Xu Wei, Wang Lu-Wen, Shi Jin-Zhi, Gong Zuo-Jiong
Department of Infectious Diseases, State Key Laboratory of Virology, Renmin Hospital of Wuhan University, Wuhan, China.
Hepatobiliary Pancreat Dis Int. 2009 Jun;8(3):300-8.
Previous studies have shown that transforming growth factor-beta 1 (TGF-beta1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice.
Three short hairpin RNAs targeting different positions of TGF-beta1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta1-m1, pGenesil-TGF-beta1-m2 and pGenesil-TGF-beta1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta1-m1, pGenesil-TGF-beta1-m2 or pGenesil-TGF-beta1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR.
The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta1-m1 was the highest among the treatment groups.
Specific siRNA targeting of TGF-beta1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells.
既往研究表明，转化生长因子-β1（TGF-β1）是肝星状细胞来源的肌成纤维细胞样细胞刺激肝纤维化形成的最有效方式。因此，TGF-β1可能成为治疗肝纤维化的靶点。本研究旨在探讨特异性TGF-β1小干扰RNA（siRNA）对刀豆蛋白A（Con A）诱导的小鼠免疫性肝纤维化的抑制作用。
设计针对TGF-β1不同位置的3条短发夹RNA，并克隆至质粒pGenesil-1，获得3种重组表达载体（pGenesil-TGF-β1-m1、pGenesil-TGF-β1-m2和pGenesil-TGF-β1-m3）。30只雄性昆明小鼠随机分为6组：正常组、模型组、对照组和3个治疗组。通过尾静脉注射Con A，8 mg/kg，每周1次，共6周，构建免疫性肝纤维化模型。在第2、4和6周，3个治疗组在注射Con A后24小时内，通过尾静脉采用基于流体动力学的转染方法分别注射pGenesil-TGF-β1-m1、pGenesil-TGF-β1-m2或pGenesil-TGF-β1-m3，注射量为0.8 ml/10 g。对照组小鼠注射相同剂量的对照质粒pGenesil-HK。所有小鼠在第7周处死。采用生化方法检测肝组织中羟脯氨酸水平。通过Van Gieson染色评估肝脏组织病理学。采用免疫组化法检测肝组织中TGF-β1、Smad3和Smad7的表达水平及定位。采用半定量RT-PCR法评估肝脏中TGF-β1、Smad3、Smad7和α-平滑肌肌动蛋白（α-SMA）mRNA的表达。
治疗组肝组织中羟脯氨酸水平低于模型组（P<0.01）。组织病理学检测显示，与模型组相比，治疗组肝纤维化明显改善。治疗组肝组织中TGF-β1和Smad3的表达水平也明显低于模型组（P<0.01），而治疗组Smad7水平高于模型组（P<0.01）。RT-PCR进一步显示，与模型组相比，治疗组TGF-β1、Smad3和α-SMA mRNA的表达明显受到抑制，而Smad7水平升高。3个治疗组之间或对照组与模型组之间上述参数无差异（P>0.05），但pGenesil-TGF-β1-m1在治疗组中的抑制作用最强。
靶向TGF-β1的特异性siRNA显著抑制Con A诱导的小鼠免疫性肝纤维化的纤维化形成。siRNAs的抗纤维化机制可能与下调肝组织中TGF-β1、Smad3和α-SMA的表达以及上调Smad7的表达有关，从而抑制肝星状细胞的激活。
