基于抑制小胶质细胞介导的炎症反应探讨淫羊藿总黄酮保护髓鞘的机制  

Mechanism of myelin protection by total flavonoids from Epimedium based on inhibition of microglia-mediated inflammatory response

作　　者：韩庆贤 丁智斌 李晓慧[1] 宋丽娟 王青 柴智[1] 尉杰忠 马存根 HAN Qing-xian;DING Zhi-bin;LI Xiao-hui;SONG Li-juan;WANG Qing;CHAI Zhi;YU Jie-zhong;MA Cun-gen(Shanxi University of Traditional Chinese Medicine,Jinzhong 030619,China;Shanxi Medical University,Jinzhong 030619,China;Shanxi Dotong University,Datong 037009,China)

机构地区：[1]山西中医药大学,晋中030619 [2]山西医科大学,晋中030619 [3]山西大同大学,大同037009

出　　处：《中华中医药杂志》2022年第3期1357-1361,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金面上项目(No.81473577);中国科学院分子发育生物学国家重点实验室开放课题(No.2020-MDB-KF-09);山西省自然科学基金项目(No.201805D111009);山西省应用基础研究计划项目(No.201901D211538);大同市平台基地计划项目(No.2019198);山西省回国留学人员重点科研资助项目(No.2014-重点7)。

摘　　要：目的:研究淫羊藿总黄酮(TFE)能否通过抑制小胶质细胞介导的炎症反应保护髓鞘。方法:BV2细胞、原代小胶质细胞及BMDMs随机分为正常组、LPS组、TFE组,并以1×10;/孔密度接种于6孔板。细胞贴壁后,TFE组细胞给予TFE预处理2 h后,LPS组和TFE组再给予LPS处理24 h。提取培养细胞上清液经ELISA法检测炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和抗炎因子IL-10、转化生长因子-β(TGF-β)分泌;18只C57BL/6小鼠按平均体质量随机分为正常组、CPZ组、TFE组,每组6只。正常组小鼠给予正常饲料饲喂养,另两组小鼠用含0.2%双环己酮草酰二腙(CPZ)的饲料饲育6周。第4周末开始,TFE组小鼠经腹腔注射TFE 2周。同时正常组和CPZ组每日给予等量0.9%氯化钠溶液腹腔注射。采用免疫荧光染色观察MBP、Iba-1、Iba-1;/iNOS;、Iba-1;/Arg1;、Iba-1;/TLR4;、Iba-1;/NF-κB;和PDGF-Rα/Ki67/DAPI表达。结果:体外实验观察到TFE能显著抑制促炎因子TNF-α和IL-1β分泌(P<0.05,P<0.01),增加抗炎因子IL-10与TGF-β分泌(P<0.05)。体内实验经MBP免疫荧光染色发现TFE能显著增加胼胝体尾部MBP荧光强度(P<0.01)。经免疫荧光染色发现TFE降低胼胝体区Iba-1;小胶质细胞数量,并能诱导Arg-1表达增多(P<0.01),降低iNOS表达(P<0.01);并且能降低胼胝体部位TLR4与NF-κB的表达(P<0.01);同时研究还发现TFE能促进侧脑室高表达PDGF-Rα(P<0.01),促进少突胶质前体细胞的增殖。结论:TFE能通过抑制小胶质细胞介导的炎症反应保护髓鞘,可能的机制是通过降低TLR4/NF-κB炎症信号通路发挥作用。Objective: To investigate whether total flavonoids from Epimedium (TFE), which can promote myelin by inhibiting microglia-mediated inflammatory response. Methods: BV2 cells, primary microglia and BMDMs were randomly divided into normal group, LPS group and TFE groups, and seeded in 6-well plates at a density of 1×10;. After cell adherent,cells in the TFE group were pretreatment with TFE for 2 h. Then the LPS group and TFE group were given LPS for another24 h. The supernatant of cultured cells were analyzed for the secretion of the inflammatory factors TNF-α, IL-1β and antiinflammatory factors TGF-β, IL-10 by ELISA;Eighteen C57BL/6 mice were randomly divided into normal group, CPZ group,and TFE group according to average body mass, with 6 mice in each group. The mice in normal group were fed normal feed and the other 2 groups of mice were fed with feed containing 0.2% CPZ for 6 weeks. At the end of weekend 4, mice in the TFE group were intraperitoneally injected with TFE for two weeks. At the same time, the normal group and the CPZ group were given the same amount of 0.9% sodium chloride solution intraperitoneally every day. Expressions of MBP, Iba-1, Iba-1;/iNOS;, Iba-1;/Arg1;, Iba-1;/TLR4;, Iba-1;/NF-κB;and PDGF-Rα/Ki67/DAPI were observed by immunofluorescencestaining.Results: In vitro experiments, TFE was observed to significantly inhibit the secretion of BV2/primary microglia/BMDMs the pro-inflammatory factors TNF-α and IL-1β (P<0.05, P<0.01), and increased the secretion of anti-inflammatory factors IL-10and TGF-β (P<0.05). In vivo experiments with immunofluorescence staining of MBP found that TFE significantly increased the fluorescence intensity of MBP (P<0.01) at the tail of the corpus callosum. Immunofluorescence staining revealed that TFE decreased the number of Iba-1;microglia in the callosal region and induced an increase in Arg-1 expression (P<0.01) and decreased iNOS expression (P<0.01);It could also reduce the expression of TLR4 and NF-κB in the corpus callosum (P<0.01);It was also found th

关 键 词：淫羊藿总黄酮 髓鞘保护 小胶质细胞 炎症反应 Toll样受体4/核因子-κB 

分 类 号：R285[医药卫生—中药学]