基于网络药理学解析临床用中药抗肝纤维化的作用机制和药效物质基础

Mechanism of action and pharmacodynamic material basis of traditional Chinese medicine clinical use for anti-hepatic fibrosis based on network pharmacology analysis

目的基于网络药理学解析临床常用中药抗肝纤维化主要活性成分及其作用机制。方法通过中药系统药理学分析平台TCMSP和Swiss Target Prediction获得21味临床常用中药的有效成分和潜在作用靶点;用GEO数据库解析乙型肝炎病毒(HBV)感染、酒精性肝病和非酒精性脂肪性肝病致肝纤维化差异基因,结合肝纤维化细胞外基质生成和逆转关键基因预测中药治疗靶点;采用Cytoscape软件构建“药物-治疗靶点-通路”网络解析中药抗纤维化成分及其作用机制;体外实验考察核心成分对巨噬细胞(THP-1)和肝星状细胞(LX2)的影响。首先对数据进行正态性检验和方差齐性检验,然后对数据两组间的比较采用t检验,多组间比较行One-Way ANOVA法进行分析。结果21味中药含355个单体化合物,对应315个已知药物靶点,基于肝纤维化发生和消退的关键环节结果显示其中57个基因为中药的抗纤维化治疗靶点;“药物-靶点-通路网络”关联分析结果显示槲皮素和山奈酚是多种复方中药最为核心的抗肝纤维化成分,主要通过调控PI3K-Akt、AGE-RAGE、MAPK、肿瘤坏死因子(TNF)和白细胞介素(IL)-17等信号通路中核心基因TNF、蛋白激酶B1、基质金属蛋白酶9、B淋巴细胞瘤2、趋化因子受体2、半胱氨酸天冬氨酸蛋白酶3、C-X-C模体趋化因子受体8、V-rel网状内皮细胞病毒癌基因同源物A、MYC和信号传导和转录激活蛋白1改善HBV、酒精性肝病和非酒精性脂肪性肝病疾病进程和纤维化进展;细胞实验显示槲皮素较山奈酚具有更强的抑制炎症型THP-1细胞释放促炎因子IL-1β、IL-6和TNF-α的能力,而山奈酚降低趋化因子趋化因子受体2更为明显。槲皮素和山奈酚明显抑制THP-1介导的LX2细胞增殖,伴随α-平滑肌肌动蛋白、胶原蛋白IA、转化生长因子-β显著降低和半胱氨酸天冬氨酸蛋白酶3、Cleaved-半胱氨酸天冬氨酸蛋白酶3的增加,二者具有协同作用。结论槲皮素和山奈酚是构成治疗肝纤维化复方中药的基石,为后续多靶点抗肝纤维化药物的研发提供参考。

ObjectiveTo analyze the main active ingredients and mechanisms of action of clinically commonly used traditional Chinese medicine for anti-hepatic fibrosis based on network pharmacology.MethodsThe traditional Chinese medicine system pharmacology analysis platform(TCMSP)and Swiss Target Prediction were employed to obtain the active ingredients and potential targets of 21 clinically commonly used traditional Chinese medicines.The GEO database was used to analyze the differential genes of liver fibrosis that resulted from hepatitis B virus(HBV)infection,alcoholic liver disease,and non-alcoholic fatty liver disease.The therapeutic targets of traditional Chinese medicine were predicted by combining the key genes for the production and reversal of the extracellular matrix in liver fibrosis.The"drug-therapeutic target pathway"network was constructed using Cytoscape software to analyze the anti-fibrosis ingredients and mechanisms of action of traditional Chinese medicine.The effects of core ingredients were investigated in vitro on macrophages(THP-1)and hepatic stellate cells(LX2).The data were initially examined for normality and homogeneity of variance,and then a t-test was used to compare the data between the two groups.The one-way ANOVA method was used for multi-ingredient comparisons.ResultsThe 21 traditional Chinese medicines contained 355 monomer compounds,which corresponded to 315 recognized drug targets.The results showed that 57 genes were anti-fibrotic therapeutic targets based on the key links between liver fibrosis occurrence and regression.The results of the"drug-target-pathway network"association analysis showed that quercetin and kaempferol were the most core anti-liver fibrosis ingredients of various traditional Chinese medicine compounds,which mainly improved HBV,alcoholic liver disease,and non-alcoholic fatty liver disease and fibrosis progression by regulating and controlling PI3K-Akt,AGE-RAGE,MAPK,TNF,and IL-17 signaling pathways core genes such as TNF,AKT1,MMP9,BCL2,CCL2,CASP3,CXCL8,RELA,MYC,and STAT1.Cellular experiments showed that quercetin had a stronger ability to inhibit the release of proinflammatory factors IL-1β,IL-6,and TNF-αfrom inflammatory THP-1 cells than kaempferol.In contrast,kaempferol had markedly reduced the chemokine CCL2.Quercetin and kaempferol significantly inhibited THP-1-mediated LX2 cell proliferation,which was accompanied by significant decreases inα-SMA,collagen IA,and TGF-β,as well as increases in CASP3 and cleaved-CASP3,indicating both had a synergistic effect.ConclusionQuercetin and kaempferol are the basic forms of traditional Chinese medicine compounds for liver fibrosis treatment,and it serve as a reference for the subsequent research and development of multi-target anti-hepatic fibrosis drugs.