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

Chinese Journal of Contemporary Pediatrics ›› 2024, Vol. 26 ›› Issue (4) : 385-393.

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Chinese Journal of Contemporary Pediatrics ›› 2024, Vol. 26 ›› Issue (4) : 385-393. DOI: 10.7499/j.issn.1008-8830.2312014
EXPERIMENTAL RESEARCH

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
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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.

Key words

Bilirubin / Chaperone-mediated autophagy / Neurotoxicity / Microglia

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PAN Zhi-Fan, LI Si-Yu, LI Ling, ZHANG Yan, HUA Zi-Yu. Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells[J]. Chinese Journal of Contemporary Pediatrics. 2024, 26(4): 385-393 https://doi.org/10.7499/j.issn.1008-8830.2312014

References

1 Olusanya BO, Kaplan M, Hansen TWR. Neonatal hyperbilirubinaemia: a global perspective[J]. Lancet Child Adolesc Health, 2018, 2(8): 610-620. PMID: 30119720. DOI: 10.1016/S2352-4642(18)30139-1.
2 Gottimukkala SB, Lobo L, Gautham KS, et al. Intermittent phototherapy versus continuous phototherapy for neonatal jaundice[J]. Cochrane Database Syst Rev, 2023, 3(3): CD008168. PMID: 36867730. PMCID: PMC9979775. DOI: DOI: 10.1002/14651858.CD008168.pub2.
3 Vodret S, Bortolussi G, Iaconcig A, et al. Attenuation of neuro-inflammation improves survival and neurodegeneration in a mouse model of severe neonatal hyperbilirubinemia[J]. Brain Behav Immun, 2018, 70: 166-178. PMID: 29458193. DOI: 10.1016/j.bbi.2018.02.011.
4 Kaushik S, Cuervo AM. The coming of age of chaperone-mediated autophagy[J]. Nat Rev Mol Cell Biol, 2018, 19(6): 365-381. PMID: 29626215. PMCID: PMC6399518. DOI: 10.1038/s41580-018-0001-6.
5 Muller S, Brun S, René F, et al. Autophagy in neuroinflammatory diseases[J]. Autoimmun Rev, 2017, 16(8): 856-874. PMID: 28572049. DOI: 10.1016/j.autrev.2017.05.015.
6 Qiao L, Ma J, Zhang Z, et al. Deficient chaperone-mediated autophagy promotes inflammation and atherosclerosis[J]. Circ Res, 2021, 129(12): 1141-1157. PMID: 34704457. PMCID: PMC8638823. DOI: 10.1161/CIRCRESAHA.121.318908.
7 Zhang J, Huang J, Gu Y, et al. Inflammation-induced inhibition of chaperone-mediated autophagy maintains the immunosuppressive function of murine mesenchymal stromal cells[J]. Cell Mol Immunol, 2021, 18(6): 1476-1488. PMID: 31900460. PMCID: PMC8167126. DOI: 10.1038/s41423-019-0345-7.
8 Tang J, Zhan MN, Yin QQ, et al. Impaired p65 degradation by decreased chaperone-mediated autophagy activity facilitates epithelial-to-mesenchymal transition[J]. Oncogenesis, 2017, 6(10): e387. PMID: 28991259. PMCID: PMC5668883. DOI: 10.1038/oncsis.2017.85.
9 Chen J, Mao K, Yu H, et al. p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease[J]. J Neuroinflammation, 2021, 18(1): 295. PMID: 34930303. PMCID: PMC8686293. DOI: 10.1186/s12974-021-02349-y.
10 Li M, Song S, Li S, et al. The blockade of NF-κB activation by a specific inhibitory peptide has a strong neuroprotective role in a Sprague-Dawley rat kernicterus model[J]. J Biol Chem, 2015, 290(50): 30042-30052. PMID: 26499797. PMCID: PMC4706004. DOI: 10.1074/jbc.M115.673525.
11 Li S, Huang H, Wei Q, et al. Depression of pyroptosis by inhibiting caspase-1 activation improves neurological outcomes of kernicterus model rats[J]. ACS Chem Neurosci, 2021, 12(15): 2929-2939. PMID: 34296848. DOI: 10.1021/acschemneuro.1c00287.
12 黄洪梅, 何春梅, 李思宇, 等. 细胞焦亡在胆红素诱导小胶质细胞损伤中的作用研究[J]. 中国当代儿科杂志, 2020, 22(9): 1027-1033. PMID: 32933638. PMCID: PMC7499435. DOI: 10.7499/j.issn.1008-8830.2003175.
13 Schiavon E, Smalley JL, Newton S, et al. Neuroinflammation and ER-stress are key mechanisms of acute bilirubin toxicity and hearing loss in a mouse model[J]. PLoS One, 2018, 13(8): e0201022. PMID: 30106954. PMCID: PMC6091913. DOI: 10.1371/journal.pone.0201022.
14 Silva SL, Vaz AR, Barateiro A, et al. Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation[J]. Neurobiol Dis, 2010, 40(3): 663-675. PMID: 20727973. DOI: 10.1016/j.nbd.2010.08.010.
15 Huang H, Li S, Zhang Y, et al. Microglial priming in bilirubin-induced neurotoxicity[J]. Neurotox Res, 2023, 41(4): 338-348. PMID: 37058197. DOI: 10.1007/s12640-023-00643-6.
16 Li L, Li S, Pan Z, et al. Bilirubin impacts microglial autophagy via the Akt-mTOR signaling pathway[J]. J Neurochem, 2023, 167(4): 582-599. PMID: 37858960. DOI: 10.1111/jnc.15984.
17 Kaushik S, Massey AC, Mizushima N, et al. Constitutive activation of chaperone-mediated autophagy in cells with impaired macroautophagy[J]. Mol Biol Cell, 2008, 19(5): 2179-2192. PMID: 18337468. PMCID: PMC2366850. DOI: 10.1091/mbc.e07-11-1155.
18 Bourdenx M, Martín-Segura A, Scrivo A, et al. Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome[J]. Cell, 2021, 184(10): 2696-2714.e25. PMID: 33891876. PMCID: PMC8152331. DOI: 10.1016/j.cell.2021.03.048.
19 Afonina IS, Zhong Z, Karin M, et al. Limiting inflammation-the negative regulation of NF-κB and the NLRP3 inflammasome[J]. Nat Immunol, 2017, 18(8): 861-869. PMID: 28722711. DOI: 10.1038/ni.3772.
20 Wu J, Han Y, Xu H, et al. Deficient chaperone-mediated autophagy facilitates LPS-induced microglial activation via regulation of the p300/NF-κB/NLRP3 pathway[J]. Sci Adv, 2023, 9(40): eadi8343. PMID: 37801503. PMCID: PMC10558133. DOI: 10.1126/sciadv.adi8343.
21 Ercan I, Cilaker Micili S, Soy S, et al. Bilirubin induces microglial NLRP3 inflammasome activation in vitro and in vivo[J]. Mol Cell Neurosci, 2023, 125: 103850. PMID: 36965549. DOI: 10.1016/j.mcn.2023.103850.
22 du Chatinier A, Velilla IQ, Meel MH, et al. Microglia in pediatric brain tumors: the missing link to successful immunotherapy[J]. Cell Rep Med, 2023, 4(11): 101246. PMID: 37924816. PMCID: PMC10694606. DOI: 10.1016/j.xcrm.2023.101246.
23 Giunti D, Parodi B, Cordano C, et al. Can we switch microglia's phenotype to foster neuroprotection? Focus on multiple sclerosis[J]. Immunology, 2014, 141(3): 328-339. PMID: 24116890. PMCID: PMC3930371. DOI: 10.1111/imm.12177.
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