Exogenous Hydrogen Sulfide Inhibits TGF-β-induced Proliferation Of Mouse Lung Fibroblasts By Down-regulating Rho / Rock Signaling Pathway

Objective:To explore the role of Rho/Rock pathway in pulmonary fibrosis and the possible mechanism of exogenous hydrogen sulfide against pulmonary fibrosis,to provide a new experimental basis for the treatment of pulmonary fibrosis.Methods:Mice lung fibroblasts(NIH3T3 cells)were cultured with 10%DMEM medium+1%penicillin and placed in a 5%CO2incubator at 37℃.After the cells are fused to 90%,add 0.25%trypsin digestion to subculture(cell density 5×10~4)and place in the culture flask.After the cells are fused to about 70%,they are used for the experiment.NIH3T3 cells were randomly divided into A,B,C,D,and E groups.Each group is treated as follows:Group A(blank control group):no treatment;Group B(TGF-β1 group):adding TGF-β1(5μg/L);Group C(TGF-β1+NaHS group):adding TGF-β1(5μg/L)and NaHS(40μmol/L);Group D(TGF-β1+Y-27632 group):Add TGF-β1(5μg/L)and Y-27632(10μmol/L);Group E(TGF-β1+NaHS+Y-27632 group):Add TGF-β1(5μg/L),NaHS(40μmol/L)and Y-27632(10μmol/L).Both NaHS and Y-27632were pre-treated for 1 hour.Use MTT method to detect the cell proliferation level of each group;WB test the cell fibrosis level index of each group alpha smooth muscle actin(α-SMA),type I collagen and Rho/Rock pathway RhoA,Rock1,Rock2 Protein expression level;cell scratch test to detect the migration distance of each group of cells.Results:1.The cell survival rate was detected by MTT and found that the cell survival rates of cells in group B at 12h,24h and 48h were significantly higher than those in group D,group C,group E and group A,and the differences were statistically significant(P<0.05);D The cell survival rates of cells in groups 12h,24h and 48h were higher than those in groups C and E.2.Western blot results showed that the expression levels of α-SMA,type I collagen,RhoA,Rock1,and Rock2 proteins in group B were significantly higher than those in groups A,D,C,and E(P<0.05).Theα-SMA and type I collagen in group D were higher than those in groups C and E,the difference was statistically significant(P<0.05).RhoA,Rock1,Rock2 protein in group D was higher than that in group C,the difference was statistically significant(P<0.05).3.Results of cell scratch test:The migration distance of cells in TGF-β1 group at 12h,24h and 36h was greater than that in group A,group C,D and E,and the difference was statistically significant(P<0.05).Conclusion:Exogenous hydrogen sulfide inhibits TGF-β-induced proliferation of mouse lung fibroblasts,and its mechanism may be related to the inhibition of Rho/Rock signaling pathway.