基于TGR5及肠黏膜屏障功能探讨“疏肝健脾”疗法对肝损伤大鼠的影响及作用机制  

Effect and Mechanism of "Liver-Soothing and Spleen-Invigorating" Therapies Against Liver Injury in Rats:An Exploration Based on TGR5 and Intestinal Mucosal Barrier Function

作　　者：魏伟莉 高艺宁 史兴华 郑智礼 康倩君 邓秀兰 WEI Wei-li;GAO Yi-ning;SHI Xing-hua;ZHENG Zhi-li;KANG Qian-jun;DENG Xiu-lan(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学中医学院,北京100029

出　　处：《中国实验方剂学杂志》2022年第1期131-140,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81573963);北京中医药大学校级科研纵向发展基金项目(2180072120082)。

摘　　要：目的:探讨"疏肝、健脾、疏肝健脾、先疏肝再疏肝健脾、先健脾再疏肝健脾"不同治疗方法对肝郁叠加肝损伤大鼠的影响及其作用机制。方法:制造6周肝郁叠加肝损伤大鼠模型,采用束缚叠加四氯化碳(CCl4)皮下注射法造模(5.89 g·kg^(-1),1次/3 d),同时灌胃给药。48只清洁级雄性SD大鼠用随机数字表法分为8组:正常组,模型组,双环醇组(0.2 g·kg^(-1)),四逆散组(4.32 g·kg^(-1)),六君子汤组(9.26 g·kg^(-1)),柴芍六君子汤甲组(柴甲组;疏肝健脾,13.57 g·kg^(-1)),柴芍六君子汤乙组(柴乙组;先疏肝,再疏肝健脾,13.57 g·kg^(-1)),柴芍六君子汤丙组(柴丙组;先健脾,再疏肝健脾,13.57 g·kg^(-1)),每组6只。光镜、电镜下观察各组大鼠肝、结肠组织切片;全自动生化分析仪检测血清肝功能生化指标;逆转录聚合酶链式反应(RT-PCR)检测肝脏、结肠组织G蛋白偶联受体5(TGR5),肠黏膜闭锁连接蛋白-1(ZO-1),闭合蛋白(Occludin),紧密连接蛋白-1(Claudin-1)mRNA相对表达量;免疫组化法检测结肠增殖细胞核抗原(PCNA)阳性表达率。结果:与正常组比较,模型组大鼠血清碱性磷酸酶(ALP),丙氨酸氨基转移酶(ALT),天冬氨酸氨基转移酶(AST),总胆红素(TBIL),直接胆红素(DBIL)水平显著升高(P<0.01),肝脏组织TGR5 mRNA表达显著降低,结肠组织TGR5 mRNA表达明显升高(P<0.05,P<0.01),结肠ZO-1,Occludin,Claudin-1 mRNA及PCNA表达显著下降(P<0.01);与模型组比较,双环醇、柴丙组血清ALP,ALT,AST,TBIL,DBIL水平明显降低(P<0.05,P<0.01),六君子汤组、柴甲、柴乙、柴丙组肝脏组织TGR5 mRNA肝脏表达显著升高,结肠组织TGR5 mRNA的表达显著降低(P<0.01),双环醇组、柴甲、柴乙、柴丙组结肠ZO-1,Claudin-1 mRNA表达明显升高(P<0.05,P<0.01),双环醇组、四逆散组、柴丙组PCNA表达显著升高(P<0.01);与柴丙组比较,双环醇组、四逆散组肝脏TGR5,结肠ZO-1 mRNA表达显著降低且结肠TGR5mRNA表达显著升高(P<0.01),六�Objective: To explore the effects of different treatment methods of "soothing liver,invigorating spleen,soothing liver and invigorating spleen,soothing liver first and then soothing liver and invigorating spleen,as well as invigorating spleen first and then soothing liver and invigorating spleen" on liver depression combined with liver injury in rats and their action mechanisms. Method: A six-week rat model of liver depression combined with liver injury was established by restraint stress and subcutaneous injection of carbon tetrachloride(CCl4,5.89 g·kg^(-1),once every three days). At the same time,the drugs were given by gavage. Forty-eight male SD rats of clean grade were randomly divided into eight groups,namely the normal group,model group,bicyclol(0.2 g·kg^(-1))group,Sinisan(4.32 g·kg^(-1))group,Liu Junzitang(9.26 g·kg^(-1))group, Chaishao Liu Junzitang A(Chai A, soothing liver and invigorating spleen,13.57 g·kg^(-1))group,Chaishao Liu Junzitang B(Chai B, soothing liver first and then soothing liver and invigorating spleen,13.57 g·kg^(-1))group,and Chaishao Liu Junzitang C(Chai C,invigorating spleen first and then soothing liver and invigorating spleen,13.57 g·kg^(-1))group,with six rats in each group. The pathological changes in liver and colon tissues of each group were observed under light microscope and electron microscope. The serum biochemical indexes of the liver were detected using an automatic biochemical analyzer. The relative mRNA expression levels of Takeda G protein-coupled receptor 5(TGR5)and intestinal mucosal zona occluden-1(ZO-1),Occludin,and Claudin-1 in the liver and colon were detected by reverse-transcription polymerase chain reaction(RT-PCR). The positive expression rate of proliferating cell nuclear antigen(PCNA)in the colon was detected by immunohistochemistry. Result: Compared with normal group, the model group exhibited significantly elevated serum alkaline phosphatase(ALP), alanine aminotransferase(ALT), aspartate aminotransferase(AST),total bilirubin(TBIL),and direct biliru

关 键 词：肝病传脾 疏肝健脾 G蛋白偶联受体5(TGR5) 闭锁连接蛋白-1(ZO-1) 闭合蛋白(Occludin) 紧密连接蛋白-1(Claudin-1) 结肠增殖细胞核抗原(PCNA) 

分 类 号：R2-0[医药卫生—中医学] R33