分子伴侣介导的自噬对胆红素诱导的小鼠小胶质细胞损伤的影响  

Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells

作　　者：潘知繁 李思宇 李玲 张燕[1] 华子瑜[1] PAN Zhi-Fan;LI Si-Yu;LI Ling;ZHANG Yan;HUA Zi-Yu(Department of Neonatology,Children's Hospital of Chongqing Medical University/National Clinical Research Center for Child Health and Disorders/Ministry of Education Key Laboratory of Child Development and Disorders/China International Science and Technology Cooperation Base of Child Development and Critical Disorders/Chongqing Key Laboratory of Child Infection and Immunity,Chongqing 400013,China)

机构地区：[1]重庆医科大学附属儿童医院新生儿科/国家儿童健康与疾病临床医学研究中心/儿童发育疾病研究教育部重点实验室/儿童发育重大疾病国家国际科技合作基地/儿童感染免疫重庆市重点实验室,重庆400013

出　　处：《中国当代儿科杂志》2024年第4期385-393,共9页Chinese Journal of Contemporary Pediatrics

基　　金：国家自然科学基金项目(81971426);重庆市技术创新与应用发展专项重点项目(CSTB2022TIAD-KPX0147)。

摘　　要：目的探讨分子伴侣介导的自噬(chaperone-mediated autophagy,CMA)对未结合胆红素(unconjugated bilirubin,UCB)诱导的小鼠小胶质细胞BV2损伤的影响。方法BV2细胞实验分为两部分。(1)CMA激活实验分为:对照组(等体积二甲基亚砜处理)、QX77组(20μmol/L QX77处理24 h)、UCB组(40μmol/L UCB处理24 h)、UCB+QX77组(20μmol/L QX77和40μmol/L UCB共处理24 h)。(2)细胞转染实验分为:LAMP2A沉默对照组(等体积二甲基亚砜处理)、LAMP2A沉默对照+UCB组(40μmol/L UCB处理24 h)、LAMP2A沉默组(等体积二甲基亚砜处理)、LAMP2A沉默+UCB组(40μmol/L UCB处理24 h)。采用改良MTT法检测细胞存活率,蛋白免疫印迹法检测p65、核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)、半胱氨酸天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,caspase-1)蛋白表达水平,实时荧光定量聚合酶链反应法检测炎症因子白细胞介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)mRNA相对表达量,ELISA法检测细胞培养上清液中炎症因子IL-6和TNF-α水平,免疫荧光法检测细胞内p65、NLRP3与热休克同源蛋白70的荧光共定位。结果与UCB组相比,UCB+QX77组细胞存活率升高,炎症相关蛋白p65、NLRP3、caspase-1表达水平降低,炎症因子IL-1β、IL-6、TNF-αmRNA相对表达量降低以及IL-6、TNF-α水平降低(P<0.05)。与对照组相比,UCB组和UCB+QX77组热休克同源蛋白70与p65、NLRP3存在共定位。沉默LAMP2A基因后,与LAMP2A沉默对照+UCB组相比,LAMP2A沉默+UCB组炎症相关蛋白p65、NLRP3、caspase-1蛋白表达水平升高,炎症因子IL-1β、IL-6、TNF-αmRNA相对表达量升高以及IL-6、TNF-α水平升高(P<0.05)。结论CMA在UCB诱导的BV2细胞损伤中被抑制,激活CMA可能通过降低p65和NLRP3蛋白水平,抑制炎症反应,拮抗胆红素神经毒性。Objective To investigate the effect of chaperone-mediated autophagy(CMA)on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin(UCB).Methods The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide),QX77 group (treated with 20 μmol/L QX77 for 24 hours), UCB group (treated with 40 μmol/L UCB for 24 hours), andUCB+QX77 group (treated with both 20 μmol/L QX77 and 40 μmol/L UCB for 24 hours). (2) For the cell transfectionexperiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencingcontrol+UCB group (treated with 40 μmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volumeof dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 μmol/L UCB for 24 hours). The cellviability was assessed using the modified MTT method. The expression levels of p65, nucleotide-bindingoligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) weredetected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1β, IL-6,and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF- α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognateprotein 70 with p65 and NLRP3 was detected by immunofluorescence. Results Compared to the UCB group, the cellviability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3,and caspase-1, as well as the mRNA relative expression levels of IL-1β, IL-6, and TNF-α and levels of IL-6 and TNF-αdecreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2Asilencing control+UCB group, the

关 键 词：胆红素 分子伴侣介导的自噬 神经毒性 小胶质细胞 

分 类 号：R722.17[医药卫生—儿科]