TLR4单克隆抗体对过敏性紫癜小鼠损伤的保护作用及其机制  

Protective Effect of TLR4 Monoclonal Antibody Against Injury of Henoch-schonlein Purpura Mice and the Mechanism

作　　者：贵琳[1] 朱松柏[1] 郑荣浩[1] 舒岚[1] 朱宏飞 王勇[3] 吴晓林[1] Gui Lin;Zhu Songbai;Zheng Ronghao(Department of Pediatric Kidney Disease,Maternal and Child Health Hospital of Hubei Province,Wuhan 430070,China)

机构地区：[1]湖北省妇幼保健院儿童肾病风湿免疫科,武汉430070 [2]湖北省中医药研究院,湖北省中医院麻醉科,武汉430061 [3]武汉大学动物实验中心,武汉430071

出　　处：《华中科技大学学报（医学版）》2020年第1期39-44,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省卫生计生委科研基金资助项目(No.WJ2017M132)。

摘　　要：目的探究Toll样受体4(Toll like receptor 4,TLR4)单克隆抗体(mAb)对过敏性紫癜(Henoch-Schonlein purpura,HSP)小鼠损伤的保护作用及其机制。方法将50只C57BL/6健康小鼠随机分为正常对照组、HSP模型组、同型对照组、TLR4 mAb组和西咪替丁阳性对照组。正常对照组不做处理,其余4组小鼠均进行为期14周的HSP模型构建。在模型构建成功后对各组小鼠进行对应药物干预,持续3周。干预结束后,采用碳粒廓清法进行小鼠网状内皮系统(reticuloendothelial system,RES)功能检测;流式细胞术检测小鼠外周血单核细胞中Th17及Treg细胞占比;ELISA法检测小鼠血清中分泌型免疫球蛋白E(secretory-IgE,S-IgE)、IL-17、IL-6、IL-10以及TGF-β的水平;qRT-PCR检测各组小鼠皮肤和肾脏中视黄酸相关性孤儿受体(retinoic acid associated orphan receptorγt,RORγt)及叉头样转录因子3(forkhead helix transcription factor 3,Foxp3)的表达;Western blot检测各组小鼠皮肤和肾脏中TLR4及IL-17蛋白表达;苏木精-伊红染色观察小鼠皮肤和肾脏的组织病理学变化。结果与模型组比较,TLR4 mAb显著增强HSP小鼠RES功能(吞噬指数K及吞噬系数α)(均P<0.01),降低血清S-IgE、IL-17、IL-6水平(均P<0.01),增加血清IL-10及TGF-β水平(均P<0.01)。此外,TLR4 mAb显著改善了HSP小鼠皮肤及肾脏的病理学变化,抑制RORγt在mRNA水平的表达,促进Foxp3在mRNA水平的表达(均P<0.01);同时降低TLR4、IL-17蛋白的表达量。结论 TLR4 mAb对HSP小鼠损伤具有显著的保护作用,其作用机制可能与下调TLR4和IL-17蛋白的表达进而介导Th17/Treg免疫平衡调节有关。Objective To investigate the protective effect of toll like receptor 4(TLR4)on monoclonal antibody(mAb)on the injury of Henoch-Schonlein purpura(HSP)mice and the mechanism.Methods Fifty C57 BL/6 healthy mice were randomly divided into normal control group,HSP model group,isotype control group,TLR4 mAb group and cimetidine positive control group.The normal control group was left untreated,and the remaining 4 groups of mice were subjected to a 14-week HSP model construction.After the model was successfully established,the corresponding drug intervention was performed on the mice in each group for 3 weeks.After the intervention,the function of reticuloendothelial system(RES)was detected by the carbon particles clearance method.The proportion of Th17 and Treg cells in peripheral blood mononuclear cells of mice was detected by flow cytometry.The levels of secretory IgE(S-IgE),IL-17,IL-6,IL-10 and TGF-β in serum of mice were detected by ELISA.The expression of retinoic acid associated orphan receptor γt(RORγt)and Forkhead helix transcription factor 3(Foxp3)in the skin and kidney of mice were detected by qRT-PCR.Western blotting was used to detect the expression of TLR4 and IL-17 protein in the skin and kidney of each group of mice.Hematoxylin-eosin staining was used to evaluate the histopathological changes in the skin and kidney of mice.Results Compared with the model group,TLR4 mAb significantly enhanced HRES mice RES function(phagocytic index K and phagocytic coefficient α)in HSP mice(all P<0.01),reduced serum S-IgE,IL-17 and IL-6 levels(all P<0.01),and increased serum IL-10 and TGF-β levels(all P<0.01).In addition,TLR4 mAb significantly improved the histopathological changes in skin and kidney of HSP mice,inhibited the expression at RORγt in mRNA level,and promoted the expression of Foxp3 at mRNA level(all P<0.01).Meanwhile,the expression levels of TLR4 and IL-17 protein were decreased.Conclusion TLR4 mAb has a significant protective effect on HSP mice,and its mechanism may be related to the down-regulation t

关 键 词：TLR4单克隆抗体 过敏性紫癜 小鼠 Th17/Treg平衡 

分 类 号：R593.1[医药卫生—内科学]