Effect Of SiRNA-mediated Smad3 Silence On Proliferation And Apoptosis In Activated Hepatic Stellate Cells

ObjectiveTo investigate the effects of siRNA mediated Smad3 silence on proliferation, apoptosis inactivated hepatic stellate cells and the mechanisms was involved.MethodsHSC-T6 cells were cultured in vitro and divided into three groups: blank group, negativecontrol group, siRNA-Smad3 transfection group. The siRNA-Smad3 was transfected intoHSC-T6 cells. After 24h, 48h, 72h, 96h of transfection, cell proliferation was measured byCCK-8. After 24h, 48h, 72h of transfection, cell apoptosis was detected by flow cytometry. After48h of transfection, P53 and Bcl-2 protein expression was detected by immunocytochemistry.Results1 After 24h, 48h, 72h of transfection, HSC proliferation was significantly inhibited in thetransfection group(0.27±0.03,0.53±0.03,0.97±0.09)compared with the blank(0.37±0.02,0.68±0.03,1.13±0.09)and negative(0.33±0.06,0.66±0.01,1.17±0.04)control group(P<0.01). The proliferation inhibition rate were19%,20%,18%. After 96h of transfection, thereis no significant change in the proliferation of HSC in the transfection group(1.40±0.08)compared with the blank(1.45±0.05)and negative(1.44±0.03)control group(P>0.05).2 After 24h, 48h, 72h of transfection, the apoptotic tate of HSC was increased significantly inthe transfection group(8.76±1.41,24.25±2.82,12.71±1.53)% compared with the blank(3.36±0.94,3.12±1.3,4.35±0.58)% and negative(4.46±0.33,4.17±0.58,4.99±0.77)% control group(P<0.01).3 After 48h of transfection, each group had positive staining in nucleus(P53)and cytoplasm(Bcl-2)showing some brown particles. The results of image analysis showed that theexpression of P53 protein was increased significantly and Bcl-2 protein was significantlydecreased in the transfection group(1.79±0.16,0.66±0.13)compared with the blank(0.58±0.18,5.02±0.52)and negative(0.59±0.13,4.89±0.49)control group(P<0.01). Conclusions1 The Smad3-siRNA mediated Smad3 silence can significantly inhibit the proliferation of HSC.Its inhibitory effect was disappeared after 96h of transfection.2 The Smad3-siRNA mediated Smad3 silence can significantly induce the apoptosis of HSC.After 48h of transfection, the rate of HSC apoptosis was highest.3 Smad3 silence inducing HSC apoptosis may be associated with up-regulating expression ofP53 and down-regulating expression of Bcl-2 on HSC.4 For the treatment of liver fibrosis, Smad3 silence may provide a new pathway.