Effect of TGF-b1 siRNA-mediated silencing on Smad proteins in hepatic fibrosis rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the changes in Smad 2, 3, 4 and 7 of the transforming growth factor-beta 1 (TGF-b1)/Smad signaling pathways in carbon tetrachloride (CCL4)-induced hepatic fibrosis rats treated with TGF-b1 small interfering (si)RNA.</p><p><b>METHODS</b>Rats were randomly divided among five groups: non-fibrotic (normal); fibrosis-induced (model); fibrotic treated with 0.125 mg/kg TGF-b1 siRNA; fibrotic treated with 0.250 mg/kg TGF-b1 siRNA; and fibrotic treated with negative control TGF-b1 siRNA. The expression of Smad 2, 3, 4 and 7 was detected by real-time polymerase chain reaction (for mRNA), immunohistochemistry and Western blotting (for protein).</p><p><b>RESULTS</b>The mRNA and protein levels of Smad 2, 3 and 4 were significantly lower in the the fibrotic rats treated with either 0.250 mg/kg or 0.125 mg/kg TGF-b1 siRNA than in the fibrotic model or the negative control TGF-b1 siRNA rats (P less than 0.01). Moreover, the mRNA and protein expression levels of Smad 2, 3 and 4 were significantly lower in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05). Comparing the 0.250 mg/kg and 0.125 mg/kg TGF-b1 siRNA groups to the model group and the TGF-b1 siRNA negative control group showed significantly increased levels of mRNA and protein expression of Smad 7 (P less than 0.01). In addition, the expression levels of Smad 7 were significantly higher in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05).</p><p><b>CONCLUSION</b>siRNA-mediated silencing of TGF-b1 in rats led to significantly reduced expression of Smad 2, 3 and 4, but significantly increased expression of Smad 7. TGF-b1 regulation of Smad signaling molecules may contribute to hepatic fibrosis in rats and represent a target of future therapeutic intervention.</p>

Construction and identification of siRNA eukaryotic expression vectors targeting on TGFβ1, TIMP-1 and TIMP-2 genes in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct the siRNA eukaryotic expression vectors targeting on TGFβ1, TIMP-1 and TIMP-2 and to investigate the inhibitory efficiency of target genes expression on rat hepatic stellate cell in vitro.</p><p><b>METHODS</b>The siRNA cDNA sequences of TGFβ1, TIMP-1 and TIMP-2 were designed, synthesized and inserted into plasmid pGenesil-1 respectively to generate eukaryotic expression plasmids. The plasmids were transfected into HSC T6 cells in vitro and the inhibitory efficiency of target genes expression was observed with real-time PCR and Western blot.</p><p><b>RESULTS</b>The eukaryotic expression vectors were constructed successfully. The expressions of TGFβ1 mRNA, TIMP-1 mRNA and TIMP-2mRNA in siRNA-transfected groups were decreased by 63.4% ± 8.0%, 64.5% ± 9.0% and 55.0% ± 17.0% respectively and the expressions of TGFβ1 protein, TIMP-1 protein and TIMP-2 protein were decreased by 57.8% ± 3.0%, 55.1% ± 5.0%, 49.3% ± 1.0% respectively as compared to the control groups.</p><p><b>CONCLUSIONS</b>The siRNA eukaryotic expression vectors constructed targeting on TGFβ1, TIMP-1 and TIMP-2 could reduce the expressions of target genes and they might be able to used for the exploration of new anti-fibrosis drugs genetically.</p>