Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells
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
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)-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.
PAN Zhi-Fan,LI Si-Yu,LI Ling et al. Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells[J]. CJCP, 2024, 26(4): 385-393.
PAN Zhi-Fan,LI Si-Yu,LI Ling et al. Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells[J]. CJCP, 2024, 26(4): 385-393.
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