基于网络药理学探讨健脾益气方治疗DEN肝癌大鼠的分子机制及关键通路的验证  

Using network pharmacology to study the molecular mechanism and validate the key pathway of Jianpi Yiqi Recipe in treating diethylinitrosamine-induced rat hepatocellular carcinoma

作　　者：音金萍 岳紫晨 王超 卓少元 Yin Jinping;Yue Zichen;Wang Chao;Zhuo Shaoyuan(School of Basic Medical Science,Guangxi University of Chinese Medicine,Guangxi 530000,China)

机构地区：[1]广西中医药大学基础医学院,广西530000

出　　处：《北京中医药大学学报》2021年第10期917-927,共11页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金地区科学基金项目(No.81860824,No.81660775);广西中医药大学研究生教育创新计划项目(No.YCSZ2020024)。

摘　　要：目的利用网络药理学分析方法探讨健脾益气方治疗肝癌的活性成分与靶点;并在此基础上利用二乙基亚硝胺(DEN)诱导的肝癌大鼠,对其中筛选出的关键通路白介素-6/信号转导和转录激活因子3(IL-6/STAT3)进行验证。方法采用中药系统药理学数据库和分析平台(TCMSP)数据库、中国知网和Swiss Target Prediction平台获取健脾益气方的活性成分和对应靶点;基于Gene Cards、CTD以及gene数据库获取肝细胞癌(HCC)相关靶点;将疾病相关靶点和药物靶点取交集后得出潜在靶点,导入Cytoscape软件绘制活性成分-靶点网络图;并利用STRING数据库对潜在靶点进行蛋白相互作用(PPI)网络构建与分析,以获取核心靶点。采用腹腔注射DEN[70 mg/(kg·d)]制备肝癌大鼠模型。将大鼠随机分为正常组,模型组,健脾益气方低、中、高剂量组(5.25、10.5、21 g/kg),STAT3抑制组(C188-9,4.5 mg/kg)和糖蛋白130(gp130)抑制组(SC144,4.5 mg/kg),均连续干预4周。采用HE染色法观察大鼠肝脏组织病理形态学变化;采用RT-qPCR法检测大鼠肝脏组织IL-6、gp130、Janus激酶1(JAK1)、Janus激酶2(JAK2)和STAT3的mRNA表达水平;采用ELISA法检测大鼠血清IL-6含量;采用Western Blot法检测大鼠肝脏组织gp130、JAK1、p-JAK1、JAK2、p-JAK2、STAT3和p-STAT3蛋白表达水平。结果通过网络药理学分析获得64个活性成分和146个潜在靶点,这些分子参与的信号通路主要与IL-6/STAT3有关,且STAT3是其中潜在的核心靶点。动物实验结果显示,与正常组比较,模型组大鼠IL-6和STAT3 mRNA和蛋白质水平,及p-JAK1/JAK1、p-JAK2/JAK2和p-STAT3/STAT3水平均明显上调(P<0.05或P<0.01)。与模型组比较,健脾益气方中、高剂量组,及STAT3抑制组和gp130抑制组大鼠血清IL-6含量,肝脏组织IL-6、gp130和STAT3 mRNA表达水平,gp130和STAT3蛋白水平,及p-JAK1/JAK1、p-JAK2/JAK2、p-STAT3/STAT3水平均降低(P<0.05或P<0.01)。结论健脾益气方可通过多成分、多靶Objective To investigate the mechanism of Jianpi Yiqi Recipe(JYR)in treating liver cancer,we explore the active components of the recipe,and their targets.Furthermore,the effect of JYR on the IL-6/STAT3 signaling pathway in diethylinitrosamine(DEN)-induced rat hepatocellular carcinoma was studied.Methods The TCMSP database,CNKI database and Swiss Target Prediction platform were used to obtain the active components of JYR and their corresponding targets.HCC-related genes were acquired by Searching Gene Cards,CTD,and Gene databases.The potential cancer-related drug targets were obtained,and the active component-target network diagram was mapped by using Cytoscape software.The protein-protein interaction(PPI)network of potential targets was constructed and analyzed by String database to obtain the core targets.The rats were intraperitoneally injected with DEN[70 mg/(kg·d)]to establish the HCC model.The rats were randomly divided into seven groups:control group,model group,low-,mid-and high-dose JYR group[5.25 g/(kg·d),10.5 g/(kg·d),21 g/(kg·d)],STAT3 inhibition group[4.5 mg/(kg·d)]and gp130 inhibition group[4.5 mg/(kg·d)].All groups were treated for 4 weeks.After HE staining,the pathological changes in liver tissue were observed under the microscope.The mRNA levels of IL-6,gp130,JAK1,JAK2 and STAT3 in liver cancer tissue were detected with RT-qPCR.The serum level of IL-6 was measured with ELISA.The protein levels of gp130,JAK1,p-JAK1,JAK2,p-JAK2,STAT3 and p-STAT3 were examined with Western blot.Results 64 active components and 146 potential targets were identified,which were found to be involved in several key pathways such as IL-6/STAT3.Moreover,STAT3 may be the core target of JYR in the treatment of HCC.The results of animal experiments showed that the IL-6 and STAT3 mRNA expression levels,the levels of p-JAK1/JAK1,p-JAK2/JAK2 and p-STAT3/STAT3,and the serum IL-6 levels in the model group were significantly increased,compared with the control group(P<0.05 or P<0.01).After treated with mid-dose,high-dose JYR

关 键 词：健脾益气方 肝细胞癌 网络药理学 IL-6/STAT3炎症信号通路 大鼠 

分 类 号：R285.5[医药卫生—中药学]