Effect Of TGF-β1/smad3 On The Expression Of IL-1R1 And IL-1β In Hepatic Stellate Cells

Objective To study the effect of TGF-β1 in hepatic stellate cells on the expression of IL-1R1 and IL-1β through smad3.Methods Construct a short hairpin RNA(shRNA)of transforming growth factor TGF-β1 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-β1-KD group and Ctrl-KD group,respectively.The cytokine TGF-β1 12ng/ml was used to stimulate HSC cells and TGF-β1-KD cells.The experiment was divided into five groups: A: HSC group,B: HSC + TGF-β1 group,C: Ctrl-KD group,D: TGF-β1-KD group,E: TGF-β1-KD + TGF-β1 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-1β and α-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-1β were detected by Western-blot.To further explore the effects of TGF-β1 and IL-1β on HSC,12 ng/ml TGF-β1 and IL-1β cytokines were used to treat cells in groups A and D,respectively,and Western-blot was used to detect α-SMA and Col-Ⅰ 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-β1 shRNA lentivirus infected HSC,the protein levels of IL-1R1 and IL-1β were increased by 36.7% and 34.9%(P=0.0087,P=0.0029,respectively),the protein level of α-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-β1 cytokine stimulated HSCs,the opposite trend appeared.The protein levels of IL-1R1 and IL-1β were down-regulated by 83.0% and 28.5%(P<0.001,P=0.0014,respectively),the protein levels of α-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-β1 on IL-1R1 and IL-1β was weaker than that of TGF-β1 alone(P=0.003).After stimulating HSC with cytokines TGF-β1 and IL-1β,the expression levels of α-SMA and Col-Ⅰ both increased(P<0.05),and TGF-β1-KD cells stimulated showed the same upregulation effect.Conclusions In rat hepatic stellate cells,TGF-β1 can suppress the expression of IL-1R1 through smad3,or directly inhibit the expression of IL-1β.