基于网络药理学探讨清肝化瘀颗粒对非酒精性脂肪性肝病和肝癌“异病同治”的作用机制

目的:基于网络药理学研究方法,探讨清肝化瘀颗粒治疗非酒精性脂肪性肝病(NAFLD)和肝癌的有效成分、作用靶点及可能作用机制,并为其对NAFLD和肝癌"异病同治"的合理性阐释提供依据。方法:通过多个中药与疾病数据库,运用网络药理学筛选清肝化瘀颗粒治疗NAFLD和肝癌的主要有效成分与作用靶点。通过STRING 11.0分析作用靶点之间的相互作用,使用cytoHubba插件从作用靶点中筛选出核心作用靶点。使用Metascape数据库对作用靶点进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析。同时,利用体外试验验证清肝化瘀颗粒主要有效成分之一山柰酚对肝癌细胞模型与NAFLD细胞模型的作用。结果:共筛选出清肝化瘀颗粒治疗NAFLD及肝癌的作用靶点43个,对应8味中药中的136种有效成分。富集分析结果显示作用靶点共涉及20种生物过程,13种分子功能,9种细胞组分及15条信号通路。利用构建的"中药-成分-靶点-疾病"网络图发现清肝化瘀颗粒主要通过以山柰酚、槲皮素及木犀草素为代表的主要有效成分对胱天蛋白酶-3(CASP3),肿瘤蛋白p53(TP53),血管内皮生长因子A(VEGFA)等枢纽基因参与细胞凋亡抑制等与肝癌有关的肿瘤生物学行为进行调节,同时对氧化应激等与NAFLD相关的生物学行为进行调节。体外试验发现山柰酚可以剂量依赖性地抑制肝癌细胞的增殖,并对NAFLD细胞模型氧化应激标志物丙二醛(MDA)及谷胱甘肽过氧化物酶(GPx)的浓度进行调节;同时,山柰酚还可对肝癌与NAFLD细胞模型中枢纽基因CASP3蛋白水平的表达量进行调节。结论:清肝化瘀颗粒对NAFLD与肝癌"异病同治"的主要机制涉及以槲皮素、木犀草素及山柰酚为代表的多成分,以VEGFA,TP53及CASP3为代表的多靶点,以及以氧化应激及细胞凋亡为代表的多通路。

An Exploration on Mechanisms of Treating Different Diseases with Same Method of Qinggan Huayu Granules in Treating NAFLD and Liver Cancer Based on Network Pharmacology

Objective: To reveal the effective components,targets and possible mechanisms of Qinggan Huayu granules in the treatment of non-alcoholic fatty liver disease(NAFLD)and liver cancer based on network pharmacology and experimental verification,and to provide a basis for its rational interpretation of treating different diseases with same method for NAFLD and liver cancer. Method: Based on databases of traditional Chinese medicine and disease,the network pharmacology was used to screen main active compounds and potential targets of Qinggan Huayu granules for NAFLD and liver cancer. STRING 11.0 was used to analyze the interaction between potential targets. The core targets were selected from the interaction targets by cyto Hubba plug-in. The gene ontology(GO) function and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on the target by Metascape database. At the same time,in vitro experiments were conducted to validate the effect of kaempferol,one of the main active ingredients of Qinggan Huayu granules,on hepatocellular carcinoma cell model and NAFLD cell model. Result:A total of 43 potential targets of Qinggan Huayu granules for for NAFLD and liver cancer were screened,corresponding to 136 active ingredients in 8 herbal medicines. Through enrichment analysis of potential targets,there were 20 biological processes,13 molecular functions,9 cellular components and 15 signaling pathways. Qinggan Huayu granules regulated biological behaviors of tumors related to liver cancer and NAFLD(such as apoptosis inhibition and oxidative stress)mainly through kaempferol,quercetin,luteolin and other active ingredients for Caspase-3(CASP3),tumor protein p53(TP53),vascular endothelial growth factor A(VEGFA)and other hub genes. In vitro experiments revealed that kaempferol could inhibit cell proliferation in a dose-dependent manner in hepatocellular carcinoma cell model. And kaempferol could modulate the levels of malondialdehyde(MDA)and glutathione peroxidase(GPx),which were the molecular markers of oxidative stress of NAFLD cell model.Kaempfero also regulated the expression level of CASP3 in hepatocellular carcinoma cell model and NAFLD cell model. Conclusion: The main mechanism of Qinggan Huayu granules in treating liver cancer and NAFLD with concept of treating different diseases with same method is related to systematic synergy effect of multiple compounds(represented by quercetin,luteolin and kaempferol),multiple targets(represented by VEGFA,TP53 and CASP3)and multiple signaling pathways(represented by oxidative stress and cell apoptosis).