基于网络药理学与实验验证的丹葛解酲汤干预酒精性肝病机制研究

Study on the mechanism of Dange Jiecheng Decoction on alcoholic liver disease based on network pharmacology and experimental verification

目的:基于网络药理学探讨丹葛解酲汤治疗酒精性肝病(ALD)的可能作用机制,并进行实验验证。方法:采用中药系统药理学分析平台(TCMSP)筛选出丹葛解酲汤的有效成分与靶点。利用GeneCards、OMIM、PharmGkb、TTD、DrugBank五个数据库检索ALD的相关基因。取药物靶点与疾病基因交集,筛选丹葛解酲汤治疗ALD的潜在治疗靶点。利用Cytoscape 3.8.0软件构建“成分-靶点-疾病”网络图,用R软件4.1.1进行GO和KEGG富集分析。通过动物实验对核心治疗靶点进行验证。采用白酒灌胃法建立ALD大鼠模型,经丹葛解酲汤干预4周后,HE染色观察大鼠肝脏组织形态学变化,蛋白印迹法(Western Blot)检测各组大鼠肝脏组织中AKT、p-AKT1、PI3K、p-PI3K、HIF1A蛋白表达量。结果:预测丹葛解酲汤靶点276个,ALD相关基因2682个,丹葛解酲汤治疗ALD的靶点170个,筛选得到CCND1、FOS、AKT1、CREB1、TP53、MAPK3、MAPK14、JUN、MYC、BELA、NFKBIA为核心蛋白靶点。GO功能富集主要涉及氧化应激反应、活性氧代谢过程、丝氨酸/苏氨酸蛋白激酶复合物等。KEGG通路主要富集在AGE-RAGE信号通路、PI3K-Akt信号通路、IL-17信号通路、TNF信号通路等。HE染色结果显示ALD模型组大鼠部分肝细胞出现脂肪变性,排列紊乱,出现肝细胞坏死,有炎性细胞浸润并伴有大量的脂滴空泡;与ALD模型组相比,各治疗组大鼠肝组织病变均有不同程度的改善。与空白对照组相比,ALD模型组大鼠肝组织p-AKT1、p-PI3K、HIF1A蛋白表达水平显著降低(P<0.01);与ALD模型组相比,各治疗组大鼠肝组织p-AKT1、p-PI3K、HIF1A蛋白表达水平显著升高(P<0.05,P<0.01)。结论:丹葛解酲汤通过多成分、多靶点、多信号通路治疗ALD,该方可能通过上调p-AKT1、p-P13K、HIF1A的表达,进而起到治疗ALD的作用。

Objective:To explore the possible mechanism of Dange Jie male decoction in the treatment of alcoholic liver disease(ALD)based on network pharmacology,and to verify it experimentally.Methods:The active components and targets of Dange Jiecheng Decoction were screened by Traditional Chinese Medicine System Pharmacology analysis platform(TCMSP).Five databases including Gene Cards,OMIM,Pharm Gkb,TTD and Drug Bank were used to search ALD related genes.The intersection of drug targets and disease genes was used to screen potential therapeutic targets of Dange Jiecheng decoction for ALD.Cytoscape 3.8.0 software was used to construct the"component-target-disease"network map,and R language was used for GO and KEGG enrichment analysis.The core therapeutic targets were validated by animal experiments.The rat model of ALD was established by gavage of liquor.Liver histological changes were observed by HE staining after 4 weeks of Dange Jiecheng decoction intervention.Protein expressions of AKT,p-AKT1,PI3K,p-PI3K and HIF1A in liver tissues were detected by Western blotting.Results:276 targets of Dange Jiecheng decoction,2682 genes related to ALD,and 170 targets of Dange Jiecheng decoction for treating ALD were predicted.CCND1,FOS,AKT1,CREB1,TP53,MAPK3,MAPK14,JUN,MYC,BELA,NFKBIA were selected as core protein targets.GO functional enrichment mainly involved oxidative stress response,reactive oxygen species metabolism process,serine/threonine protein kinase complex,etc.KEGG pathways were mainly enriched in AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,IL-17 signaling pathway and TNF signaling pathway.HE staining showed that some hepatocytes in the ALD model group had steatosis,disordered arrangement,hepatocyte necrosis,inflammatory cell infiltration,and a large number of lipid droplets and vacuoles.Compared with the ALD model group,the pathological changes of liver tissue in each treatment group were improved to varying degrees.Compared with blank control group,the protein expression levels of p-AKT1,p-PI3K and HIF1A in ALD model group were significantly decreased(P<0.01).Compared with ALD model group,the protein expression levels of p-AKT1,p-PI3K and HIF1A in each treatment group were significantly increased(P<0.05,P<0.01).Conclusion:Dange Jiecheng decoction treats ALD through multi-component,multi-target and multi-signal pathways.It may play a therapeutic role in ALD by up-regulating the expression of p-AKT1,p-PI3K and HIF1A.