Department of Pharmacy, Nanjing Drum Tower Hospital, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China; Department of Physiology, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China.
Department of Physiology, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China.
Int Immunopharmacol. 2024 Jan 25;127:111396. doi: 10.1016/j.intimp.2023.111396. Epub 2023 Dec 21.
Microglia, resident brain immune cells, is critical in inflammation, apoptosis, neurogenesis and neurological recovery during cerebral ischemia/reperfusion (I/R) injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF), a novel identified endoplasmic reticulum stress-inducible neurotrophic factor, can alleviate I/R injury by reducing the inflammatory reaction, but its specific regulatory mechanism on microglia after ischemic stroke has not been fully clarified. To mimic the process of ischemia/reperfusion in vivo and in vitro, middle cerebral artery occlusion/reperfusion (MCAO/R) was induced in C57BL/6J mice and oxygen glucose deprivation/reoxygenation (OGD/R) model was established in BV-2 cells. Moreover, MANF small interfering RNA (siRNA) was used to silence the expression of endogenous MANF, while recombination human MANF protein (rhMANF) acted as an exogenous supplement. Seventy-two hours after MCAO/R, 2,3,5-triphenyltetrazolium staining, neurological scores, brain water content, immunohistochemical staining, immunofluorescent staining, flow cytometry, hematoxylin and eosin staining, quantitative real-time PCR and western blot are applied to evaluate the protective effect and possible mechanism of MANF on cerebral I/R injury. In vitro, cell viability, inflammatory cytokines and the expression of MANF, A20, NF-κB and the markers of microglia were analyzed. The results showed that MANF decreased brain infarct volume, neurological scores, and brain water content. In addition, MANF promoted the polarization of microglia to an anti-inflammatory phenotype both in vivo and in vitro, which are related to A20/NF-κB pathway. In summary, MANF may offer novel therapeutic approaches for ischemic stroke in the process of microglia polarization.
小胶质细胞是驻留于脑内的免疫细胞，在脑缺血/再灌注（I/R）损伤过程中的炎症、细胞凋亡、神经发生和神经功能恢复中起关键作用。脑源性神经营养因子（MANF）是一种新发现的内质网应激诱导的神经营养因子，通过减轻炎症反应，减轻 I/R 损伤，但它在缺血性脑卒中后对小胶质细胞的具体调节机制尚未完全阐明。为了模拟体内和体外的缺血/再灌注过程，在 C57BL/6J 小鼠中诱导大脑中动脉闭塞/再灌注（MCAO/R），并在 BV-2 细胞中建立氧葡萄糖剥夺/再氧合（OGD/R）模型。此外，使用 MANF 小干扰 RNA（siRNA）沉默内源性 MANF 的表达，同时重组人 MANF 蛋白（rhMANF）作为外源性补充。MCAO/R 后 72 小时，通过 2,3,5-三苯基四氮唑染色、神经学评分、脑水含量、免疫组织化学染色、免疫荧光染色、流式细胞术、苏木精和伊红染色、实时定量 PCR 和 Western blot 评估 MANF 对脑 I/R 损伤的保护作用及可能机制。体外，分析细胞活力、炎症细胞因子以及 MANF、A20、NF-κB 和小胶质细胞标志物的表达。结果表明，MANF 降低了脑梗死体积、神经学评分和脑水含量。此外，MANF 促进了小胶质细胞在体内和体外向抗炎表型的极化，这与 A20/NF-κB 通路有关。综上所述，MANF 可能为小胶质细胞极化过程中的缺血性脑卒中提供新的治疗方法。
