柴胡疏肝散治疗肝纤维化核心靶基因筛选及对肝星状细胞活化的影响  

Screening of core target genes of Chaihu Shugan powder in treatment of hepatic fibrosis and its effect on activation of hepatic stellate cells

作　　者：王帅[1] 葛茂旭 刘安昌[1] WANG Shuai;GE Maoxu;LIU Anchang(Department of Pharmacy,Qilu Hospital of Shandong University,Jinan 250012,China;不详)

机构地区：[1]山东大学齐鲁医院药剂科(临床药学部),山东济南250012

出　　处：《山东医药》2025年第3期1-6,共6页Shandong Medical Journal

基　　金：山东省自然科学基金青年基金资助项目(ZR2021QH204)。

摘　　要：目的利用网络药理学方法筛选柴胡疏肝散治疗肝纤维化的核心靶基因,分析柴胡疏肝散对肝星状细胞活化的影响及机制。方法将柴胡疏肝散有效活性成分的作用靶基因与肝纤维化疾病相关靶基因取交集,得到柴胡疏肝散治疗肝纤维化的潜在靶基因,进行基因本体论(GO)功能及KEGG、Reactome通路富集分析;利用STRING平台构建潜在靶基因的蛋白质相互作用网络图,根据度值筛选核心靶基因。将人肝星状细胞系LX-2分为转化生长因子β_(1)(TGF-β_(1))组、低剂量组、中剂量组、高剂量组(分别加入2 ng/mL TGF-β_(1)及0、12.5、25、50 mg/mL柴胡疏肝散作用24 h)和Control组(不予处理),采用实时荧光定量PCR法及Western blotting法检测细胞纤维化标志物Ⅰ型胶原α1链(COL1A1)、波形蛋白(vimentin)、α平滑肌肌动蛋白(α-SMA)mRNA及蛋白表达,免疫荧光法检测细胞纤连蛋白(Fibronectin)表达(以绿色荧光强度表示)。将LX-2细胞随机分为pcDNA3.1组、信号传导转录激活因子3(STAT3)-Flag组(分别转染pcDNA3.1质粒、pcDNA3.1-STAT3-Flag质粒)和对照组(不予处理),pcDNA3.1组和STAT3-Flag组转染后均分为TGF-β_(1)及TGF-β_(1)+低、中、高剂量柴胡疏肝散四部分,分别给予2 ng/mL TGF-β_(1)及0、12.5、25、50 mg/mL柴胡疏肝散作用24 h,采用Western blotting法检测p-STAT3及COL1A1、vimentin、α-SMA蛋白表达。结果共得到柴胡疏肝散治疗肝纤维化潜在靶基因45个[核心靶基因12个,度值最大的是STAT3],富集于细胞迁移正调控、对缺氧的响应、转录因子结合、蛋白激酶活性等功能,以及白细胞介素信号转导、JAK-STAT信号通路等。与Control组比较,TGF-β_(1)组细胞COL1A1、vimentin、α-SMA mRNA及蛋白相对表达量均升高(P均<0.05)。与TGF-β_(1)组比较,低、中、高剂量组COL1A1 mRNA及蛋白相对表达量均降低,且中、高剂量组COL1A1 mRNA相对表达量降低更明显(P均<0.05);与TGF-β_(1)组比较,高剂量Objective To screen core target genes of Chaihu Shugan powder(CSP)in treatment of hepatic fibrosis via network pharmacology and to analyze its effect and mechanism on the activation of hepatic stellate cells.Methods The target genes of the active ingredients in CSP were intersected with the target genes of liver fibrosis to obtain the potential anti-liver fibrosis target genes of CSP.Gene Ontology(GO),KEGG,and Reactome pathway analyses were performed.The protein-protein interaction network of the potential anti-liver fibrosis target genes of CSP was constructed using STRING platform,and the core target genes were screened by degree value.Human hepatic stellate cells LX-2 were divided into the transforming growth factor-β_(1)(TGF-β_(1))group,low-dose group,medium-dose group,high-dose group(treated with 2 ng/mL TGF-β_(1) and 0,12.5,25,and 50 mg/mL CSP for 24 h),and the Control group(untreated).Real-time PCR and Western blotting were employed to detect the mRNA and protein levels of fibrotic markers,including COL1A1,vimentin,and α-SMA.Immunofluorescence was used to detect the expression of Fibronectin(represented by green fluorescence intensity).LX-2 cells were divided into the pcDNA3.1 group(transfected with pcDNA3.1 plasmid),the signal transducer and activator of transcription 3(STAT3)-Flag group(transfected with pcDNA3.1-STAT3-Flag plasmid),and the Control group(untreated).After transfection,the pcDNA3.1 group and STAT3-Flag group were further divided into four parts,which were treated with 2 ng/mL TGF-β_(1) and 0,12.5,25,50 mg/mL CSP for 24 h,respectively.Western blotting was used to detect the protein levels of p-STAT3 Tyr705,COL1A1,vimentin,andα-SMA.Results Forty-five potential anti-liver fibrosis target genes of CSP were obtained,among which 12 were core target genes,with STAT3 having the highest degree.These genes were enriched in cell migration,response to hypoxia,transcription factor binding,protein kinase activity,and other functions,and interleukin signal transduction and the JAK-STAT signal pathwa

关 键 词：柴胡疏肝散 网络药理学 肝纤维化 肝星状细胞 信号传导转录激活因子3信号通路 

分 类 号：R575.2[医药卫生—消化系统]