大黄灵仙方调控TAK1与TRAF6相互作用及共定位对胆管细胞炎症反应的影响  

Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells

作　　者：甘苡榕 桂雄斌 俞渊 许斌[2] 戴建业[1] 常明 陈金梅 李承积 GAN Yi-rong;GUI Xiong-bin;YU Yuan;XU Bin;DAI Jian-ye;CHANG Ming;CHEN Jin-mei;LI Cheng-ji(Guangxi University of Chinese Medicine,Nanning 530001,China;The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,China)

机构地区：[1]广西中医药大学,南宁530001 [2]广西中医药大学第一附属医院,南宁530023

出　　处：《中国实验方剂学杂志》2022年第6期92-99,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：广西自然科学基金项目(2019JJA140301,2018JJA140857,2019JJB140120);广西中医药大学“岐黄工程”高层次人才团队培育项目(2021006);广西中医药大学研究生创新课题项目(YCSZ2020006)。

摘　　要：目的:观察大黄灵仙方(DHLX)对大鼠胆管上皮细胞的修复作用,探讨其作用机制是否通过调节转化生长因子-β(TGF-β)激活激酶1(TAK1)与肿瘤坏死因子受体相关因子6(TRAF6)的相互结合,调控核转录因子-κB(NF-κB)/丝裂原活化蛋白激酶(MAPK)信号通路活化而发挥作用。方法:将20只SD大鼠随机分为正常组和DHLX组,分别予生理盐水及DHLX(320 mg·kg^(-1)·d^(-1))灌胃8 d,制备正常血清及含DHLX血清;从正常SD大鼠中提取胆管上皮细胞,取胆管上皮细胞分为9组:正常组、模型组(20 mg·L^(-1))、脂多糖(LPS)+DHLX组(20 mg·L^(-1)+10%含药血清)、LPS+PDTC组(20 mg·L^(-1)+200μmol·L^(-1))、LPS+SB203580组(20 mg·L^(-1)+0.5μmol·L^(-1))、LPS+PDTC+SB203580组(20 mg·L^(-1)+200μmol·L^(-1)+0.5μmol·L^(-1))、LPS+PDTC+DHLX组(20 mg·L^(-1)+200μmol·L^(-1)+10%含药血清)、LPS+SB203580+DHLX组(20 mg·L^(-1)+0.5μmol·L^(-1)+10%含药血清)、LPS+PDTC+SB203580+DHLX组(20 mg·L^(-1)+200μmol·L^(-1)+0.5μmol·L^(-1)+10%含药血清);显微镜下观察药物干预后各组细胞的形态学变化,采用酶联免疫吸附测定法(ELISA)检测各组细胞中白细胞介素(IL)-1β与IL-6的表达量;蛋白免疫印迹法(Western blot)检测各组细胞中TAK1与TRAF6蛋白表达水平,免疫共沉淀技术检测TAK1与TRAF6的相互作用,激光共聚焦显微镜观察TAK1与TRAF6在细胞中的分布及共定位情况。结果:LPS作用后,细胞突触减少,胞体变圆变小,用药后各组细胞形态趋向正常;与正常组比较,模型组IL-1β和IL-6表达量明显上升(P<0.05),TAK1表达量降低而TRAF6的表达量升高(P<0.05),TAK1-TRAF6蛋白质复合物含量呈降低趋势;与模型组比较,LPS+DHLX组IL-1β和IL-6表达量明显降低(P<0.05),TAK1表达量升高而TRAF6表达量降低(P<0.05),TAK1-TRAF6蛋白质复合物含量显著增加(P<0.01),两蛋白共定位于细胞质中;与LPS+DHLX组比较,其余各组IL-1β和IL-6表达量均明显降低(P<0.05,P<0.01),通路阻滞剂干预后各组Objective: To observe the repair effect of Dahuanglingxian prescription(DHLX)on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor-β(TGF-β)activated kinase 1(TAK1)and tumor necrosis factor receptor-associated factor 6(TRAF6),and regulate the activation of the nuclear transcription factor-κB(NF-κB)/mitogenactivated protein kinase(MAPK)signaling pathway. Method: The 20 SD rats were randomly divided into normal group and DHLX group,10 rats in each group,were given saline and DHLX(320 mg·kg^(-1)·d^(-1))for 8 days,to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups: Normal group, model group(20 mg·L^(-1)),LPS+DHLX group(20 mg·L^(-1)+10%DHLX),LPS+PDTC group(20 mg·L^(-1)+200 μmol·L^(-1)),LPS+SB203580 group(20 mg·L^(-1)+0.5 μmol·L^(-1)),LPS+PDTC+SB203580 group(20 mg·L^(-1)+200 μmol·L^(-1)+0.5 μmol·L^(-1)), LPS+PDTC+DHLX group(20 mg·L^(-1)+200 μmol·L^(-1)+10% DHLX serum),LPS+SB203580+DHLX group(20 mg·L^(-1)+0.5 μmol·L^(-1)+10%DHLX serum),LPS+PDTC+SB203580 +DHLX group(20 mg·L^(-1)+200 μmol·L^(-1)+0.5 μmol·L^(-1)+10% DHLX serum). The microscopic observation of morphological changes in each group of cells after drug intervention.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of(IL)-1β and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells,Co-IP detected the interaction between TAK1 and TRAF6, and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope. Result:After the action of LPS,the cell synapses are reduced,the cell body becomes significantly rounded and smaller,but the cell morphology of each group tends to be normal after medication. Compared with normal group,the expression levels of IL-1β and IL-6 in model group were significantly increased(P<0.05),while the expression level of T

关 键 词：胆管炎症 大黄灵仙方 转化生长因子-β(TGF-β)激活激酶1(TAK1) 肿瘤坏死因子受体相关因子6(TRAF6) 相互作用 核转录因子-κB(NF-κB)/丝裂原活化蛋白激酶(MAPK)信号通路 

分 类 号：R2-0[医药卫生—中医学] R285