IGFBPrP1 SiRNA Induced Apoptosis In Rat Hepatic Stellate Cells And Its Mechanism

ObjectiveTo investigate the influence of inhibiting insulin-like growth factor binding protein related protein1(IGFBPrP1)by chemically synthesized small interfering RNA(siRNA)on apoptosis of HSC-T6 cells and to explore the possible mechanism.MethodsDivide the hepatic stellate cells into three groups as following: normal control group ,negative control group and IGFBPrP1 siRNA transfection group.The IGFBPrP1 siRNA was transfected into hepatic stellate cells. After transfecting with different time, cell proliferation was measured by CCK-8 assay, cell apoptosis was detected by flow cytometry with AnnexinV/PI double staining, both P53 and Bcl-2 protein expressions were detected by immunocytochemistry.Results(1)CCK-8 assay showed tha(t24h, 48h, 72h)after IGFBPrP1 siRNA transfection,the value of OD of each IGFBPrP1 siRNA transfection group was decreased compared with the normal control group and the negative control group, respectively (P<0.01);(24h, 48h, 72h)after IGFBPrP1 siRNA transfection,the inhibitory ratio of IGFBPrP1 siRNA transfection groups were increased compared with the negative control group, respectively(P <0.01);(2)Flow cytometry with AnnexinV/PI double staining showed that(24h, 48h, 72h)after IGFBPrP1 siRNA transfection, the apoptotic rate of IGFBPrP1 siRNA transfection group was increased compared with the normal control group and the negative control group, respectively (P<0.01), the apoptotic rates of the normal control group and the negative control group were no different(P >0.05);(3)Immunocytochemistry showed that 48h after IGFBPrP1 siRNA transfection, the expression of P53 protein:Each group had positive staining in nucleolus showing some buffy or brown particles.The expression of Bcl-2 protein:Each group had positive staining in cytoplasm showing some buffy or brown particles. The expression of P53 protein was significantly increased and the expression of Bcl-2 protein was significantly decreased compared with normal control group and negative control group, respectively(P <0.01).Conclusion(1)IGFBPrP1 siRNA can result in significant inhibition of the hepatic stellate cells proliferation and can induce apoptosis of hepatic stellate cells effectively;(2) Up-regulation of P53 expression and down-regulation of Bcl-2 expression may be one of the ways for the apoptosis of hepatic stellate cells induced by IGFBPrP1 siRNA;(3) siRNA targeting IGFBPrP1 may be potential to prevent and treat liver fibrosis.