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.
目的: 探讨细胞焦亡是否参与胆红素诱导原代培养大鼠大脑皮质小胶质细胞的损伤。
方法: 原代培养大鼠大脑皮质小胶质细胞，随机分为胆红素组（30 μmol/L胆红素刺激）、VX-765+胆红素组（30 μmol/L VX-765预处理1 h，再用30 μmol/L胆红素刺激）、对照组（等体积二甲基亚砜处理）。改良MTT法检测小胶质细胞存活率；Western blot法检测焦亡相关蛋白半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）、gasdermin D（GSDMD）表达；乳酸脱氢酶（LDH）释放实验检测细胞毒性作用；不同分子量染料EtBr/EthD2（分子量394 Da/1 293 Da）染色检测细胞膜孔大小；ELISA法检测细胞培养上清液中炎症因子IL-1β水平。
结果: 胆红素刺激后，小胶质细胞存活率随时间依赖性下降；LDH释放呈时间依赖性增加；胆红素组小分子染料EtBr通过细胞膜阳性率明显高于对照组（P < 0.001），但各组大分子染料EthD2通过率比较差异无统计学意义（P > 0.05）；胆红素刺激后0.5 h活化型Caspase-1、6 h活化型GSDMD表达增加（P < 0.05）；IL-1β水平在胆红素刺激后6 h明显增加，24 h达高峰（P < 0.001）。与胆红素组相比，VX-765+胆红素组细胞存活率升高（P < 0.05），活化型GSDMD表达、EtBr通过率、LDH及IL-1β释放均减少（P < 0.05）。
结论: 细胞焦亡参与胆红素诱导的原代培养小胶质细胞损伤。