雷公藤甲素双向调控Nrf2/p65产生毒/效作用的机制研究  

Bidirectional regulation of triptolide on Nrf2/P65 and related toxic/therapeutic effects

作　　者：刘灵 张慧民 郭林 杨艳 曹蓉 罗欢 邹攀 龚慧[2] 江志超[1,4] 颜苗 LIU Ling;ZHANG Hui-min;GUO Lin;YANG Yan;CAO Rong;LUO Huan;ZOU Pan;GONG Hui;JIANG Zhi-chao;YAN Miao(Hunan University of Chinese Medicine,Changsha 410208;The Second Xiangya Hospital,Central South University,Changsha 410011;Wuzhou Gongren Hospital,Wuzhou Guangxi 543001;Hunan Province Directly Affiliated TCM Hospital,Zhuzhou Hunan 410016;Nanxishan Hospital of Guangxi Zhuang Autonomous Region,Guilin Guangxi 541002)

机构地区：[1]湖南中医药大学,长沙410208 [2]中南大学湘雅二医院,长沙410011 [3]梧州市工人医院,广西梧州543001 [4]湖南省直中医医院,湖南株洲410016 [5]广西壮族自治区南溪山医院,广西桂林541002

出　　处：《中南药学》2024年第3期660-667,共8页Central South Pharmacy

基　　金：国家自然科学基金面上项目(No.81974532);湖南省中医药管理局重点课题(No.A2023032);中国药学会医院药学专委会人才专项资助项目(No.CPA-Z05-ZC-2021-003);广西壮族自治区中医药局自筹经费科研课题(No.GXZYC20230219)。

摘　　要：目的探索雷公藤甲素(TP)双向调控Nrf2/p65产生毒性与保护的双重作用及机制。方法通过小分子与蛋白质对接模拟,分析TP与Nrf2/p65的潜在作用关系。在细胞实验里,采用不同浓度的TP分别干预人正常肝脏细胞(L02)与小鼠肝库普弗细胞(KC),采用MTT法检测细胞存活率,Western blot检测Nrf2、HO1、p65、IL-6的蛋白表达;在动物实验里,采用TP干预糖尿病肾病(DN)模型大鼠,HE染色法检测大鼠肾脏病理,靛酚蓝比色法检测血清BUN,ELISA法检测血清炎症因子TNF-α、IL-6与IL-1β的含量变化,RT-qPCR检测肾组织中p65、podocin和Kim-1的mRNA水平,Western blot检测肾组织中p65的蛋白表达,免疫组化法观察肾组织细胞p65与Nrf2的蛋白表达情况。结果分子对接结果表明,TP与Nrf2及p65具有良好结合活性;体外实验表明,TP可以降低L02细胞存活率,呈剂量和时间依赖性降低Nrf2蛋白表达,降低KC细胞存活率,抑制Nrf2及其下游HO-1的表达,上调p65及IL-6的表达;动物实验表明,TP对DN大鼠有明显保护作用,降低大鼠肾组织中p65的mRNA及蛋白的表达,上调Nrf2蛋白的表达,下调血清中TNF-α、IL-6、IL-1β炎症因子水平。结论TP可以通过下调Nrf2蛋白表达,上调p65蛋白的表达对肝细胞造成毒性损伤;同时也可以上调Nrf2蛋白表达,下调p65蛋白与mRNA的表达而对DN大鼠产生保护作用,即对Nrf2/p65分子产生双向调控而表现毒性或药性。Objective To determine the dual role of triptolide(TP),in regulating both the toxicity and protection through the Nrf2/P65 pathway and related mechanism.Methods Molecular docking simulations were performed to analyze the potential interaction between TP and Nrf2/P65.In the cell experiments,different concentrations of TP were used to intervene human normal liver cells(L02)and mouse Kupffer cells(KC).The cell viability was assessed with the MTT assay,and the protein expression of Nrf2,HO1,P65,and IL-6 was examined by Western blot.In the animal experiments,TP was administered for model rats with diabetic nephropathy.The kidney pathology was evaluated with HE staining,the serum BUN level was measured by indophenol blue colorimetric method,and changes in the serum levels of inflammatory cytokines TNF-α,IL-6,and IL-1β were determined by ELISA.The mRNA levels of P65,podocin,and Kim-1 in the kidney tissue were measured with RTqPCR,and protein expression of P65 in the kidney tissue with Western blot.Immunohistochemistry was performed to observe the protein expression of P65 and Nrf2 in the kidney tissue.Results The molecular docking showed good binding between TP with Nrf2 and P65.In vitro experiments demonstrated that TP reduced the cell viability in L02 cells,decreased Nrf2 protein expression in a dose-and time-dependent manner,reduced the cell viability in KC cells,inhibited the expression of Nrf2 and its downstream target HO-1,and upregulated the expression of P65 and IL-6.The animal experiments showed that TP had obvious protective effect on DN rats,namely reducing the mRNA and protein expression of P65 in the kidney tissue,upregulating Nrf2 protein expression,and downregulating the levels of inflammatory cytokines TNF-α,IL-6,and IL-1β in the serum.Conclusion TP can induce toxic damage to liver cells by downregulating Nrf2 protein expression and upregulating P65 protein expression.Meanwhile,it can also exert a protective effect on DN rats by upregulating Nrf2 protein expression and downregulating the expression o

关 键 词：双向调控 分子对接 雷公藤甲素 P65 NRF2 

分 类 号：R285[医药卫生—中药学]