基于网络药理学探讨肝豆扶木汤治疗肝豆状核变性肝纤维化的作用机制及实验验证  

Mechanism of Gandou Fumu Decoction in Treating Liver Fibrosis Mice with Wilson’s Disease Based on Network Pharmacology and Experimental Proof

作　　者：魏涛华[1,2] 杨文明 唐露露[1] 郝文杰 陈永华 张荣信[1] 董薇[1] 江海林[1] 杨悦 杨玉龙 WEI Tao-hua;YANG Wen-ming;TANG Lu-lu;HAO Wen-jie;CHEN Yong-hua;ZHANG Rong-xin;DONG Wei;JIANG Hai-lin;YANG Yue;YANG Yu-long(Department of Neurology,The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei,230031;Graduate School,Anhui University of Chinese Medicine,Hefei,230038)

机构地区：[1]安徽中医药大学第一附属医院脑病科,合肥230031 [2]安徽中医药大学研究生院,合肥230038

出　　处：《中国中西医结合杂志》2021年第8期981-990,共10页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上资助项目(No.81973825);安徽省自然科学基金面上资助项目(No.1808085MH263);安徽中医药大学新安医学教育部重点实验室开放基金项目资助(No.2020xayx12)。

摘　　要：目的基于网络药理学方法研究肝豆扶木汤(GDFMD)治疗肝豆状核变性肝纤维化(LF)的作用机制。方法基于TCMSP数据库及Uniprot数据库筛选出GDFMD有效成分及靶点基因。通过GeneCards数据库及OMIM数据库筛选出LF的疾病靶点基因,并用Cytoscape软件构建"活性-成分-靶点"相互作用网络图。将有效成分靶标与疾病靶点上传到STRING数据库,构建蛋白互作网络图(PPI),使用R语言进行核心基因的筛选并对关键靶点进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。基于网络分析结果,选择PI3K/Akt信号通路进行动物实验验证。将20只肝豆状核变性LF模型TX小鼠随机分为模型组、GDFMD组,10只相同遗传背景的野生型小鼠作为对照组。GDFMD组按6.96 g生药/kg进行灌胃,模型组和对照组给予等容积生理盐水,每天1次,连续灌胃4周。观察肝脏组织病理学改变。采用ELISA法检测血清肝纤四项[透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原蛋白(PC-Ⅲ)、Ⅳ型胶原蛋白(C-Ⅳ)]、羟脯氨酸(Hyp)含量,采用qRT-PCR法检测肝组织α-平滑肌肌动蛋白(α-SMA)、1型胶原蛋白(Col-1)、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)、Bcl-2相关X蛋白(Bax)和B淋巴细胞瘤-2 (Bcl-2)mRNA的表达,采用Western Blot法检测肝组织α-SMA、Col-1、PI3K、Akt、Bax、Bcl-2蛋白表达。并通过TUNEL法检测肝细胞凋亡水平。结果 (1)预测得到GDFMD治疗肝豆状核变性LF的有效成分55个及共作有效靶点95个。有效成分中度值较高的为槲皮素(quercetin),β-谷甾醇(beta-sitosterol)及山柰酚(kaempferol),作用靶点中度值较高的为丝裂原激活蛋白激酶8(MAPK8),表皮生长因子受体(EGFR)与白介素6(IL-6)。PPI网络中核心基因为MAPK8及EGFR等;GO富集分析显示会影响基因的转录、核受体活性等;KEGG通路富集分析显示磷脂酰肌醇3激酶/蛋白激酶B信号通路、凋亡信号通路、低氧诱导因子-1�Objective To study the therapeutic mechanism of Gandou Fumu Decoction(GDFMD) of liver fibrosis(LF) mice with Wilson’s disease based on network pharmacology. Methods Based on TCMSP database and Uniprot database, the active constituents and target genes of flavored GDFMD were screened. Target genes of LF were screened by GeneCards database and OMIM database, and Cytoscape software was used to construct active-component-target interaction network diagram. The active component targets and disease targets were uploaded to the STRING database, the protein interaction network map(PPI) was constructed, and the characteristic values were calculated, and core genes were screened by using R language. Finally, R language was used to analyze gene ontology(GO) enrichment and Kyoto encyclopedia of genes and genome(KEGG) pathway enrichment of key targets. Based on the results of network pharmacology analyses, 20 toxic milk(TX) mice were randomly divided into model group and GDFMD group, and 10 wild type mice with the same genetic background served as control group. GDFMD at 6.96 g crude drugs/kg body weight was administered to mice in GDFMD group. Equal volume of normal saline was administered to mice in the model group and the control group. The intragastric administration was performed once daily for 4 successive weeks. The effects on four serum liver fibrosis indices [hyaluronic acid(HA), laminin(LN), type Ⅲ procollagen(PC-Ⅲ), type Ⅳ collagen(C-Ⅳ)] were detected using ELISA. Hydroxyproline(HYP) content was also detected.qRT-PCR was used to detect contents of liver tissue α-smooth muscle actin (α-SMA), type 1 collagen(Col-1), phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), and Bcl-2 related X protein(Bax) and Bcl-2 mRNA expressions. Protein expressions of α-SMA, Col-1, PI3 K, Akt, Bax, and Bcl-2 were detected using Western Blot. Apoptotic level of liver cells were detected by TUNEL method. Results(1) The 55 active components and 95 effective targets of GDFMD in the treatment of LF were predicted. Querc

关 键 词：肝豆扶木汤 肝豆状核变性 肝纤维化 网络药理学 PI3K/AKT信号通路 

分 类 号：R285[医药卫生—中药学]