转化生长因子β1的小干扰RNA对肝纤维化大鼠Smad蛋白表达的影响

Effect of TGF-β1 siRNA-mediated silencing on Smad proteins in hepatic fibrosis rats

目的研究转化生长因子（TGF）β1的小干扰RNA（siRNA）对CCl4诱导肝纤维化大鼠TGFβ1／Smad信号通路的影响。方法以前期研究的TGFβ siRNA治疗前后肝纤维化大鼠肝脏组织作为研究对象，分为正常组，模型组、TGFβ1 siRNA0．125mg／kg组、TGFβ1 siRNA0．25mg／kg组和阴性对照组（0．25mg／kg非特异性靶序列）。用免疫组织化学和Western blot检测Smad2、3、4、7的蛋白表达；Real-time PCR法检测Smad2、3、4、7的mRNA表达。多组间数据比较用方差分析，两两比较采用q检验。结果免疫组织化学结果显示TGF-β siRNA0．25mg／kg组及0．125mg／kg组Smad2／3、4的蛋白表达量较模型组、阴性对照组明显减少（F值分别为115．538，117．178，P〈0．01），TGFβ1 siRNA 25mg／kg组表达量较0．125mg／kg组少（P〈0．05）。TGFβ1 siNA0．25mg／kg组及0．125mg／kg组Smad7的蛋白表达量较模型组、阴性对照组增加（F=125．34，P〈0．01），TGFβ1 siRNA0．25mg／kg组表达量较0．125mg／kg组多（P〈0．05）。Western blot结果与之相似。Real-time PCR结果显示，与模型组和阴性对照组比较，TGFβ1 siRNA可以明显抑制Smad2、3、4基因的表达，且TGFβ1 siRNA0．25mg／kg组Smad2、3、4的mRNA表达量（相对表达量分别为2．870±0．705、2．904±0．414、2．667±0．466）较TGFβ1积NA0．125mg／kg组（相对表达量分别为5．998±0．329、5．827±0．781、5．102±0．102）明显减少（P〈0．05）。TGFβ1 siRNA0．25mg／kg组及0．125rng／kg组Smad7的mRNA表达量较模型组、阴性对照组增加（F=29．615，P〈0．01），TGFβ1 siRNA0．25mg／kg组表达量较0．125mg／kg组多（P〈0．05）。结论TGFβ1 siRNA干扰大鼠TGFβ1表达后，Smad2、3、4表达量明显降低，而Smad7表达仍维持在较高水平，这利于肝纤维化的改善。

Objective To investigate the changes in Smad 2, 3, 4 and 7 of the transforming growth factor-beta 1 （TGF-β1）/Smad signaling pathways in carbon tetrachloride （CCL4）-induced hepatic fibrosis rats treated with TGF-β1 small interferring （si）RNA. Methods Rats were randomly divided among five groups： non-fibrotic （normal）; fibrosis-induced （model）; fibrotic treated with 0.125 mg/kg TGF-β1 siRNA; fibrotic treated with 0.250 mg/kg TGF-β1 siRNA; and fibrotic treated with negative control TGF-β1 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）. Results 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-β1 siRNA than in the fibrotic model or the negative control TGF-β1 siRNA rats （P 〈 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-β1 siRNA group than in the 0.125 mg/kg group （P 〈 0.05）. Comparing the 0.250 mg/kg and 0.125 mg/kg TGF-β1 siRNA groups to the model group and the TGF-β1 siRNA negative control group showed significantly increased levels of mRNA and protein expression of Smad 7 （P 〈 0.01）. In addition, the expression levels of Smad 7 were significantly higher in the 0.250 mg/kg TGF-β1 siRNA group than in the 0.125 mg/kg group （P 〈 0.05）. Conclusions siRNA-mediated silencing of TGF-β1 in rats led to significantly reduced expression of Smad 2, 3 and 4, but significantly increased expression of Smad 7. TGF-β1 regulation of Smad signaling molecules may contribute to hepatic fibrosis in rats and represent a target of future therapeutic intervention.