Anisodine hydrobromide alleviates hypoxia/reoxygenation (H/R)‐induced brain microvascular endothelial cell injury via muscarinic acetylcholine receptor 4

Abstract

Anisodine hydrobromide (Ah) could delivery to the brain across the blood-brain barrier, exerting its protective effect in cerebral ischemia/reperfusion injury. Metabolic alterations under hypoxic conditions lead to endothelial dysfunction. In the present study, we tested that whether Ah regulates the production of nitric oxide and reactive oxygen species (ROS) and the glycolytic pathway via muscarinic receptors under hypoxia/reoxygenation (H/R) conditions. Under H/R conditions, brain microvascular endothelial cells were treated with Ah. Then, the expression of muscarinic acetylcholine receptors was detected by western blot. The production of nitric oxide was detected by Greiss reaction. ROS was measured by DCFH-DA probes. The expression of hypoxia-inducible transcription factor 1 (HIF-1α) was detected. Specific inhibitor of M2 and M4 muscarinic acetylcholine receptors were used to assess the role of muscarinic receptors in Ah protects against endothelial cell dysfunction. Results showed that H/R induced the expression of M2- and M4-AchRs, but not M1-, M3-, and M5-AchRs. Ah significantly suppressed the H/R-upregulated M2- and M4-AchRs. H/R induced production of NO and ROS. Ah and M4-AchRs inhibitor significantly inhibited the H/R-induced production of NO and ROS. HIF-1α was significantly induced by H/R, whereas it was significantly suppressed by Ah. Therefore, Ah protects against H/R injury in brain microvascular endothelial cells via activating HIF-1α, producing NO and ROS, which is dependent on M4-AchR.