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[Role of pyroptosis in bilirubin-induced microglial injury]
Hong-Mei Huang1, Chun-Mei He, Si-Yu Li
1Department of Neonatology, Children's Hospital of Chongqing Medical University/Ministry of Education Key Laboratory of Child Development and Disorders/National Clinical Research Center for Child Health 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. h_ziyu@163.com.
Objective:
To study whether pyroptosis is involved in the bilirubin-induced injury of primary cultured rat cortical microglial cells.
Methods:
Primary cultured rat cortical microglial cells were randomly administered with 30 μmol/L bilirubin (bilirubin group), 30 μmol/L bilirubin following 30 μmol/L VX-765 pretreatment (VX-765+bilirubin group), or an equal volume of dimethyl sulfoxide (control group). Modified MTT assay was used to measure the viability of microglial cells. Western blot was used to measure the expression of the pyroptosis-related proteins Caspase-1 and gasdermin D (GSDMD). Lactate dehydrogenase (LDH)-release assay was used to evaluate the cytotoxicity of microglial cells. EtBr/EthD2 with different molecular weights (394 Da/1 293 Da) was used to measure the size of plasma membrane pores. ELISA was used to measure the level of the inflammatory factor interleukin-1β (IL-1β) in culture supernatant.
Results:
After bilirubin stimulation, the viability of microglial cells decreased and LDH release increased, both in a time-dependent manner. Compared with the control group, the bilirubin group had a significantly higher positive rate of small-molecule EtBr passing through the cell membrane (P<0.001), while there was no significant difference in the pass rate of large-molecule EthD2 between groups (P>0.05). The expression of activated Caspase-1 significantly increased at 0.5 hour after bilirubin stimulation (P<0.05), and that of activated GSDMD significantly increased at 6 hours after bilirubin stimulation (P<0.05). The release of IL-1β significantly increased at 6 hours after bilirubin stimulation and reached the peak at 24 hours (P<0.001). Compared with the bilirubin group, the VX-765+bilirubin group had a significant increase in cell viability (P<0.05) and significant reductions in the expression of activated GSDMD, the pass rate of EtBr, and the release of LDH and IL-1β (P<0.05).
Conclusions:
Pyroptosis is involved in bilirubin-induced injury of primary cultured microglial cells.
Insights
Bilirubin induces injury in rat microglial cells by activating pyroptosis, a cell death pathway. Inhibiting pyroptosis with VX-765 protected these cells from bilirubin-induced damage.
Area of Science:
- Neuroscience
- Cell Biology
- Toxicology
Background:
- Microglial cells are crucial for brain immunity and can be injured by bilirubin.
- Pyroptosis is an inflammatory form of programmed cell death.
- The role of pyroptosis in bilirubin-induced microglial injury remains unclear.
Purpose of the Study:
- To investigate the involvement of pyroptosis in bilirubin-induced injury of primary cultured rat cortical microglial cells.
Main Methods:
- Primary rat microglial cells were treated with bilirubin, with or without VX-765 (a pyroptosis inhibitor).
- Cell viability was assessed using MTT assay.
- Pyroptosis markers (Caspase-1, GSDMD), cytotoxicity (LDH release), membrane pore formation (EtBr/EthD2 assay), and IL-1β release were measured.
Main Results:
- Bilirubin exposure decreased cell viability and increased LDH release and IL-1β secretion in a time-dependent manner.
- Bilirubin induced pyroptosis, evidenced by increased Caspase-1 and GSDMD activation and plasma membrane pore formation.
- VX-765 pretreatment significantly attenuated bilirubin-induced cell injury and pyroptosis markers.
Conclusions:
- Pyroptosis plays a significant role in bilirubin-induced injury to primary cultured microglial cells.
- Targeting pyroptosis may offer a therapeutic strategy for bilirubin-related neurological damage.
