基于网络药理学及细胞实验验证软肝饮治疗肝癌的可能

Verification of Possibility of Ruangan Yin(软肝饮)Treating Liver Cancer Based on Network Pharmacology and Cell Experiments

目的基于网络药理学及细胞实验研究软肝饮治疗肝细胞癌(HCC)的作用机制。方法基于中药系统药理学数据库与分析平台(TCMSP)、BATMAN数据库及Drugbank数据库筛选出软肝饮有效成分及靶点基因。通过GeneCards、OMIM数据库及Liverome、OncoDB.HCC数据库筛选出HCC的疾病靶点基因,运用Cytoscape构建化合物-基因靶点网络;并将有效成分靶目与疾病靶点上传到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GO富集分析和KEGG富集分析。基于网络分析结果,使用体外细胞实验方法对药物治疗关键靶点作用进行验证。结果(1)预测得到软肝饮治疗肝癌的有效成分59个及共作靶点1301个。有效成分中度值较高的为新隐丹参酮Ⅱ、脱氧新胰蛋白酶酮、次甲丹参醌、反式刚酸、槲皮素、茯苓甾醇、β谷甾醇等;共作靶点及PPI网络图核心基因为AKT1、TP53、PIK3CA、EGFR、JUN等;GO分析有血管收缩舒张调节、分泌颗粒腔、磷脂酶C活性等;KEGG通路分析有氨基酸代谢、神经活性配体-受体相互作用、MAPK信号通路、癌症关连通路等显著通路。(2)细胞实验表明,软肝饮含药血清处理肝癌Hu-7、HepG2细胞后,细胞增殖、迁移能力显著降低;Western Blot检测肝癌细胞经软肝饮含药血清处理后,细胞中Cleaved Caspase-3、Cleaved PARP凋亡相关蛋白表达相较肝癌细胞组和正常血清对照组升高;p-c-Raf、p-c-MEK、p-c-Myc、p-c-Fos蛋白表达相较肝癌细胞组和正常血清对照组下降。结论(1)生信学提示软肝饮和肝癌具有丰富的关联靶点,并通过多通路调控肝癌细胞的发生过程;(2)软肝饮可能借升高Cleaved Caspase-3、Cleaved PARP凋亡蛋白表达,下调p-c-Raf、p-c-MEK、p-c-Myc、p-c-Fos肿瘤相关基因表达,从而促使肝癌Hu-7、HepG2细胞株凋亡,抑遏肝癌Hu-7、HepG2细胞株的增殖迁移侵袭能力,进而起到治疗肝癌的作用。

Objective Based on network pharmacology and cell experiment,the possibility of Ruangan Yin(软肝饮,RGY)in the treatment of hepatocellular carcinoma was studied.Methods The active ingredients and target genes of RGY were screened based on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),BATMAN database and Drugbank database.Through GeneCards,OMIM database and Liverome,OncoDB.HCC database,the disease target genes of hepatocellular carcinoma(HCC)were screened and Cytoscape was used to construct a compound gene target network.And the active ingredient targets and disease targets were uploaded to string database to construct protein interaction network maps(PPI),and the screening of core genes was performed using R language and GO enrichment analysis and KEGG enrichment analysis of key targets were performed.Based on the network analysis results,an in vitro cell experimental approach was used to validate the drug treatment key target effects.Results(1)It is predicted that there were 59 effective components and 1301 targets of RGY in the treatment of liver cancer.The moderately high values of active components wereneocryptotanshinone Ⅱ,deoxyneotrypsinone,demethyltanshinone,trans congo acid,quercetin,pachymesterolβsitosterol,etc.The core genes were AKT1,TP53,PIK3CA,EGFR,JUN,etc.GO analysis included vasoconstriction and relaxation regulation,secretory granule cavity,phospholipase C activity,etc.KEGG pathway analysis included amino acid metabolism,neuroactive ligand receptor interaction,MAPK signaling pathway,cancer related pathway and other significant pathways.(2)Cell experiment showed that the proliferation and migration of hepatoma Hu-7 and HepG2 cells treated with serum containing RGY decreased significantly.Western Blot assay showed that the expressions of cleaved caspase-3 and cleaved PARP apoptosis related proteins in hepatoma cells treated with serum containing RGY were higher than those in hepatoma cell group and normal serum control group.The protein expressions of p-c-RAF,p-c-MEK,p-c-MYC and p-c-FOS decreased compared with those of the hepatoma cell group and normal serum control group.Conclusion(1)Bioinformatics suggests that RGY has rich related targets with liver cancer,and regulates the carcinogenesis of liver cancer cells through multiple pathways.(2)RGY may increase the expressions of cleaved caspase-3 and cleaved PARP apoptotic proteins and regulate the expressions of p-c-RAF,p-c-MEK,p-c-MYC and p-c-FOS tumor related genes,so as to promote the apoptosis of liver cancer Hu-7 and HepG2 cell lines,inhibit the proliferation,migration and invasion of liver cancer Hu-7 and HepG2 cell lines,and then plays a role in the treatment of liver cancer.