肝豆扶木汤对Wilson病小鼠肝纤维化和上皮-间质转化的干预作用  

Interventional effect of Gandou Fumu Decoction on liver fibrosis and epithelial-mesenchymal transition in Wilson's disease mice

作　　者：曾靖喻 唐露露 陈洁 张宇 杨文明[2] 张静[2] ZENG Jingyu;TANG Lulu;CHEN Jie;ZHANG Yu;YANG Wenming;ZHANG Jing(First Clinical Medical College,Anhui University of Chinese Medicine,Hefei,Anhui 230038;Department of Neurology,The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei,Anhui 230031)

机构地区：[1]安徽中医药大学第一临床医学院,安徽合肥230012 [2]安徽中医药大学第一附属医院神经内科,安徽合肥230031

出　　处：《河北中医》2024年第10期1654-1660,共7页Hebei Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金青年科学基金项目(编号:82104783、82205076);安徽省卫生健康委员会“江淮名医培育工程”(编号:ahsjhmypygc20230076)。

摘　　要：目的观察肝豆扶木汤(GDFMD)对Wilson病(WD)模型小鼠肝纤维化及上皮-间质转化(EMT)的影响。方法挑选30只tx-j小鼠,6只小鼠为一组,随机分为模型组、GDFMD低剂量组、GDFMD中剂量组、GDFMD高剂量组、青霉胺组,对照组由相同遗传背景下6只野生型小鼠组成。采用酶联免疫吸附试验(ELISA)检测小鼠血清透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原蛋白(PC-Ⅲ)、Ⅳ型胶原蛋白(C-Ⅳ)、羟脯氨酸(Hyp)。采用苏木素-伊红(HE)、马松(Masson)染色观察小鼠肝脏病理形态情况及肝组织损害程度。采用免疫组织化学法检测转化生长因子-β1(TGF-β1)、E-上皮钙黏附素(E-cadherin)蛋白表达水平。采用实时荧光定量聚合酶链反应(PCR)检测肝组织TGF-β1、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)、E-cadherin、基质金属蛋白酶-9(MMP-9)mRNA表达水平。结果与对照组比较,模型组血清LN、HA、PC-Ⅲ、C-Ⅳ、Hyp含量均显著升高(P<0.05),与模型组相比,GDFMD各组血清LN、HA、PC-Ⅲ、C-Ⅳ、Hyp含量明显下降(P<0.05)。肝脏组织病理形态学结果显示,与对照组比较,模型组肝损害明显,表现为肝细胞肿胀、脂肪变性、坏死,炎性细胞浸润,胶原纤维沉积增多等。GDFMD治疗后肝脏损害均有不同程度的改善。免疫组化检测结果:与对照组比较,模型组TGF-β1蛋白表达水平明显升高(P<0.05),E-cadherin蛋白表达水平明显下降(P<0.05);用药干预后,发现GDFMD可下调TGF-β1蛋白表达水平(P<0.05),上调E-cadherin蛋白表达水平(P<0.05)。实时荧光定量PCR检测结果:与对照组比较,模型组TGF-β1、α-SMA、Vimentin mRNA表达水平均呈升高(P<0.05),E-cadherin、MMP-9 mRNA表达水平呈下降(P<0.05),GDFMD组可下调TGF-β1、α-SMA、Vimentin mRNA表达水平(P<0.05),上调E-cadherin、MMP-9 mRNA表达水平(P<0.05)。结论GDFMD能够改善WD肝纤维化,其作用机制可能与调节肝脏EMT有关。Objective To explore the effects of Gandou Fumu Decoction(GDFMD)on hepatic fibrosis and epithelial-mesenchymal transition(EMT)in the mouse model of Wilson's disease(WD).Methods The model group,GDFMD low,medium and high dose groups,penicillamine group consisted of randomized 30 tx-j mice,with 6 per group.In additiona,six wild mice with same genetic background for the control group.Hyaluronicacid(HA),laminin(LN),typeⅢprocollagen(PC-Ⅲ),collagenⅣ(C-Ⅳ),hydroxyproline(Hyp)were detected by enzyme linked immunosorbent assay(ELISA).The hematoxylin-eosin(HE)staining and Masson staining were used to observe the pathology of liver and the degree of liver tissue damage in mice.The transforming growthfactor-β1(TGF-β1),E-cadherin proteins expressions were measured by immunohistochemistry.TGF-β1,alpha-smooth muscle actin(α-SMA),vimentin,E-cadherin and matrix metallproteinase-9(MMP-9)mRNA expressions in liver tissue were detected using real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Results Compared to the normal group,the model group had significantly higher LN,HA,PC-Ⅲ,C-Ⅳ,Hyp levels(P<0.05),Compared to the model group,GDFMD group presented significantly lower LN,HA,PC-Ⅲ,C-Ⅳ,Hyp levels(P<0.05).In pathomorphology results,compared to the control group,the model group presented significant liver damage with liver cell swelling,fatty degeneration and necrosis,inflammatory cell infiltration,increased collagen fiber deposition,while GDFMD groups had improved liver damage at varying degrees.In immunohistochemical results,compared with the control group,the model group had significantly increased TGF-β1 protein expression,and decreased E-cadherin protein expression(P<0.05).GDFMD groups presented significantly downregulated TGF-β1 protein expression,and upregulated E-cadherin protein expression levels after drug intervention(P<0.05).In qRT-PCR results,compared with the control group,the model group had significantly increased the expression levels of TGF-β1,α-SMA and vimentin mRNA,and decrea

关 键 词：肝豆扶木汤 WILSON病 上皮-间质转化 肝纤维化 

分 类 号：R241[医药卫生—中医诊断学]