Xinxiang Key Laboratory of Forensic Toxicology, School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China.
The Second Affiliated Hospital of Xinxiang Medical University, Henan Mental Hospital, Xinxiang, Henan, China.
PLoS One. 2022 Sep 1;17(9):e0274057. doi: 10.1371/journal.pone.0274057. eCollection 2022.
The present study explored whether pyroptosis is involved in the injury process of PC12 cells induced by glucocorticoid (GC) and the regulatory relationship between endoplasmic reticulum stress (ERS) and pyrolysis.
LDH leakage of PC12 cells was detected by LDH assay. The number of dead cells was detected by SYTOX green nucleic acid staining. The levels of IL-1β and IL-18 in the supernatants was detected by ELSIA assay. The expression levels of glucose regulated protein 78 (GRP78), cleaved gasdermin D-NT (cleaved-GSDMD-NT), NLR-pyrin domain-containing 3 (NLRP3) and cleaved-caspase-1 were observed by immunofluorescence staining and western blot.
The LDH assay revealed that GC exposure significantly increased the release of LDH. The results of SYTOX green acid staining showed that GC exposure significantly increased the number of SYTOX green acid-positive cells. The ELSIA assay revealed that GC exposure significantly increased the levels of IL-1β and IL-18 in the supernatants. The results of immunofluorescence staining and western blot showed that GC exposure significantly increased the expression of GRP78, cleaved-GSDMD-NT, NLRP3 and cleaved caspase-1. Treatment with the ERS inhibitor tauroursodeoxycholate (TUDCA) and siRNA GSDMD attenuated related damage and downregulated the expression of the abovementioned proteins.
The present study clearly demonstrated that GC exposure can induce GSDMD-dependent pyrolysis, and ERS is involved in the above damage process.
本研究探讨了细胞焦亡是否参与了糖皮质激素（GC）诱导的 PC12 细胞损伤过程，以及内质网应激（ERS）与细胞焦亡之间的调控关系。
通过 LDH 检测试剂盒检测 PC12 细胞 LDH 漏出情况，SYTOX 绿色核酸染色法检测死亡细胞数量，ELISA 试剂盒检测上清液中白介素-1β（IL-1β）和白介素-18（IL-18）的水平，免疫荧光染色和 Western blot 观察葡萄糖调节蛋白 78（GRP78）、天冬氨酸特异性半胱氨酸蛋白酶-1 剪切体（cleaved-GSDMD-NT）、NLR 家族含pyrin 结构域蛋白 3（NLRP3）和 cleaved-caspase-1 的表达水平。
LDH 检测试剂盒结果显示，GC 暴露显著增加了 LDH 的释放。SYTOX 绿色酸染色结果显示，GC 暴露显著增加了 SYTOX 绿色酸阳性细胞的数量。ELISA 试剂盒结果显示，GC 暴露显著增加了上清液中 IL-1β和 IL-18 的水平。免疫荧光染色和 Western blot 结果显示，GC 暴露显著增加了 GRP78、cleaved-GSDMD-NT、NLRP3 和 cleaved-caspase-1 的表达。用内质网应激抑制剂牛磺熊脱氧胆酸（TUDCA）和 GSDMD siRNA 处理可减轻相关损伤，并下调上述蛋白的表达。
本研究明确表明，GC 暴露可诱导 GSDMD 依赖性细胞焦亡，ERS 参与了上述损伤过程。
