联合应用TGF-β、TGF-βR siRNA对小鼠急性肝损伤TGF-β/Smad信号传导通路相关基因表达的抑制

Combined use of siRNAs specific for TGF-β1,TGF-β1 RI and TGF-β1 RII synergistically inhibits the TGF-β/Smad signaling pathway in rats with acute liver injury

目的:探讨TGF-β1、TGF-β1RsiRNA联合应用对小鼠急性肝损伤TGF-β/Smad信号传导通路的干预作用.方法:清洁级健康大鼠36只分为2组:正常对照组(n=6,注射生理盐水)与实验组(n=30).实验组动物在实验第1天和第5天腹腔注射CCl4花生油溶液6mL/kg,造成急性肝损伤并启动TGF-β/Smad信号传导通路.实验分为TGF-β1shRNA干预组(T);TGF-β1siRNA+TβRⅠshRNA干预组(T+R1);TGF-β1shRNA+TβRⅡsiRNA干预组(T+R2);TGF-β1siRNA+TβRⅠshRNA+TβRⅡshRNA干预组(T+R1+R2)和模型对照组(M).观察各实验组血清ALT、AST、HA;肝组织学RT-PCR和免疫组织化学检测肝组织中α-SMA、COL-1、COL-3、TGF-β1、Smad3、PCNA和TGF-αmRNA及蛋白的表达.结果:经shRNA质粒DNA干预的4个治疗组与模型组比较,均能显著降低血清ALT,AST及HA的水平(ALT:592.80±98.4IU/L,440.80±91.9IU/L,461.61±120.0IU/L,284.00±49.0IU/Lvs949.5±196.1IU/L;AST:686.80±112.3IU/L,591.00±99.87IU/L,607.50±84.8IU/L,398.30±61.9IU/Lvs985.67±274.8IU/L;HA:5682.80±824.14μg/L,2871.26±394.68μg/L,3004.29±354.25μg/L,1982.12±402.71μg/Lvs8444.65±812.15μg/L,P<0.05)的测定值;4个治疗组两两比较,T+R1+R2组疗效明显高于其他3组(P<0.01).与模型组比较.shRNA质粒DNA干预能明显抑制TGF-β1,Smad3,α-SMA,COL-1及COL-3的mRNA与蛋白的表达(均P<0.05).其抑制效果以T+R1+R2组最明显.对蛋白水平表达的作用也有相似趋势.结论:针对TGF-βRⅠ及其Ⅰ型受体(TβRⅠ)、Ⅱ型受体(TβRⅡ)的siRNA联合治疗对小鼠肝损伤中TGF-β/Smad信号传导通路相关的基因表达的抑制具有协同作用.

AIM： To investigate the inhibitory effect of combined use of small interfering RNAs （siRNAs） specific for transforming growth factor beta I （TGF-β1） and transforming growth factor beta 1 receptors （TGF-β1 RI and TGF-β1 RII） on the TGF-β/Smad signaling pathway in rats with acute liver injury. METHODS： Thirty rats were given two intraper- itoneal injections of carbon tetrachloride （CC14; 6 mL/kg） to induce acute liver injury and initiate the TGF-β/Smad signaling pathway. The rats were then divided into five groups： TGF-β1 siR- NA intervention group （T）, TGF-β1 siRNA plus TGF-β1 RI siRNA intervention group （T±R1）, TGF-β1 siRNA plus TGF-β1 RII siRNA interven- tion group （T±R2）, TGF-β1 siRNA plus TGF-β1 RI siRNA plus TGF-β1 RII siRNA intervention group （T±R1±R2） and model control group （M）. Serum samples were taken to determine serum ALT, AST and HA. The pathological changes in the liver were evaluated by HE staining. Quantitative detection of α-SMA, collagen I and III, TGF-β1, Smad3, PCNA and TGF-α mRNAs was performed by RT-PCR. The expression of α-SMA, collagen I and III, TGF-β1, Smad3, PCNA and TGF-α in the liver was detected by immunohistochemistry. RESULTS： After acute liver injury rats were transfected with plasmids harboring specific siR- NA, serum ALT, AST and HA levels compared with that of model control group decreased sig- nificantly （ALT： 592.80 ± 98.4 IU/L, 440.80 ± 91.9 IU/L, 461.61 ± 120.0 IU/L, 284.00± 49.0 IU/L vs 949.5 ± 196.1 IU/L; AST： 686.80 ± 112.3 IU/L, 591.00 ± 99.87 IU/L, 607.50 ± 84.8 IU/L, 398.30 ± 61.9 IU/L vs 985.67 ± 274.8 IU/L and HA： 5682.80 ± 824.14μg/L, 2871.26 ± 394.68μg/L, 3004.29 ± 354.25 μg/L, 1982.12 ± 402.71μg/L vs 8444.65 ± 812.15 μg/L, respectively; all P 〈 0.05）. The best effect was achieved in the T±R±R2 group. Compared with nonspecific siRNA trans- fection, siRNAs specific for TGF-β1, TGF-β1 RI and TGF-β1 RII could significantly inhibit the expression of TGF-β1, Smad3, α-SMA, collagen I and collagen III mRNAs in the liver （all P 〈 0.05）. The best effect was achieved in the T±RI±R2 group. Similar results were observed for protein expression. CONCLUSION： Combined use of siRNAs spe- cific for TGF-β1, TGF-β1 RI and TGF-β1 RII can synergistically inhibit the TGF-~/Smad signal- ing pathway and reduce liver injury in rats with acute liver injury.