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信号通路为显著富集的通路。(2)动物实验表明,GDFMD组能改善肝豆状核变性肝损伤尤其是肝纤维化。与模型组比较,GDFMD组血清HA、LN、PC-Ⅲ、C-Ⅳ、Hyp含量降低(P<0.05,P <0.01),α-SMA、Col-1mRNA及蛋白表达降低(P <0.05,P <0.01);PI3K、AktmRNA及蛋白表达升高(P<0.01,P<0.05);肝组织凋亡细胞数目降低(P<0.05),BaxmRNA和蛋白表达降低(P<0.05),Bcl-2mRNA和蛋白表达升高(P<0.05)。结论通过网络药理学的方法预测GDFMD防治肝纤维化的关键作用靶点及相关通路,其不仅具有抗肝纤维化的作用,尚有抗凋亡、抗炎、调节脂代谢等生物学功能。
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. Quercetin, beta-sitosterol, and kaempferol were the most effective components, while mitogen-activated protein kinase 8(MAPK8), epidermal growth factor receptor(EGFR), and interleukin-6(IL-6) were the most effective targets. Core genes in PPI network were EGFR and MAPK8, etc. GO enrichment analysis showed that it affected gene transcription, nuclear receptor activity, etc. Enrichment analysis of KEGG pathway showed that PI3 K-Akt signaling pathway, apoptosis signaling pathway, and HIF-1 signaling pathway were the most significant pathways.(2) Animal experiments showed that LF, especially liver fibrosis were improved in GDFMD group. Compared with the model group, the serum levels of HA, LN, and PC-Ⅲ decreased significantly(P<0.01), the contents of C-IV and Hyp decreased(P<0.05,P<0.01), mRNA expressions of α-SMA and Col-1 significantly decreased(P<0.05,P<0.01), protein expressions of α-SMA protein and Col-1 decreased(P<0.05, P<0.01), mRNA expressions of PI3K and Akt in liver tissue significantly increased(P<0.01, P<0.05), protein expressions of PI3K and Akt in liver tissue significantly increased(P<0.01, P<0.05), the number of apoptotic liver cells decreased(P<0.05), mRNA and protein expressions of Bax decreased(P<0.05), mRNA and protein expressions of Bcl-2 increased(P<0.05) in the GDFMD group. Conclusions The key targets and related pathways of GDFMD in preventing and treating LF were predicted by the methods of network pharmacology. It not only had anti-liver fibrosis effects, but also had biological functions such as anti-apoptosis, anti-inflammation, and regulating lipid metabolism.
作者
魏涛华
杨文明
唐露露
郝文杰
陈永华
张荣信
董薇
江海林
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)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
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)。
作者简介
通讯作者:杨文明,Tel:0551-62838502,E-mail:yangwm8810@126.com。
