Sun Yashu, Luo Lan, Li XiaoYan, Zhang Bing
Department of Anesthesiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China.
Neural Plast. 2025 Aug 28;2025:6776608. doi: 10.1155/np/6776608. eCollection 2025.
Astrocytes play a crucial role in ensuring neuronal survival and function. In stroke, astrocytes trigger the unfolded protein response (UPR) to restore endoplasmic reticulum homeostasis. Mesencephalic astrocyte-derived neurotrophic factor (MANF), a newly identified endoplasmic reticulum stress-induced neurotrophic factor, attenuates cerebral ischemic injury by reducing inflammatory responses. The mechanisms by which astrocytes regulate MANF expression and the role of MANF in modulating inflammation remain to be elucidated. In this study, we constructed middle cerebral artery occlusion (MCAO)/reperfusion model in C57BL/6J mice and an oxygen glucose deprivation/reoxygenation model in a neuronal and astrocyte coculture system. The present study utilized an intraventricular injection of adeno-associated virus (AAV) to effectively block the PERK pathway in astrocytes. Moreover, MANF-siRNA was employed to suppress endogenous MANF expression, while rhMANF was used as an exogenous supplement. 2,3,5-Triphenyltetrazolium chloride (TTC), modified neurological severity score (mNSS), adhesive removal test, Golgi staining, hematoxylin-eosin (HE) staining, western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to evaluate the protective effects of PERK pathway and the expression of MANF in astrocytes. In vitro experiments, ELISA, cell counting kit-8 (CCK-8), and western blot were used to detect the mechanisms by which MANF regulates neuroinflammation. The results showed that blocking the astrocytic PERK pathway decreased MANF expression, aggravated synaptic loss, and exacerbated infarct volume and neurological outcomes. Conversely, cellular experiments showed that activation of PERK increased MANF expression, promoted synaptic protein expression, and increased neuronal cell viability. Additionally, increasing exogenous MANF inhibited STAT3 phosphorylation, reduced the release of inflammatory factors, and improved neuronal cell viability. In conclusion, our study demonstrates that after stroke, astrocytes activate PERK and upregulate MANF expression, which inhibits STAT3 phosphorylation, reduces proinflammatory cytokine release, rescues neuronal synapse loss, and promotes the recovery of neurological function in mice.
星形胶质细胞在确保神经元存活和功能方面发挥着关键作用。在中风中，星形胶质细胞触发未折叠蛋白反应（UPR）以恢复内质网稳态。中脑星形胶质细胞源性神经营养因子（MANF）是一种新发现的内质网应激诱导的神经营养因子，通过减少炎症反应减轻脑缺血损伤。星形胶质细胞调节MANF表达的机制以及MANF在调节炎症中的作用仍有待阐明。在本研究中，我们构建了C57BL/6J小鼠大脑中动脉闭塞（MCAO）/再灌注模型以及神经元和星形胶质细胞共培养系统中的氧糖剥夺/复氧模型。本研究采用脑室内注射腺相关病毒（AAV）有效阻断星形胶质细胞中的PERK通路。此外，使用MANF-siRNA抑制内源性MANF表达，而重组人MANF（rhMANF）用作外源性补充剂。应用2,3,5-三苯基氯化四氮唑（TTC）、改良神经功能缺损评分（mNSS）、黏附去除试验、高尔基染色、苏木精-伊红（HE）染色、蛋白质免疫印迹法和酶联免疫吸附测定（ELISA）来评估PERK通路的保护作用以及星形胶质细胞中MANF的表达。在体外实验中，使用ELISA、细胞计数试剂盒-8（CCK-8）和蛋白质免疫印迹法检测MANF调节神经炎症的机制。结果表明，阻断星形胶质细胞的PERK通路会降低MANF表达，加重突触损失，并加剧梗死体积和神经功能结果。相反，细胞实验表明，激活PERK会增加MANF表达，促进突触蛋白表达，并提高神经元细胞活力。此外，增加外源性MANF可抑制信号转导和转录激活因子3（STAT3）磷酸化，减少炎症因子释放，并提高神经元细胞活力。总之，我们的研究表明，中风后，星形胶质细胞激活PERK并上调MANF表达，这抑制了STAT3磷酸化，减少促炎细胞因子释放，挽救神经元突触损失，并促进小鼠神经功能的恢复。
