中国当代儿科杂志2024，Vol.26Issue(4)：385-393,9.DOI:10.7499/j.issn.1008-8830.2312014
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
摘要
Abstract
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),and UCB+QX77 group(treated with both 20 μmol/L QX77 and 40 μmol/L UCB for 24 hours).(2)For the cell transfection experiment:LAMP2A silencing control group(treated with an equal volume of dimethyl sulfoxide),LAMP2A silencing control+UCB group(treated with 40 μmol/L UCB for 24 hours),LAMP2A silencing group(treated with an equal volume of dimethyl sulfoxide),and LAMP2A silencing+UCB group(treated with 40 μmol/L UCB for 24 hours).The cell viability was assessed using the modified MTT method.The expression levels of p65,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),and cysteinyl aspartate specific proteinase-1(caspase-1)were detected by Western blot.The relative mRNA expression levels of the inflammatory cytokines interleukin(IL)-l β,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 cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence.Results Compared to the UCB group,the cell viability 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 p65 and NLRP3 in both the UCB and UCB+QX77 groups.After silencing the LAMP2A gene,compared to the LAMP2A silencing control+UCB group,the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65,NLRP3,and caspase-1,as well as increased mRNA relative expression levels of IL-1 β,IL-6,and TNF-α and levels of IL-6 and TNF-α(P＜0.05).Conclusions CMA is inhibited in UCB-induced BV2 cell damage,and activating CMA may reduce p65 and NLRP3 protein levels,suppress inflammatory responses,and counteract bilirubin neurotoxicity.[Chinese Journal of Contemporary Pediatrics,2024,26(4):385-393]关键词
胆红素/分子伴侣介导的自噬/神经毒性/小胶质细胞Key words
Bilirubin/Chaperone-mediated autophagy/Neurotoxicity/Microglia引用本文复制引用
潘知繁,李思宇,李玲,张燕,华子瑜..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[J].中国当代儿科杂志,2024,26(4):385-393,9.基金项目
国家自然科学基金项目(81971426) （81971426）
重庆市技术创新与应用发展专项重点项目(CSTB2022TIAD-KPX0147). （CSTB2022TIAD-KPX0147）
