Effect Of TGF-?1/smad3 On The Expression Of IL-1R1 And IL-1? In Hepatic Stellate Cells

Objective To study the effect of TGF-?1 in hepatic stellate cells on the expression of IL-1R1 and IL-1? through smad3.Methods Construct a short hairpin RNA(shRNA)of transforming growth factor TGF-?1 and a negative control plasmid to assemble into a lentiviral vector.Then transfected into rat hepatic stellate cell line HSC-T6 cells and selected stable interfering cell lines,named TGF-?1-KD group and Ctrl-KD group,respectively.The cytokine TGF-?1 12ng/ml was used to stimulate HSC cells and TGF-?1-KD cells.The experiment was divided into five groups: A: HSC group,B: HSC + TGF-?1 group,C: Ctrl-KD group,D: TGF-?1-KD group,E: TGF-?1-KD + TGF-?1 group.After 48 hours of cell culture,total RNA and total protein of each group were extracted to detect the expression of IL-1R1,IL-1? and ?-SMA by q-PCR and Western-blot.In order to explore whether smad3 is involved,group B cells were treated with smad3 inhibitor(SIS3)2 hours in advance,and IL-1R1 and IL-1? were detected by Western-blot.To further explore the effects of TGF-?1 and IL-1? on HSC,12 ng/ml TGF-?1 and IL-1? cytokines were used to treat cells in groups A and D,respectively,and Western-blot was used to detect ?-SMA and Col-? expression.The t test was used to compare the mean between the two groups,and one-way ANOVA was used to compare the multiple groups.Result After the TGF-?1 shRNA lentivirus infected HSC,the protein levels of IL-1R1 and IL-1? were increased by 36.7% and 34.9%(P=0.0087,P=0.0029,respectively),the protein level of ?-SMA was decreased by 22.3%(P=0.0117),the relative level of IL-1R1 mRNA was increased by 106.6%(P=0.0027).However,after TGF-?1 cytokine stimulated HSCs,the opposite trend appeared.The protein levels of IL-1R1 and IL-1? were down-regulated by 83.0% and 28.5%(P<0.001,P=0.0014,respectively),the protein levels of ?-SMA were up-regulated by 49.5%(P=0.0049),the relative level of IL-1R1 mRNA was significantly reduced by 45.7%(P=0.0011).After the inhibitor SIS3 was treated in advance,the inhibitory effect of TGF-?1 on IL-1R1 and IL-1? was weaker than that of TGF-?1 alone(P=0.003).After stimulating HSC with cytokines TGF-?1 and IL-1?,the expression levels of ?-SMA and Col-? both increased(P<0.05),and TGF-?1-KD cells stimulated showed the same upregulation effect.Conclusions In rat hepatic stellate cells,TGF-?1 can suppress the expression of IL-1R1 through smad3,or directly inhibit the expression of IL-1?.