Exogenous Hydrogen Sulfide Inhibits Proliferation Of Mouse Pulmonary Fibroblasts By Down-Regulating TGF-β1/P38MAPK/NF-κB Signaling Pathway

Objective:To investigate whether exogenous hydrogen sulfide inhibits the growth and differentiation of fibroblasts by inhibiting the TGF-β1/P38MAPK/NF-κB signaling pathway,and thus plays an anti-pulmonary fibrosis role.Methods:NIH3T3 cells were cultured in vitro in the incubator,the temperature of the incubator was set to 37℃,containing 5%CO₂,and the appropriate humidity was ensured.NIH3T3 cell culture medium DMEM was cultured in incubator after adding 10%FBS and 1% double antibody.The NIH3T3 cells cultured in vitro were randomly divided into group A,group B,group C,group D and group E.after subculture,the cells in the above groups were treated as follows: group A（blank control group）: no drug treatment,only cultured in the incubator for 24 hours.Group B（TGF-β1）: cells were cultured and treated with TGF-β1（5μg/L）for24 hours.Group C（TGF-β1+NaHS group）: the drugs were added in the following order: pretreated with Na HS（40μmol/L）for 1hour,and then treated with TGF-β1（5μg/L）for 24 hours.Group D（TGF-β1+SB203580 group）: the drugs were added in the following order:first pretreated with SB203580 at the concentration of10μmol/L for 1 hour,then added with TGF-β1（5μg/L）for 24 hours.Group E（TGF-β1+Na HS+SB203580 group）: the drugs were added in the following order: first pretreated with SB203580 at the concentration of 10μmol/L for 1h,then treated with Na HS（40μmol/L）for 1h,and finally treated with TGF-β1（5μg/L）for 24 h.The level of cell proliferation in each group was detected by CCK8 method.The protein expression levels of α-SMA,Collagen Ⅰ,P38 MAPK,NF-κB,ρ-P38 MAPK and ρ-NF-κB were detected by Western blot method.the expression levels of P38 MAPK and NF-κB in each group were detected.by RT-qPCR.Results:1、Morphological changes of cells under phase contrast microscope:a large number of fibroblasts could be seen in group A: their morphology was fusiform or star-shaped flat structure,and the nucleus was oval.The myofibroblasts with flat fiber structure were found in other groups,and the myofibroblasts in group B,C,D and E were more obvious than those in group A,especially in group B.2、CCK8 results: By detecting the cell activity of each group,it was found that after drug treatment,the cell activity of group B was higher than that of the remaining groups,The remaining cell activity in each group was D > C > E > A,with statistically significant differences（P<0.05）.3、Western blotting results: the protein expression levels ofα-SMA and Collagen Ⅰ were B > D > C > E > A from high to low,and the difference was statistically significant.Compared with group C and group E,the protein expression of α-SMA and Collagen Ⅰ in group D was higher than that in group C and group E.The protein expressions of P38 MAPK,ρ-P38 MAPK,NF-κB andρ-NF-κB in group B were higher than those in the other groups（P< 0.05）.4、qRT-PCR Results: the relative expression levels of P38 MAP K and NF-κB were B>C>D>E>A from high to low,with statisticall y significant differences（P<0.05）.Conclusion:One of the mechanisms by which exogenous hydrogen sulfide inhibits proliferation and differentiation of mouse lung fibroblasts may be by inhibiting the activity of TGF-β1/P38MAPK/NF-κB signaling pathway.