Exogenous Hydrogen Sulfide Inhibits TGF-β1 Induced NIH3T3 Cell Proliferation And CTGF Generation By Down-regulation ERK1/2 Signaling Pathway

Objective:To investigate whether exogenous H₂S can inhibit TGF-β1-induced fibroblast proliferation through ERK1/2 signaling pathway,and to explore the relationship between CTGF generation in fibroblasts and ERK1/2 pathway.Methods:In normal cells incubator（DMEM medium with concentration of 10%fetal bovine serum and the concentration of 1%of antibiotic,adjust the incubator temperature of 37℃,and contains 5%CO₂,95%air and saturated humidity）cultivation of NIH3T3 cells,NIH3T3 cells were randomly divided into 5 groups:group A as the control group,B group as TGF-β1,C group as TGF-β1+Na HS,group D as TGF-β1+U0126,group E as TGF-β1+U0126+Na HS.Group A was cultured in an incubator for 24h without adding drugs.Group B was treated with 5μg/L TGF-β1 for 24h.Group C was pretreated with 40μmol/L Na HS for 1h,and then treated with 5μg/L TGF-β1 for 24h.Group D was pretreated with U0126（10μmol/L）for 1h,and then TGF-β1（5μg/L）for 24h.In group E,U0126（10μmol/L）was added for pretreatment for 1h,followed by Na HS（40μmol/L）for 1h,and TGF-β1（5μg/L）for further treatment for 24h.Morphological changes of cells in each group were observed at 12h,24h and 48h,respectively.Cell proliferation was detected by CCK-8.The expression levels of Collagen I,α-SMA,total ERK1/2,ρ‐ERK1/2 and CTGF in each group were determined by q RT-PCR,and the expression levels of Collagen I,α-SMA,total ERK1/2,ρ‐ERK1/2 and CTGF were analyzed by Western Blot.Results:1.Morphological observation results of cells:under the microscope of group A,many cell bodies were fusiform,flat or spindle shaped fibroblasts,and the above structures were seen in the other groups,especially in group B,which was the most widely distributed and numerous.In addition to the fibroblasts,there were more myofibroblasts with flat fibrous structure in group B,C,D and E,and the flat fibrous structure was the most obvious in group B.2.CCK-8 results:The cell proliferation of each group at 12,24 and48h was the highest in group B,and the cell proliferation of the other four groups was lower than that of group B,followed by C>D>E>A（P<0.05）,which was statistically significant.3.q RT-PCR results:The m RNA expression of CTGF,α-SMA and Collagen I was the highest in group B,and the expression level of other groups was lower than that in group B.the expression level of each group from high to low was C>D>E>A（P<0.05）.4.WB results:The protein expression levels ofα-SMA,Collagen I,CTGF andρ‐ERK1/2 in group A,B,C,D and E were the strongest in group B,and the protein expression levels in the other groups were weaker than that in group B.The protein expression levels in the four groups from strong to weak were C>D>E>A（P<0.05）.There was no significant difference in total ERK1/2 expression level among groups A,B,C,D and E（P>0.05）.Conclusion:1.Exogenous hydrogen sulfide inhibits the phosphory-lation of ERK1/2 and the proliferation of NIH3T3 cells by down-regulating TGF-β-ERK1/2 signaling pathway.2.TGF-βcan induce CTGF synthesis through the ERK1/2 signaling pathway.Exogenous hydrogen sulfide inhibit TGF-βinduced CTGF synthesis by down-regulating the ERK1/2 signaling pathway.