罗汉果山萘酚抑制HepG2细胞增殖潜在分子机制的探析

Study on the Potential Molecular Mechanism of Kaempferol from Siraitia Grosvenorii in Inhibiting HepG2 Cell Proliferation

利用网络药理学–分子对接结合细胞实验探讨罗汉果抑制肝细胞癌(hepatocellular carcinoma,HCC)的潜在分子机制。应用中药系统药理学数据库(TCMSP)筛选罗汉果活性成分与靶点,Genecards数据库检索肝细胞癌相关靶点,二者靶点经Venny平台取交集,得到罗汉果治疗肝细胞癌的潜在靶点。以潜在作用靶点为基础,利用DAVID数据库进行基因功能和通路(GO/KEGG)富集分析后,采用Cytoscape软件构建罗汉果治疗肝细胞癌的“药物–成分–疾病–靶点”和“药物成分–靶点–通路–疾病”网络图,经生物分子功能注释系统数据库和Cytoscape软件获得核心作用靶点。选择药物活性成分(山萘酚)和关键靶点,运用AutoDock Vina软件进行分子对接。以山萘酚为实验药物、HepG2为研究对象,采用CCK-8法筛选其抑制HepG2细胞增殖的有效浓度,流式细胞术检测细胞凋亡率,Western blot检测潜在作用靶点的蛋白表达水平。筛选出罗汉果有效成分11个、作用靶点77个,肝细胞癌的疾病靶点7773个,二者共有靶点62个;GO与KEGG功能富集分析得到275个生物功能单位和87条信号通路;筛选出罗汉果抑制肝癌细胞增殖的潜在核心靶点p65、JNK1、Caspase-3、AKT1等10个;20μg/mL以上的罗汉果山萘酚能显著降低HepG2细胞的增殖活性和提高细胞凋亡率,显著上调p65、Caspase-3表达水平和下调JNK1、AKT1表达水平。罗汉果山萘酚具有抑制肝癌细胞增殖和促进其凋亡的活性,然而罗汉果具有多成分、多靶点、多通路的功效和作用特点,提示罗汉果治疗肝细胞癌的机制较为复杂。

Exploring the potential molecular mechanism of Siraitia grosvenorii in inhibiting HCC(hepatocellular carcinoma)by network pharmacology molecular docking combined with cell experiment.The TCMSP database was used to screen the active components and targets of Siraitia grosvenorii,and the Genecards database was used to search the relevant targets of hepatocellular carcinoma.The potential targets of Siraitia grosvenorii for the treatment of hepatocellular carcinoma were obtained by intersection of these two targets on the Venny platform.DAVID database was used to enrich analysis for gene function and their working pathways(GO/KEGG)of potential action targets,and the“drug component-disease-target”and“drug component-target-pathway-disease”network maps of Momordica grosvenorii for the treatment of hepatocellular carcinoma were established by Cytoscape software.The core targets were obtained through the database of biological molecular function annotation system and the software of Cytoscape.Molecular docking between proposed drug active products(kaempferol)and key targets was performed in AutoDock Vina software.Using naphthol as the experimental drug and HepG2 as the research object,CCK-8 method was used to screen the concentration of kaempferol inhibiting HepG2 cell proliferation;flow cytometry was used to detect the apoptosis rate,and Western blot was used to analyze protein levels of potential targets identified through screening.Totally 11 active components and 77 related targets of Siraitia grosvenorii and 7773 disease targets of hepatocellular carcinoma and 62 common targets were screened.Totally 275 biological functions and 87 signal pathways were obtained by functional enrichment analysis of GO and KEGG.Ten potential core targets of Siraitia grosvenorii for the treatment of hepatocellular carcinoma were screened,such as p65,JNK1,Caspase-3 and AKT1.Kaempferol above 20μg/mL could significantly reduce the proliferation activity of HepG2 cells,increase its apoptosis rate,and significantly up regulate p65,Caspase-3 expression levels and down regulate JNK1 and AKT1 expression levels in HepG2 cells.Kaempferol isolated from Siraitia grosvenorii has the activity in inhibiting the proliferation and promoting apoptosis of liver cancer cells.However,Siraitia grosvenorii has the efficacy and characteristics of multi-component,multi-target,and multi-pathway,which reveals that the mechanism of Siraitia grosvenorii in the treatment of hepatocellular carcinoma is more complex.