芍药苷对高糖刺激的小鼠骨髓来源的巨噬细胞TLR4信号通路的影响  

Effects of paeoniflorin on TLR4 signal pathway in bone marrow derived macrophages of mice induced by high glucose

作　　者：杨雯雯 段分分 邵云侠[1] 王坤[1] 吴永贵[1] Yang Wenwen;Duan Fenfen;Shao Yunxia(Dept of Nephrology,The First Affiliated Hospital of Anhui Medical University,Hefei 23002)

机构地区：[1]安徽医科大学第一附属医院肾脏内科,合肥230022

出　　处：《安徽医科大学学报》2018年第7期1037-1044,共8页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81374034)

摘　　要：目的探讨芍药苷(PF)对高糖刺激的小鼠骨髓来源的巨噬细胞(BMDMs)Toll样受体4(TLR4)信号通路的影响。方法分离骨髓来源的巨噬细胞作为研究对象,高糖作为刺激因素,芍药苷作为干预因素分组。将BMDMs分为7组:正常糖对照组(LG组)、正常糖对照+PF组(LG+PF组)、高糖刺激组(HG组)、高糖刺激+PF组(HG+PF组)、TLR4-/-对照组(TLR4-/-组)、TLR4-/-对照+高糖刺激组(TLR4-/-+HG组)、TLR4-/-对照+高糖刺激+PF组(TLR4-/-+HG+PF组)。流式细胞术鉴定巨噬细胞的纯度及成熟度;CCK-8检测PF对BMDMs活力的影响;Transwell检测各组BMDMs的趋化功能;激光共聚焦法检测各组的TLR4和诱生型一氧化氮合酶(i NOS)的协同表达;qRT-PCR测定各组细胞中的肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、单核细胞趋化因子-1(MCP-1)及i NOS mRNA的转录表达;Western blot法检测各组总蛋白中i NOS、TLR4、髓样分化因子88(My D88)、TIR结构域衔接蛋白(Trif)、磷酸化白介素-1受体相关激酶(p-IRAK1)、磷酸化干扰素调节因子3(p-IRF3)、核因子κB(NF-κB)p65和NF-κBp-p65蛋白的表达;ELISA法检测各组细胞培养上清液中促炎因子TNF-α、IL-1β和MCP-1的分泌情况。结果与LG组比较,高糖刺激可以增加巨噬细胞的趋化功能;明显上调细胞TNF-α、IL-1β、MCP-1及i NOS mRNA的转录表达(P<0.01);同时HG组的i NOS及TLR4、My D88、Trif、p-IRF3、NF-κBp65和NF-κBpp65等信号通路蛋白表达水平明显增强(P<0.01);细胞培养上清液中分泌的TNF-α、IL-1β及MCP-1水平增高(P<0.01)。PF和敲除TLR4基因均可以抑制高糖刺激导致的巨噬细胞的激活效应。结论高糖可以诱导BMDMs细胞内的TLR4及下游信号传导通路表达上调,同时使巨噬细胞激活导致促炎因子表达上调;PF和敲除TLR4基因均可抑制TLR4信号通路的激活,并且使巨噬细胞促炎因子的表达下调。Objective To investigate the effect of paeoniflorin（PF） on Toll-like receptor 4（TLR4） signaling pathway in bone marrow-derived macrophages（BMDMs）.Methods Macrophages derived from bone marrow were isolated from C57 mice and TLR4 knockout mice,high glucose was used as stimulus factor and paeoniflorin as intervention factor.BMDMs were divided into seven groups：normal control group（LG）,normal control group ＋ PF group（LG ＋ PF）,high glucose group（HG）,high glucose ＋ PF group（HG ＋ PF）,TLR4-/-control group（TLR4-/-）,TLR4-/-control ＋ high glucose group（TLR4-/-＋ HG）,TLR4-/-control ＋ high glucose group ＋PFgroup（TLR4-/-＋ HG ＋ PF）.The antigens expressed on cell surface including F4/80,CD11 b and CD11 c were detected by flow cytometry analyses.CCK-8 method was used to determine whether PF influenced macrophage viability.The chemotactic function of BMDMs in each group was detected by Transwell assay.The synergistic expression of TLR4 and inducible nitric oxide synthase（i NOS） was detected by laser scanning confocal microscopy.The expressions of tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）,monocyte chemoattractant protein 1（MCP-1） and i NOS mRNA in each group were detected by quantitative real-time PCR.The expressions of TLR4,My D88,TIR-domain-containing adapter-inducing interferon-β（Trif）,phospho-interferon regulatory factor 3（pIRF3）,NF-κB p65,NF-κB p-p65 and i NOS protein were detected by Western blot.Expressions of inflammatory cytokines TNF-α,IL-1β and MCP-1 in cell supernatant were determined by ELISA.Results Compared with the LG group,high glucose can promote macrophage polarization.The transcripts of TNF-α,IL-1β,MCP-1 and i NOS mRNA were significantly increased.The expression of TLR4,My D88,Trif,p-IRF3,IRF3,NF-κBp65,NF-κBpp65 and i NOS proteins in HG group were significantly increased.The secretion of TNF-α,IL-1β and MCP-1 levels increased.Both PF and TLR4 knockout inhibited the macrophage activation induced by high

关 键 词：芍药苷 巨噬细胞 TOLL样受体4 炎症 糖尿病肾病 

分 类 号：R364.5[医药卫生—病理学] R329.24[医药卫生—基础医学]