[Role of pyroptosis in bilirubin-induced microglial injury].
[Role of pyroptosis in bilirubin-induced microglial injury].
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DOI：
10.7499/j.issn.1008-8830.2003175
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发表时间：
2020-09
期刊：
影响因子：
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通讯作者：
Hongjian Huang;Chun-mei He;Si-Yu Li;Yan Zhang;Zi-Yu Hua
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文献类型：
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作者：
Hongjian Huang;Chun-mei He;Si-Yu Li;Yan Zhang;Zi-Yu Hua
目的研究焦亡是否参与了胆红素对原代培养的大鼠皮层小胶质细胞的损伤。方法原代培养的大鼠皮质小胶质细胞随机分为3组：30 μ mol/L胆红素组（胆红素组）、30 μ mol/L胆红素加30 μmol/LVX-765预处理组（VX-765+胆红素组）和等体积二甲基亚砜组（对照组）。采用改良MTT法检测小胶质细胞活力。Western blot检测细胞内Caspase-1和Gasdermin D（GSDMD）的表达。采用乳酸脱氢酶（LDH）释放法检测小胶质细胞的细胞毒性。用不同分子量（394 Da/1 293 Da）的EtBr/EthD 2测定质膜孔的大小。ELISA法检测培养上清中炎症因子白细胞介素1β（IL-1β）的含量。结果胆红素刺激后，小胶质细胞活力下降，LDH释放增加，两者均呈时间依赖性。与对照组相比，胆红素组小分子EtBr穿膜阳性率明显增高（P0. 05）。胆红素刺激后0.5h，活化Caspase-1表达显著增加（P<0.05）;胆红素刺激后6 h，活化GSDMD表达显著增加（P<0.05）。IL-1β的释放在胆红素刺激后6 h显著增加，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显著升高（P<0.05），活化GSDMD表达、EtBr通过率、LDH和IL-1β释放显著降低（P<0.05）。结论：焦亡参与了胆红素对原代培养的小胶质细胞的损伤。
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 (P0.05). 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.