marin,using observed the change of lung tissues by hematoxylin and Eosin(HE).(2)We divided MRC-5 cells into four groups,control group,LPS group(LPS:10μg/mL),low-dose group(Fasudil:15 μmol/mL + LPS:10 μg/mL)and high-dose group(Fasudil:30 μmol/mL + LPS:10 μg/mL).Cells were treated in serum-free medium for 24 h.RhoA activity was determined by Rho pull-down analysis and the protein levels of GTP-RhoA,ROCK1,MYPT-1,p-MYPT-1 and a-SMA(a marker of myofibroblast cells differentiation)were determined by Western blot.Quantitive real-time polymerase chain reaction was used to determine the level of a-SMA mRNA.Cell proliferation rate was examined using Cell Counting Kit-8(CCK-8)and Cell-LightTM EdU Apollo(?)643 In Vitro Imaging Kit(EdU).Results(1)In the mice of LPS group we found that LPS could up-regulated RhoA activity,protein expressions of ROCK1(P<0.05),p-MYPT-1 and a-SMA(P<0.05).And Fasudil(a highly selective inhibitor of ROCK)blocked LPS-induced PF,P<0.05).In the control group,the pathological changes of lung tissue were slight,inflammatory cells were rarely found,alveolar septum was not thickened,and no significant pulmonary fibrosis.The change of lung tissue pathological of LPS group were obvious,a large number of inflammatory cells infiltrated in lung tissues,alveolar septum were markedly thickened,and change of pulmonary fibrosis were distinctly;in Fasudil group less inflammatory cells infiltrated in lung tissues,alveolar septum thickened slightly,and slight change of fibrosis compared with LPS group.(2)LPS up-regulated RhoA activity,protein expressions of ROCK1(P<0.05),p-MYPT-1 and a-SMA(P<0.05)as well as proliferation rate(P<0.05).Fasudil(a highly selective inhibitor of ROCK)blocks LPS-induced cells proliferation and differentiation,and the significant higher inhibitory effects in high-dose Fasudil group were observed compared with low-dose Fasudil(high-dose group vs low-dose group,P<0.05)Conclusion(1)Fasudil has certain treatment on lipopolysaccharide induced pulmonary fibrosis in mice.(2)LPS promoted cells proliferation and differentiation with an involvement of RhoA/ROCK1 activation and MYPT1 phosphorylation in MRC-5 cells.Fasudil attenuated LPS-induced cells proliferation and differentiation by inhibiting RhoA/ROCK1 signaling pathway and de-phosphorylation of MYPT1.Regulating the levels of RhoA/ROCKl and p-MYPT1 could be a potential new target to treat pulmonary fibrosis.