小胶质细胞BV2支架蛋白PDLIM5对α7烟碱乙酰胆碱受体的免疫调节作用  

The immunoregulatory role of scaffold protein PDLIM5 onα7 nicotinic acetylcholine receptor in microglial cells BV2

作　　者：阿斯汝 王文慧 平苏宁 陈海莲 陈元 Asiru;WANG Wenhui;PING Suning;CHEN Hailian;CHEN Yuan(Neurobiology Research Center,School of Medicine,Sun Yat-sen University,Shenzhen,Guangdong 518107,China)

机构地区：[1]中山大学医学院神经生物研究中心,广东深圳518107

出　　处：《热带医学杂志》2025年第1期12-18,共7页Journal of Tropical Medicine

基　　金：广东省自然科学基金(2022A1515012314);深圳市科技计划项目(JCYJ20220530145615034)。

摘　　要：目的探讨支架蛋白PDLIM5在小鼠小胶质细胞系BV2中对α7烟碱乙酰胆碱受体(α7nAChRs)的免疫调节作用,为α7nAChRs的新药物靶点研发及临床治疗提供新方向。方法设置BV2细胞对照组、尼古丁(100μmol/L,12 h)处理组及α-银环蛇毒素(10 nmol/L,12 h)处理组。在BV2细胞中敲低或过表达PDLIM5,并进行尼古丁处理,利用蛋白免疫印迹、酶联免疫吸附实验(ELISA)、实时荧光定量PCR等技术检测部分相关炎症因子[抗肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、IL-1β、IL-4、IL-10]、PDLIM5及α7nAChRs的核酸及蛋白变化。结果免疫印迹结果显示,无尼古丁作用时,敲低PDLIM5后,TNF-α表达水平显著降低,IL-10水平显著升高,差异均有统计学意义(t=11.13、11.06,P均<0.001);过表达PDLIM5后,TNF-α表达水平升高,IL-10表达水平降低,差异均有统计学意义(t=11.11、11.20,P均<0.001);敲低或过表达PDLIM5,α7nAChRs总蛋白未改变。此外,单独使用α-银环蛇毒素处理(10 nmol/L,12 h)可以抑制α7nAChRs及PDLIM5蛋白表达(t=6.93、5.36,P均<0.01),并上调TNF-α表达(t=8.70,P<0.05),差异均有统计学意义。免疫印迹与ELISA结果显示,仅加入尼古丁时(1、10、100μmol/L,12 h),PDLIM5表达会随尼古丁浓度升高上调(F=80.01,P<0.0001),而TNF-α表达则随着尼古丁浓度升高下调(F=456.60,P<0.0001),细胞上清中的TNF-α水平也显著降低(F=90.24,P<0.05),差异均有统计学意义,IL-10无明显变化。实时荧光PCR显示,尼古丁处理后,IL-10 mRNA表达上调(t=3.92,P<0.001),IL-6、IL-1β、TNF-α表达下调(t=7.31、4.26、7.34,P均<0.05),差异均有统计学意义,IL-4无明显变化。敲低PDLIM5后加入尼古丁处理,TNF-α蛋白表达显著减少(F=142.00,P<0.001),细胞上清中TNF-α水平显著降低(F=137.10,P<0.001),IL-10水平升高(F=131.90,P<0.0001),差异均有统计学意义。过表达PDLIM5后加入尼古丁处理,TNF-α蛋白表达显著降低(F=296.70,P<0.0001),细胞上清中TNF-α水平显著降低(F=34Objective To investigate the immunoregulatory role of the scaffold protein PDLIM5 on alpha7 nicotinic acetylcholine receptors(α7nAChRs)in the BV2 microglial cell line of mice.Methods BV2 cells were divided into control group,nicotine treatment(100μmol/L for 12 h)group,andα-bungarotoxin treatment(10 nmol/L for 12 h)groups.PDLIM5 was knocked down or overexpressed in BV2 cells,followed by nicotine treatment.Changes in nucleic acid and protein levels of inflammatory cytokines[tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β,IL-4,IL-10],PDLIM5,andα7nAChRs were analyzed using Western blotting,ELISA,and RT-qPCR.Results Western blot results indicate that,in the absence of nicotine,knockdown of PDLIM5 resulted in a significant decrease in TNF-αprotein expression(t=11.13,P<0.001)and a significant increase in IL-10 protein expression(t=11.06,P<0.001).Conversely,PDLIM5 overexpression results in elevated TNF-αexpression(t=11.11,P<0.001)and decreased IL-10 expression(t=11.20,P<0.001).Knockdown or overexpression of PDLIM5 both did not alter the total protein levels ofα7 nicotinic acetylcholine receptor.Additionally,treatment withα-bungarotoxin(10 nmol/L,12 h)alone suppressed the protein expression of bothα7nAChRs and PDLIM5(t=6.93,5.36;all P<0.01),and upregulated the expression of TNF-α(t=8.70,P<0.05).Immunoblotting and ELISA results showed that nicotine treatment(1,10,100μmol/L,12 h)upregulated PDLIM5 expression in a dose-dependent manner(F=80.01,P<0.0001),while TNF-αexpression was downregulated with increasing nicotine concentration(F=456.60,P<0.0001).The concentration of TNF-αin the cell supernatant was also significantly reduced(F=90.24,P<0.05),with no significant change in IL-10.Real-time PCR analysis showed that nicotine treatment significantly upregulated the mRNA expression of IL-10(t=3.92,P<0.001),while no significant change was observed in IL-4,and the expressions of IL-6,IL-1β,and TNF-αwere significantly downregulated(t=7.31,4.26,7.34;all P<0.01).After knocking down PDLIM5 and treating with

关 键 词：α7烟碱乙酰胆碱受体 PDLIM5 小胶质细胞 

分 类 号：R392[医药卫生—免疫学]