Objectives: 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)-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 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-α decreased (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-1β, 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.
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（20 μmol/L QX77处理24 h）、UCB组（40 μmol/L UCB处理24 h）、UCB+QX77组（20 μmol/L QX77和40 μmol/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（40 μmol/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（40 μmol/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-α mRNA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-α mRNA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。.
Keywords: Bilirubin; Chaperone-mediated autophagy; Microglia; Neurotoxicity.