Department of Anesthesiology&Institute of Neuroscience and Brain Diseases, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Jingzhou Road 136, Xiangcheng District, Xiangyang, Hubei, 441021, China.
Xiangyang Maternal and Child Health Hospital, Chunyuan Road 12,Fancheng District, Xiangyang, Hubei, 441021, China.
Mol Biotechnol. 2024 Aug;66(8):2046-2063. doi: 10.1007/s12033-023-00823-x. Epub 2023 Aug 12.
Cerebral ischemia/reperfusion injury (CIRI) involves various pathogenic mechanisms, including cytotoxicity, apoptosis, inflammation, and pyroptosis. Stromal interactive molecule 2 (STIM2) is implicated in cerebral ischemia. Consequently, this study investigates the biological functions of STIM2 and its related mechanisms in CIRI progression. Middle cerebral artery occlusion/reperfusion (MCAO/R) mouse models and oxygen-glucose deprivation/reoxygenation (OGD/R) cellular models were established. STIM2 level was upregulated in experimental CIRI models, as shown by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunofluorescence staining. Brain infarction and edema were attenuated by STIM2 knockdown, as 2,3,5-triphenyltetrazolium chloride (TTC) staining and brain water content evaluation revealed. STIM2 knockdown relieved neuronal apoptosis, microglia activation, inflammation and pyroptosis in MCAO/R mice, as detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, enzyme-linked immunosorbent assay (ELISA) and western blotting. Results of flow cytometry, ELISA, western blotting and cell counting kit-8 (CCK-8) assays also showed that STIM2 knockdown inhibited inflammation, apoptosis and pyroptosis in OGD/R-treated BV2 cells. Moreover, STIM2 knockdown inhibited apoptosis and pyroptosis in PC12 cells incubated with conditioned medium collected from OGD/R-exposed BV2 cells. Mechanistically, lncRNA Malat1 (metastasis associated lung adenocarcinoma transcript 1) positively regulated STIM2 expression by sponging miR-30d-5p. Their binding relationship was confirmed by luciferase reporter assays. Finally, lncRNA Malat1 elevation or miR-30d-5p knockdown abolished the sh-STIM2-induced inhibition in cell damage. In conclusion, STIM2 knockdown in microglia alleviates CIRI by inhibiting microglial activation, inflammation, apoptosis, and pyroptosis.
脑缺血/再灌注损伤（CIRI）涉及多种致病机制，包括细胞毒性、细胞凋亡、炎症和细胞焦亡。基质相互作用分子 2（STIM2）与脑缺血有关。因此，本研究探讨了 STIM2 在 CIRI 进展中的生物学功能及其相关机制。建立了大脑中动脉闭塞/再灌注（MCAO/R）小鼠模型和氧葡萄糖剥夺/再氧合（OGD/R）细胞模型。通过逆转录定量聚合酶链反应（RT-qPCR）、Western blot 和免疫荧光染色显示，实验性 CIRI 模型中 STIM2 水平上调。TTC 染色和脑水含量评估显示，STIM2 敲低减轻了脑梗死和脑水肿。STIM2 敲低减轻了 MCAO/R 小鼠中的神经元凋亡、小胶质细胞激活、炎症和细胞焦亡，通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）染色、酶联免疫吸附测定（ELISA）和 Western blot 检测。流式细胞术、ELISA、Western blot 和细胞计数试剂盒-8（CCK-8）检测的结果还表明，STIM2 敲低抑制了 OGD/R 处理的 BV2 细胞中的炎症、凋亡和细胞焦亡。此外，STIM2 敲低抑制了在 OGD/R 暴露的 BV2 细胞条件培养基孵育的 PC12 细胞中的凋亡和细胞焦亡。机制上，lncRNA Malat1（转移相关肺腺癌转录本 1）通过海绵 miR-30d-5p 正向调节 STIM2 的表达。通过荧光素酶报告基因实验证实了它们的结合关系。最后，lncRNA Malat1 升高或 miR-30d-5p 敲低消除了 sh-STIM2 诱导的细胞损伤抑制作用。总之，小胶质细胞中的 STIM2 敲低通过抑制小胶质细胞激活、炎症、细胞凋亡和细胞焦亡来减轻 CIRI。
