基于GPR43/β-arrestin-2/IκBα/NF-κB通路探讨左归降糖通脉方对高糖合并LPS诱导的人脐静脉内皮细胞炎性损伤的影响  

Effect of Zuogui Jiangtang Tongmai Prescription on Inflammatory Injury of HumanUmbilical Vein Endothelial Cells Induced by High Glucose and LPS Based onGPR43/β-arrestin-2/IκBα/NF-κB Pathway

作　　者：彭岚玉 姚敬心 李钰佳 李定祥 刘迅 邓奕辉 PENG Lanyu;YAO Jingxin;LI Yujia;LI Dingxiang;LIU Xun;DENG Yihui(School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;School of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,长沙410208 [2]湖南中医药大学中医学院,长沙410208

出　　处：《中国实验方剂学杂志》2024年第3期64-74,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖南省教育厅科学研究项目(21A0228);湖南省中医药科研计划项目(E2022010);湖南中医药大学中西医结合一流学科开放基金项目(2020ZXYJH31)。

摘　　要：目的:探讨左归降糖通脉方(ZJTP)对高糖合并脂多糖(LPS)损伤后人脐静脉内皮细胞(HUVECs)的影响及作用机制。方法:通过细胞增殖与活性检测(CCK-8)法检测细胞存活率、酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α)水平确定LPS最适损伤浓度及作用时间、ZJTP含药血清的最佳作用浓度。将HUVECs分为空白组、模型组、ZJTP含药血清组、短链脂肪酸(SCFAs)混合液组。采用ELISA检测内皮素-1(ET-1)、一氧化氮(NO)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和TNF-α的含量;蛋白免疫印迹法(Western blot)检测G蛋白偶联受体43(GPR43)、β-抑制蛋白-2(β-arrestin-2)、核转录因子-κB抑制因子α(IκBα)、核转录因子-κB p65(NF-κB p65)蛋白表达;免疫荧光法(IF)观察NF-κB p65入核情况。运用小干扰核糖核酸(siRNA)的方法观察GPR43在炎性损伤调控中的作用。将干扰后的细胞分为空载体组、ZJTP含药血清组、Si-GPR43组、Si-GPR43+ZJTP含药血清组。ELISA检测IL-1β、IL-6和TNF-α含量;Western blot检测通路蛋白表达;IF观察NF-κB p65入核情况。结果:最适造模条件为1 mg·L^(-1) LPS作用24 h;最佳药物干预条件为5%的ZJTP含药血清作用24 h。与空白组比较,模型组ET-1含量显著升高,NO含量显著下降(P<0.01);炎症因子水平显著上升(P<0.01);GPR43和IκBα蛋白表达量下降,β-arrestin-2和NF-κB p65蛋白表达量显著升高(P<0.01);NF-κB p65蛋白由核外转移至核内(P<0.01)。与模型组比较,ZJTP含药血清组ET-1含量下降,NO含量升高(P<0.05);炎症因子水平下降(P<0.05);GPR43和IκBα蛋白表达升高,β-arrestin-2和NF-κB p65表达下降(P<0.05);NF-κB p65由核外转移至核内的数量减少(P<0.01)。机制研究发现,与Si-GPR43组比较,ZJTP含药血清干预后IL-1β、IL-6和TNF-α含量显著下降(P<0.01);GPR43和IκBα蛋白表达量显著上升(P<0.01),β-arrestin-2和NF-κB p65蛋白表达量显著下降(P<0.01);NF-κB p65由核外转移至核�Objective:To investigate the effects and mechanism of Zuogui Jiangtang Tongmai prescription(ZJTP)on human umbilical vein endothelial cells(HUVECs)damaged by high glucose combined with lipopolysaccharide(LPS).Method:The survival rate of cells was determined by cell counting kit-8(CCK-8),and the level of tumor necrosis factor-α(TNF-α)was determined by enzyme-linked immunosorbent assay(ELISA)to determine the optimal injury concentration and action time of LPS,as well as the optimal action concentration of ZJTP drug-containing serum.HUVECs were divided into a blank control group,a model group,a ZJTP drug-containing serum group,and an SCFA mixed liquid group.ELISA was used to detect the level of endothelin-1(ET-1),nitric oxide(NO),interleukin-1β(IL-1β),interleukin-6(IL-6),and TNF-α.Western blot was performed to detect the protein expression of G protein-coupled receptor43(GPR43),β-suppressor protein-2(β-arrestin-2),nuclear factor-κB suppressorα(IκBα),and nuclear factorκB p65(NF-κB p65).The nucleation of NF-κB p65 was observed by immunofluorescence staining(IF).The role of GPR43 in the regulation of inflammatory injury was observed by means of small interfering ribonucleic acid(siRNA).The cells after intervention were divided into an empty carrier group,a ZJTP drug-containing serum group,a Si-GPR43 group,and a Si-GPR43+ZJTP drug-containing serum group.The content of IL-1β,IL-6,and TNF-αwas detected by ELISA.The protein expression of pathways was detected by Western blot.IF was used to observe the nucleation of NF-κB p65.Result:The optimal molding condition was 1 mg·L^(-1) LPS for 24 h.The optimal drug intervention condition was 5%ZJTP drug-containing serum for 24 h.Compared with the blank control group,the content of ET-1 in the model group was significantly increased,and the content of NO was significantly decreased(P<0.01).The levels of inflammatory factors were significantly increased(P<0.01).The expressions of GPR43 and IκBαwere significantly decreased,while the protein expressions ofβ-arrestin

关 键 词：人脐静脉内皮细胞 炎性损伤 左归降糖通脉方 G蛋白偶联受体43 核转录因子-κB 

分 类 号：R2-0[医药卫生—中医学] R33R289R587.1