文献类型：期刊文献

中文题名：基于计算机辅助药物设计的慈菇消脂方调控HIF-1α信号通路干预HSC-T6细胞活化的作用及机制研究

英文题名：Molecular mechanism of Cigu Xiaozhi formula interfering with HSC-T6 cell activation by regulating HIF-la signaling pathway based on computer aided drug design

作者：任真[1];尹硕[1];王爱娣[1];王莉[1];赵秀萍[1];马燕花[1]

第一作者：任真

机构：[1]甘肃中医药大学,甘肃兰州730000

第一机构：甘肃中医药大学

年份：2023

卷号：58

期号：10

起止页码：3049

中文期刊名：药学学报

外文期刊名：Acta Pharmaceutica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

基金：国家自然科学基金资助项目(81860821)。

语种：中文

中文关键词：计算机辅助药物设计;慈菇消脂方;缺氧诱导因子-1α;大鼠肝星状细胞;刺猬信号通路;肝纤维化

外文关键词：computer aided drug design;Cigu Xiaozhi formula;hypoxia-inducible factor-la;rat hepatic stellate cell;Hedgehog signaling pathway;hepatic fibrosis

摘要：基于网络药理学及计算机辅助药物设计探讨慈菇消脂方(Cigu Xiaozhi Formula)对缺氧微环境下肝星状细胞(hepatic stellate cells,HSCs)HSC-T6活性的影响,预测并验证其可能作用的靶点及相关信号通路。通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)、中医药百科全书在线数据库(Encyclopaedia of Traditional Chinese Medicine,ETCM)及中药分子机制的生物信息学分析工具(Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine,BATMAN-TCM)数据库检索筛选出慈菇消脂方的潜在活性成分及作用靶点,并借助Gene Cards和Pharm GK数据库检索到的肝纤维化相关靶点进行交集整合,获得慈菇消脂方治疗肝纤维化的潜在作用靶点。在Omic Share平台进行GO(gene ontology)富集分析和KEGG(kyoto encyclopedia of genes and genomes)信号通路富集分析,利用Cytoscape软件构建出“潜在活性成分-关键靶点-通路”网络。利用Auto Dock软件对活性成分和靶点蛋白进行分子对接分析,根据分子动力学模拟和结合自由能计算结果,将degree值排名前5的活性成分进行对接打分,对君药山慈菇活性成分豆甾醇和β-谷甾醇进行分子对接。体外实验应用氯化钴(CoCl_(2))诱导HSC-T6细胞构建缺氧模型,通过CCK-8实验检测细胞活力,确定100μmol·L^(-1)CoCl_(2)作用24 h为构建HSC-T6细胞缺氧模型的最佳作用时间和浓度。细胞划痕愈合实验和免疫荧光检测发现,在缺氧状态下HSC-T6细胞被活化,划痕愈合率显著升高,活化标志蛋白α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)荧光信号显著增强,而6%含药血清能抑制HSC-T6细胞活化,细胞划痕愈合率显著降低,α-SMA荧光信号显著减弱。进一步研究发现,在缺氧状态下HSC-T6细胞中缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、α-SMA和刺猬信号通路(hedgehog,Hh)关键蛋白Patched-1(Ptch-1)、脑胶质瘤相关癌基因1(glioma related oncogene homology-1,Gli-1)表达上调,而6%含药血清可下调HIF-1α、α-SMA、Ptch-1和Gli-1蛋白表达;HIF-1α特异性阻断剂YC-1组和Hh信号通路特异性阻断剂环巴胺组HIF-1α、α-SMA、Ptch-1和Gli-1蛋白表达均显著下调。以上研究结果表明,HIF-1α和Hh信号通路参与HSC-T6细胞活化,中药慈菇消脂方可抑制HSC-T6细胞活化,其机制可能与抑制HIF-1α表达和阻断Hh信号通路有关。综上所述,中药慈菇消脂方通过直接作用于HIF-1α和Hh信号通路抑制HSC-T6细胞活化,从而发挥抗肝纤维化作用。实验方案经甘肃中医药大学动物实验伦理委员会批准,所有程序均严格按照动物使用和护理的伦理原则进行。
In this study,we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design,and predicted and verified its possible targets and related signaling pathways.The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Encyclopaedia of Traditional Chinese Medicine(ETCM)and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM)databases,and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis.GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform,and Cytoscape software was used to construct the"potential active ingredient-key target-pathway"network.The active components and target proteins were subjected to molecular docking analysis by Auto Dock software.According to the results of molecular dynamics simulation and binding free energy calculation,the top 5 active components with degree were scored.The active components stigmasterol andβ-sitosterol were subjected to molecular docking.CoCl_(2)was used to induce HSC-T6 cells to construct hypoxia model in vitro.The cell viability was detected by CCK-8 assay,and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100μmol·L^(-1)CoCl_(2)for 24 h.Under hypoxia condition,HSC-T6 cells were activated,the wound healing rate was significantly increased,and the fluorescence signal of activation marker proteinα-smooth muscle actin(a-SMA)was significantly enhanced.However,6%drug-containing serum could inhibit the activation of HSC-T6 cells,and the wound healing rate was significantly decreased,and the fluorescence signal of a-SMA was significantly weakened.Further studies showed that the expressions of hypoxia-inducible factor-lα(HIF-1α),α-SMA and key proteins of Hedgehog(Hh)signaling pathway in HSC-T6 cells were up-regulated under hypoxia,while the expressions of HIF-1α,a-SMA,Patched-1(Ptch-1)and glioma related oncogene homology-1(Gli-1)were down-regulated in 6%drug-containing serum group,the YC-1 group and the cyclopamine group.These results indicated that HIF-1αand Hh signaling pathways were involved in the activation of HSC-T6 cells,and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells,and the mechanism may be related to the inhibition of HIF-1αexpression and the blocking of Hh signaling pathway.In conclusion,Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1αand Hh signaling pathway,and exert an anti-hepatic fibrosis effect.The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine,in compliance with the Institutional Animal Care Guidelines.