Orexin-A (OXA) is a neuropeptide with neuroprotective effect by reducing cerebral ischemia/reperfusion injury (CIRI). Inflammation and apoptosis mediated by astrocyte activation are the key pathological mechanisms for CIRI. We thus attempted to confirm neuroprotective effects of OXA on astrocytic inflammation and apoptosis in CIRI and clarify the relative mechanisms. A middle cerebral artery occlusion and reperfusion (MCAO/R) rat model and U251 glioma cells model subjected to oxygen glucose deprivation and reperfusion (OGD/R) were established, with or without OXA treatment. Neurological deficit score was determined, and cerebral infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western Blot was used to detect the expressions of NF-κB p65, p-p65, p-ERK, p-p38, GFAP, OX1R, IL-1β, TNF-α, IL-6, iNOS, Bcl-2, Bax, CytC, cleaved caspase-9 and cleaved caspase-3 in vivo and in vitro. Pro-inflammatory cytokines in cell supernatant IL-1β, TNF-α and IL-6 were determined by ELISA. Hoechst 33342 staining was used to detect the apoptosis of astrocyte. Immunofluorescent staining was performed to assess the nuclear translocation of p65 and the expression of GFAP. The results showed that OXA significantly improved neurological deficit score and decreased the volume of infarct area in brain. OXA decreased inflammatory mediators, inhibited astrocyte activation and nuclear translocation of NF-κB and phosphorylation of NF-κB, MAPK/ERK and MAPK/p38. Besides, OXA suppressed apoptosis via upregulating the ratio of Bcl-2/Bax and downregulating cytochrome C, cleaved-caspase-9 and cleaved caspase-3. Overall, it was concluded that OXA exerts neuroprotective effect during CIRI through attenuating astrocytes apoptosis, astrocytes activation and pro-inflammatory cytokines production, by Inhibiting OX1R-mediated NF-κB, MAPK/ERK and MAPK/p38 signaling pathways. The progress in our study is helpful to elucidate the molecular mechanisms of OXA neuroprotection, which could lead to the development of new treatment strategies for ischemic stroke.

Orexin-A (OXA) 是一种神经肽，通过减少脑缺血/再灌注损伤 (CIRI) 具有神经保护作用。星形胶质细胞激活介导的炎症和凋亡是 CIRI 的关键病理机制。因此，我们试图确认 OXA 对 CIRI 中星形胶质细胞炎症和细胞凋亡的神经保护作用，并阐明相关机制。建立大脑中动脉闭塞再灌注（MCAO/R）大鼠模型和氧糖剥夺再灌注（OGD/R）大鼠模型和U251胶质瘤细胞模型，加或不加OXA治疗。确定神经功能缺损评分，并通过 2,3,5-氯化三苯基四唑 (TTC) 染色评估脑梗塞体积。Western Blot检测NF-κB p65、p-p65、p-ERK、p-p38、GFAP、OX1R、IL-1β、TNF-α、IL-6、iNOS、Bcl-2、Bax、细胞色素C，在体内和体外裂解 caspase-9 和裂解 caspase-3。通过ELISA测定细胞上清液IL-1β、TNF-α和IL-6中的促炎细胞因子。Hoechst 33342染色检测星形胶质细胞凋亡。进行免疫荧光染色以评估 p65 的核易位和 GFAP 的表达。结果表明，OXA显着改善神经功能缺损评分并减少脑梗死面积。OXA 减少炎症介质，抑制星形胶质细胞活化和 NF-κB 的核转位以及 NF-κB、MAPK/ERK 和 MAPK/p38 的磷酸化。此外，OXA 通过上调 Bcl-2/Bax 的比例和下调细胞色素 C、cleaved-caspase-9 和 cleaved caspase-3 来抑制细胞凋亡。全面的，结论是，OXA 通过抑制 OX1R 介导的 NF-κB、MAPK/ERK 和 MAPK/p38 信号通路，通过减弱星形胶质细胞凋亡、星形胶质细胞活化和促炎细胞因子的产生，在 CIRI 期间发挥神经保护作用。我们的研究进展有助于阐明 OXA 神经保护的分子机制，这可能会导致缺血性卒中新治疗策略的发展。