小青龙汤通过TLR4/MyD88/NF-κB信号通路对慢性肾小球肾炎的作用机制研究  

Mechanism of Minor Green Dragon Decoction on Chronic Glomerulonephritis via TLR4/MyD88/NF-κB Signaling Pathway

作　　者：柴溥苒 储开博 赖永健 柴希 张育敏 何丽清 CHAI Puran;CHU Kaibo;LAI Yongjian;CHAI Xi;ZHANG Yumin;HE Liqing(Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学,山西晋中030619

出　　处：《中医药学报》2024年第12期11-17,共7页Acta Chinese Medicine and Pharmacology

基　　金：国家自然科学基金面上项目(82274365);山西省自然科学基金面上项目(20210302123228);山西省中医药管理局中医药创新团队(zyytd2024024);山西省中医药管理局科研项目(2023ZYYB2038);2022年山西中医药大学科技创新团队项目(2022TD1011);山西省教育厅研究生科研创新项目(2023KY683);山西中医药大学研究生教育改革及创新创业项目(2023CX034)。

摘　　要：目的:通过建立脂多糖(LPS)诱导的RAW264.7小鼠单核巨噬细胞炎症模型以及人胚肾细胞炎症模型,探讨小青龙汤(XQLT)通过调控TLR4/MyD88/NF-κB信号通路对慢性肾小球肾炎(CGN)产生的作用机制。方法:基于课题组前期研究,采用LPS(10μg/mL)诱导RAW264.7小鼠单核巨噬细胞,建立炎症反应模型,随机分为空白对照组、模型组、小青龙汤组(1000μg/mL)。采用Elisa法检测各组巨噬细胞中TNF-α、IL-6和IL-1β水平;采用蛋白免疫印迹法(Western blot)检测各组巨噬细胞中TLR4、MyD88以及核内NF-κB蛋白表达水平;采用免疫组化技术检测各组巨噬细胞中TLR4、MyD88以及核内NF-κB水平。收集小青龙汤(1000μg/mL)干预巨噬细胞的条件培养液,根据MTT结果确定不同比例条件培养液培养HEK293细胞24 h/48 h,检测HEK293的细胞活力。根据MTT结果分为空白对照组、模型组(10μg/mL的LPS干预HEK293细胞)、炎性上清组(根据MTT情况选择参考10%和90%的炎性上清),采用Elisa法检测HEK293细胞炎症模型各组TNF-α、IL-6和IL-1β炎症因子的含量;采用免疫组化技术检测HEK293细胞炎症模型各组中TLR4、MyD88以及核内NF-κB的表达水平。结果:小青龙汤可显著降低LPS诱导的RAW264.9细胞上清液中炎性因子TNF-α、IL-6及IL-1β表达(P<0.05),通过WB法及IHC法表明小青龙汤也可显著降低LPS诱导的RAW264.9细胞中TLR4、MyD88及核内NF-κB蛋白表达(P<0.05,P<0.01)。同时,灰度分析积分光密度(IOD)结果显示,小青龙汤对LPS诱导的RAW264.9细胞中的TLR4、MyD88及核内NF-κB表达具有调控作用;与模型组比较,TLR4、MyD88及核内NF-κB蛋白IOD值均降低(P<0.01,P<0.001)。通过MTT法检测HEK293细胞活力,可以观察到随着作用时间的延长以及药物的浓度的提高,小青龙汤干预巨噬细胞后的条件培养液对HEK293细胞增殖的抑制作用不断增强,为证明小青龙汤保护HEK293细胞免受炎性损伤,选用10%及90%条件培养液(炎性上清Objective:Through the establishment of a lipopolysaccharide(LPS)-induced RAW264.7 mouse monocyte macrophage inflammation model and a human embryonic kidney cell inflammation model,this study aims to investigate the mechanism of action of Minor Green Dragon decoction(MGDD)on chronic glomerulonephritis(CGN)via regulation of the TLR4/MyD88/NF-κB signaling pathway.Methods:Based on our previous research,mononuclear macrophages of RAW264.7 mice were induced by LPS(10μg/mL)to establish an inflammatory response model and were divided into a blank control group,a model group,and a MGDD group(1000μg/mL).The levels of TNF-α,IL-6,and IL-1βin the macrophages of each group were detected using Elisa method.The expression levels of TLR4,MyD88,and NF-κB proteins in macrophages of each group were detected using Western blot.TLR 4,MyD 88 as well as nuclear NF-κB in macrophages of each group were determined by immunohistochemistry.The conditioned culture medium of MGDD(1000μg/mL)was collected for the intervention of macrophages;according to the results of MTT,HEK293 cells were cultured in medium with different proportions for 24h/48h to detect the cell viability of HEK293.According to MTT results,it was divided into blank control group,model group(10μg/mL LPS was used to interfer HEK293 cells),and inflammatory supernatant group(10%and 90%inflammatory supernatant were selected according to MTT conditions).The contents of TNF-α,IL-6 and IL-1βin HEK293 cell inflammatory model were detected by Elisa.The expression levels of TLR4,MyD88 and NF-κB in HEK293 cell inflammatory model groups were detected by immunohistochemistry.Results:MGDD could significantly reduce the expression of inflammatory factors TNF-α,IL-6 and IL-1βin the supernatant of RAW264.9 cells induced by LPS.WB and IHC methods showed that MGDD could also significantly reduce the expression of TLR4,MyD88 and NF-κB protein in the nuclear of RAW264.9 cells induced by LPS.Meanwhile,according to grayscale analysis integrated optical density(IOD)detection,MGDD had a

关 键 词：小青龙汤 HEK293细胞 炎症模型 TLR4/MyD88/NF-κB信号通路 

分 类 号：R285.5[医药卫生—中药学]