TLR2介导小胶质细胞激活和神经炎症促进血管性痴呆进展的分子机制研究  

Effect of TLR2-mediated Microglia Activation and Neuroinflammation on Vascular Dementia Progression

作　　者：孙洋子 伍贞宇 曾超胜 游咏 SUN Yangzi;WU Zhenyu;ZENG Chaosheng;YOU Yong(Department of Neurology,the Second Affiliated Hospital of Hainan Medical College,Haikou,570000,China;Department of Neurosurgery,the Second Affiliated Hospital of Hainan Medical College,Haikou,570000,China)

机构地区：[1]海南医学院第二附属医院神经内科,海口市570000 [2]海南医学院第二附属医院神经外科,海口市570000

出　　处：《医学分子生物学杂志》2024年第4期315-322,共8页Journal of Medical Molecular Biology

基　　金：海南省医药卫生科研项目(No.21A200002)。

摘　　要：目的探讨TLR2是否在血管性痴呆(vascular dementia,VaD)中参与介导小胶质细胞的病理性激活和神经损伤及其分子机制。方法体外实验使用LPS-诱导激活人小胶质细胞系HMC3细胞。CCK-8法测定HMC3细胞增殖能力。构建腺病毒Adv-TLR2 shRNA和Adv-shRNA NT载体并介导全身性敲低TLR2。通过永久性双侧颈总动脉闭塞(2VO)建立VaD大鼠模型。蛋白质印迹法测定HMC3细胞和大鼠脑白质组织中TLR2、NF-κB、Iba-1、Claudin-5和ZO-1的表达水平。ELISA法测定大鼠脑白质组织中炎症因子IL-6、IL-1β和TNF-α的水平以及铁死亡相关指标Fe2+离子、丙二醛(MDA)和谷胱甘肽(GSH)的水平。Morris水迷宫测试大鼠脑神经损伤。结果体外细胞实验,与对照组相比,脂多糖组细胞内TLR2、NF-κB和Iba-1表达水平增强(P<0.05);与脂多糖组相比,腺病毒-TLR2 shRNA敲低组逆转了上述指标的表达水平(P<0.05)。在体大鼠模型实验,与假手术组相比,模型组大鼠脑白质组织中TLR2/NF-κB和Iba-1表达水平上调,Claudin-5和ZO-1的表达水平降低(P<0.05);炎症因子IL-6、IL-1β和TNF-α水平上调(P<0.05);Fe2+离子和MDA水平升高,GSH水平降低(P<0.05);与模型组相比,模型+腺病毒-TLR2 shRNA敲低组逆转了上述指标的水平(P<0.05)。Morris水迷宫测试,与假手术组相比,模型组和模型+腺病毒-TLR2 shRNA敲低组大鼠找到目标象限(左上象限)所用时长较长(P<0.05),停留在目标象限(左上象限)的时间百分比(%)缩短(P<0.05),大鼠在MWM中4个象限的游泳路线分布较为均匀;与模型组相比,模型+腺病毒-TLR2 shRNA敲低组逆转了上述指标并且在MWM中目标象限(左上象限)的游泳路线分布较为密集。结论敲低TLR2可抑制小胶质细胞的激活及其介导的紧密连接蛋白降解和脑血管屏障网络渗漏,降低血管性痴呆大鼠模型的神经损伤。Objective To investigate the effect of TLR2 in mediating the pathological activation of microglia and neuronal injury in vascular dementia(VaD)rat model and its molecular mechanisms.Methods In vitro experiments were carried out by using LPS-induced activation of human microglia cell line HMC3 cells.The proliferation ability of HMC3 cells was determined by CCK-8 assay.Systemic TLR2 knockdown was performed by using Adv-TLR2 shRNA or Adv-shRNA NT.The VaD rat model was established by permanent bilateral common carotid artery occlusion(2VO).The expression levels of TLR2,NF-κB,Iba-1,Claudin-5 and ZO-1 in HMC3 cells and rats’white matter tissues were determined by Western blotting.The levels of inflammatory cytokines IL-6,IL-1βand TNF-α,and the ferroptosis-related indicators of Fe2+,malondialdehyde(MDA)and glutathione(GSH)in rats’white matter tissues were determined by ELISA.Morris water maze(MWM)test was used to determine rats’brain neuronal injury.Results The expression levels of TLR2,NF-κB and Iba-1 in the LPS group were enhanced when compared with those in the Control group(P<0.05).The expression levels of the above proteins in the Adv-TLR2 shRNA group were decreases when compared with those in the LPS groups(P<0.05).The expression levels of TLR2/NF-κB and Iba-1 were up-regulated,and those of Claudin-5 and ZO-1 were down-regulated in the white matter tissues of Model group rats,when compared with those of the Sham group,(P<0.05).In addition,the levels of inflammatory cytokines IL-6,IL-1βand TNF-αwere up-regulated(P<0.05),the Fe2+and MDA levels were increased,and the GSH levels were decreased(P<0.05).TLR2 knockdown had reversed the values of above indicators in the Model+Adv-TLR2 shRNA group when compared with those in the Model group(P<0.05).Morris water maze test showed that rats in the Model group and Model+Adv-TLR2 shRNA group took longer time to find the target quadrant(upper left quadrant)(P<0.05),stayed shorter time in the target quadrant(P<0.05)when compared with those in the Sham group,the swimm

关 键 词：血管性痴呆 炎症 TOLL样受体2 小胶质细胞 铁死亡 

分 类 号：R749.16[医药卫生—神经病学与精神病学]