靶向重组信号结合蛋白-Jκ干扰腺病毒对四氯化碳诱导大鼠肝纤维化影响的体内研究

Effects of adenovirus-targeting recombination signal binding protein for immunoglobulin-Jκ on liver fibrosis of rats induced by carbon tetrachloride in vivo

目的通过体内实验探讨重组信号结合蛋白-Jκ(recombination signal binding protein for immunoglobulin kappa J region,RBPJκ)在四氯化碳(CCl4)诱导的大鼠肝纤维化发生发展中的作用。方法选取雄性SD大鼠40只为研究对象,以40%CCl4腹腔注射法制备SD大鼠肝纤维化模型。将大鼠随机分为正常对照组、肝纤维化模型组、腺病毒空载体组(治疗对照组)和RBPJκ干扰腺病毒(Adsh RBPJκ)干预组(治疗组),每组各10只。采用苏木精-伊红(HE)染色、Masson染色和天狼猩红染色观察细胞外基质(extracellular matrix,ECM)含量;采用碱水解法测定大鼠肝组织羟脯氨酸含量;采用q PCR、Western blot和免疫组织化学法检测大鼠肝组织α-SMA、Ⅰ型胶原、TGF-β1、RBPJκ及其靶基因Hey2、Hey L和上皮间质转化(epithelial-mesenchymal transition,EMT)指标[蜗牛家族转录抑制因子1(snail family transcriptional repressor 1,Snail 1)、波形蛋白和E-钙连接素]的表达。结果 (1)与正常对照组相比,HE染色、Masson染色和天狼猩红染色示模型组大鼠肝细胞明显肿胀、变性,肝小叶结构被破坏,纤维组织增生明显;模型组大鼠肝内羟脯氨酸含量显著高于正常对照组[(99.35±8.12)μg/mg vs(285.12±12.32)μg/mg,t=38.193,P<0.001)]。(2)RTPCR、Western blot及免疫组织化学结果显示,模型组大鼠RBPJκ靶基因Hey2、Hey L、胶原相关基因(α-SMA、Ⅰ型胶原、TGF-β1)以及EMT相关基因Snail 1和波形蛋白转录及翻译水平的表达均显著增加(P均<0.05),但E-钙连接素表达水平降低(P<0.05)。(3)与治疗对照组相比,Adsh RBPJκ(治疗组)可改善大鼠肝纤维化程度,降低羟脯氨酸含量[(193.52±13.12)μg/mg vs(279.85±8.32)μg/mg,t=15.92,P<0.001)]。(4)RT-PCR、Western blot及免疫组织化学结果显示,与治疗对照组相比,抑制肝RBPJκ表达(治疗组)不仅可降低RBPJκ及其靶基因Hey2、Hey L的表达水平,也可降低纤维化指标(α-SMA、Ⅰ型胶原、TGF-β1)及EMT相关基因(Snail 1、波形蛋白)的表达水平,同时还可上调E-钙连接素的表达(P<0.05)。结论高表达的RBPJκ可能参与了肝纤维化的形成过程,抑制RBPJκ可能为肝纤维化的防治提供新方向。

Objective To investigate the role of recombination signal binding protein-Jκ(RBPJκ)on the progression of liver fibrosis of rats induced by carbon tetrachloride(CCl4)in vivo.Methods Total of 40 male SD rats were selected and the models of experimental liver fibrosis were established by intraperitoneal injection of 40%CCl4.The rats were randomly divided into four groups(10 rats in each group):normal control group,liver fibrosis model group,adenoviral mock-vehicler group(treatment control group)and adenoviral RBPJκ-shRNA vehicler(AdshRBPJκ)intervention group(treatment group).HE staining,Masson’s trichrome staining and sirius red staining were carried out to detect the content of extracellular matrix.The liver samples were subjected to hydrolysis to determine the amount of hydroxyproline.qPCR,Western blot and immunohistochemistry were carried out to detect the expression of RBPJκand the target gene-Hey 2 and Hey L,the fibrosis indexes(α-SMA,collagen I,TGF-β1)and the epithelial-to-mesenchymal transition(EMT)indexes(Snail 1,vimentin,E-cadherin).Results①HE staining,Masson’s trichrome staining and sirius red staining showed that the degree of fibrosis and liver cell degeneration or necrosis increased,while the hydroxyproline content decreased[(99.35±8.12)μg/mg vs(285.12±12.32)μg/mg;t=38.193,P<0.001)]in the liver fibrosis model group compared with the normal control group.②qPCR,Western blot and immunohistochemistry showed that the expression of RBPJκ,Hey2,HeyL,the fibrosis related genes(α-SMA,collagenⅠ,TGF-β1)and EMT related genes(Snail 1,Vimentin)increased,while the expression of E-cadherin decreased in the liver fibrosis model group(P<0.05).③Compared with adenoviral mock-vehicler group,AdshRBPJκintervention(treatment group)could reduce the degree of liver fibrosis and the content of hydroxyproline[(193.52±13.12)μg/mg vs(279.85±8.32)μg/mg,t=15.92,P<0.001)].④RT-PCR,Western blot and immunohistochemistry showed that the inhibition of RBPJκexpression in liver could increase the expression of RBPJκ,Hey2,HeyL,the fibrosis related genes(α-SMA,collagenⅠ,TGF-β1)and EMT related genes(Snail 1,Vimentin)increased and the expression of E-cadherin decreased(P<0.05)compared with adenoviral mock-vehicler group.Conclusions The up-regelatian of RBPJκexpression in liver may participate in the progession of liver fibrosis induced by carbon tetrachloride.The interruption of RBPJκmight be a novel strategy in liver fibrosis.