Orexin-A alleviates astrocytic apoptosis and inflammation via inhibiting OX1R-mediated NF-κB and MAPK signaling pathways in cerebral ischemia/reperfusion injury

• Published in: Biochimica et biophysica acta. Molecular basis of disease 2021-11, Vol.1867 (11), p.166230-166230, Article 166230
• Main Authors: Xu, Dandan, Kong, Tingting, Shao, Ziqi, Liu, Minghui, Zhang, Rumin, Zhang, Shengnan, Kong, Qingxia, Chen, Jing, Cheng, Baohua, Wang, Chunmei
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Apoptosis - immunology; Astrocytes - immunology; Astrocytes - pathology; Cell Line, Tumor; Cerebral Cortex - blood supply; Cerebral Cortex - cytology; Cerebral Cortex - immunology; Cerebral Cortex - pathology; Cerebral ischemia/reperfusion injury (CIRI); Disease Models, Animal; Humans; Infarction, Middle Cerebral Artery - complications; Infarction, Middle Cerebral Artery - immunology; Infarction, Middle Cerebral Artery - pathology; Inflammation; Male; MAP Kinase Signaling System - immunology; Mitogen-activated protein kinase (MAPK); NF-kappa B - metabolism; Nuclear factor-Κb (NF-κB); Orexin Receptors - metabolism; Orexin-A (OXA); Orexins - administration & dosage; Orexins - metabolism; Rats; Reperfusion Injury - immunology; Reperfusion Injury - pathology
• ISSN: 0925-4439; 1879-260X
• DOI: 10.1016/j.bbadis.2021.166230

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. •OXA is neuroprotective against CIRI.•OXA inhibited the pro-inflammatory factors in OGD/R U251 astrocytoma cells.•OXA inhibited the pro-inflammatory factors in MCAO/R rats.•OXA inhibited the excessive activation of astrocytes in MCAO/R rats.•OXA alleviates inflammation via Inhibiting NF-κB and MAPK signaling pathways.