葛根芩连汤缓解肝内质网应激改善体内外糖代谢的效应机制  

Mechanism of Gegen Qinlian Decoction in improving glucose metabolism in vitro and in vivo by alleviating hepatic endoplasmic reticulum stress

作　　者：姜月 延李科 王颖[1,2] 丁浚峰 徐中华 崔灿 涂珺 JIANG Yue;YAN Li-ke;WANG Ying;DING Jun-feng;XU Zhong-hua;CUI Can;TU Jun(Jiangxi Province Key Laboratory of Traditional Chinese Medicine Etiopathogenisis&Research Center for Differentiation and Development of Traditional Chinese Medicine Basic Theory,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Key Laboratory of Traditional Chinese Medicine Pharmacology of Jiangxi Province,Nanchang 330004,China)

机构地区：[1]江西中医药大学江西省中医病因学重点实验室&中医基础理论分化发展研究中心,江西南昌330004 [2]江西省中药药理重点实验室,江西南昌330004

出　　处：《中国中药杂志》2023年第20期5565-5575,共11页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81960809,82160838);江西中医药大学校级科技创新团队发展计划项目(CXTD22007)。

摘　　要：探讨葛根芩连汤(Gegen Qinlian Decoction,GQD)缓解内质网应激(endoplasmic reticulum stress,ERS)改善体内外糖代谢的效应机制。分子对接预测GQD入血主要药效成分与ERS相关靶点的结合活性。取冻存正常组、高脂诱导糖尿病模型组、二甲双胍组和GQD组大鼠肝组织,提取RNA和蛋白质,qPCR检测ERS标志蛋白葡萄糖调节蛋白78(glucose-regulated protein 78,Grp78)、未折叠蛋白反应(unfolded protein response,UPR)通路基因肌醇需求酶1(inositol requiring enzyme 1,Ire1)、转化因子6(activating transcription factor 6,Atf6)、Atf4、C/EBP同源蛋白(C/EBP-homologous protein,Chop)和半胱氨酸天冬氨酸蛋白酶(caspase)-12基因mRNA表达;Western blot检测GRP78、IRE1、蛋白激酶R样内质网激酶(protein kinase R-like ER kinase,PERK)、ATF6、X-盒结合蛋白1(X-box binding protein 1,XBP1)、ATF4、CHOP、caspase-12、caspase-9和caspase-3蛋白表达;比色法检测肝组织钙离子含量。体外采用衣霉素诱导6 h建立ERS-HepG2细胞模型,2.5%、5%、10%GQD含药血清(GQD-containing serum)给药9 h,葡萄糖氧化酶法检测细胞外液葡萄糖含量;流式细胞术检测细胞凋亡;糖原染色检测细胞糖原含量;免疫荧光检测ERS标志蛋白GRP78表达;比色法检测胞内钙离子含量;Western blot检测GRP78和ERS诱导IRE1、PERK、ATF6和真核翻译启动因子2α(eukaryotic translation initiation factor 2α,eIF2α)磷酸化,同时检测胰岛素降糖通路蛋白胰岛素受体底物1(insulin receptor substrate 1,IRS1)、磷脂酰肌醇3-激酶p85调控亚基(phosphatidylinositol 3-kinase regulatory subunit p85,PI3Kp85)和蛋白激酶B(protein kinase B,Akt)的磷酸化水平。此外,Western blot检测沟通ERS与胰岛素降糖通路的c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNKs)磷酸化水平。分子对接结果显示,GRP78、IRE1、PERK、ATF4与黄芩素、小檗碱、大豆黄酮、药根碱、甘草苷、棕榈碱、葛根素、汉黄芩苷有较强结合活性,提示GQD可能干�This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS).Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets.Liver tissue samples were obtained from normal rats,high-fat-induced diabetic rats,rats treated with metformin,and rats treated with GQD.RNA and protein were extracted.qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78),and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1),activating transcription factor 6(Atf6),Atf4,C/EBP-homologous protein(Chop),and caspase-12.Western blot was used to detect the protein expression of GRP78,IRE1,protein kinase R-like ER kinase(PERK),ATF6,X-box binding protein 1(XBP1),ATF4,CHOP,caspase-12,caspase-9,and caspase-3.The calcium ion content in liver tissues was determined by the colorimetric assay.The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours,and 2.5%,5%,and 10% GQD-containing serum were administered for 9 hours.The glucose oxidase method was used to measure extracellular glucose levels,flow cytometry to detect cell apoptosis,glycogen staining to measure cellular glycogen content,and immunofluorescence to detect the expression of GRP78.The intracellular calcium ion content was measured by the colorimetric assay.Whereas Western blot was used to detect GRP78 and ERS-induced IRE1,PERK,ATF6,and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation.Additionally,the phosphorylation levels of insulin receptor substrate 1(IRS1),phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85),and protein kinase B(Akt),which were involved in the insulin signaling pathway,were also measured.In addition,the phosphorylation levels of c-Jun N-terminal kinases(JNKs),which were involved in both the ERS and insulin signaling pathways,were measured by Western blot.Molecular docking

关 键 词：糖尿病 糖代谢 胰岛素抵抗 内质网应激 葛根芩连汤 

分 类 号：R285.5[医药卫生—中药学]