Effect Of The Main Components α-asarone In Volatile Oils Of Acorus Tatarrinowii Schott On Cerebral Ischemia-reperfusion Injury And Its Mechanism

Objective:To evaluate the protective effect and mechanism ofα-asarone on microglials with cerebral ischemia-reperfusion injury by measuring the expression of polar transformation and related inflammatory proteins in BV2 cells in vitro.Effect of on cerebral ischemia-reperfusion injury mice was evaluated by animal experiments,and its target and pathway were predicted by network pharmacology.Methods:The cerebral ischemia-reperfusion injury BV2 cells were pretreated byα-asarone in vitro and simulated by OGD/R model.1.Screening Dosageα-asarone and OGD/R Model by CCK-8;2.The effect ofα-asarone on the viability of damaged cells in OGD/R model was determined by CCK-8;3.The morphological changes of cells in normal group,OGD/R model group andα-sarone were observed to analyze the general morphology of OGD/R damaged cells in OGD/R model;4.The levels of proinflammatory factor IL-1β,IL-18 and anti-inflammatory factor IL-10,IL-4 secreted by BV2 cells were detected by enzyme-linked immunosorbent assay;5.Western blot detection the protein expression of proinflammatory mediator TGF-βand anti-inflammatory mediator TNF-α;6.To detect the expression of inflammatory related protein NLRP3,caspase 1,NF-κB in BV2 cells and explore the mechanism of the improvement ofα-asarone on the OGD/R damaged cells of OGD/R model;7.Detection of ROS activity of BV2 cells by ELISA。The in vivo experiment mainly through theα-asarone pre-administered C57BL/6J mice,by establishing a MCAO model to simulate cerebral ischemia-reperfusion injury.1.Longa five-point method was used to evaluate the effect ofα-asarone on neurological deficit in mice with cerebral ischemia-reperfusion injury;2.Laser speckle system was used to analyze the area of cerebral ischemia and the amount of cerebral blood perfusion in mice,and the effect ofα-asarone on the recovery of cerebral blood flow in mice with cerebral ischemia reperfusion injury was analyzed;3.Network pharmacological data mining method was used to predict the target and molecular signaling pathway ofα-asarone in stroke and cerebral ischemic reperfusion.Results:The results of in vitro experiments were as follows:1.After screeningα-asarone and the OGD/R model,it was determined that the dosageα-asarone was divided into 1μM,4μM,16μM dose group,and the OGD/R model was established with 10μM dose Na₂S₂O₄.The oxygen glucose deprivation time was 1.5 h,reperfusion time was 12 h;2.α-Asarone significantly increased the activity of damaged cells in OGD/R model,with the increase of administration dose,the cells in the low,medium and high dose groups ofα-asarone decreased the“amoeba-like”cells and the cell body gradually became stereoscopic and full.From the results of cell morphology,it can be seen thatα-asarone has a certain proliferative effect on normal cells;3.Pretreatment withα-asarone can significantly reduce the release BV2 cells proinflammatory factor IL-1β,IL-18 and TNF-αin OGD/R injury,also significantly increase the release of IL-10,IL-4 and TGF-β,with a dose-effect relationship,the high dose（16μM）was the best;4.α-Asarone pretreatment can significantly reduce the expression of inflammatory related protein NLRP3,caspase 1,NF-κB and ROS activity in injured cells of OGD/R model.The results of in vivo experiments were as follows:1.α-Asarone can significantly reduce the nerve function defect and improve its behavior in mice with cerebral ischemia-reperfusion injury;2.α-Asarone can promote the recovery of cerebral blood flow in mice with cerebral ischemia-reperfusion injury and reduce the area of cerebral ischemia;3.Network pharmacology predicts a total of 95 targets ofα-asarone to stroke/cerebral ischemic reperfusion,the mechanism mainly involves 20 molecular signaling pathways such as neuroactive ligand-receptor interaction,cell cycle,p53 signaling pathway,calcium signaling pathway,nitrogen metabolism,steroid hormone synthesis,serotonin energy synapse,etc.Conclusion:The results of in vitro and in vivo confirmed thatα-asarone had a significant protective effect on cerebral ischemia-reperfusion injury,mainly by regulating ROS activity and inhibiting phosphorylation of NF-κB,in order to reduce the excessive activation of NLRP3 inflammatory corpuscles reducing the secretion of proinflammatory factor IL-1βand IL-18,promoting the secretion of anti-inflammatory factor IL-10 and IL-4,so as to protect cerebral ischemia-reperfusion injury by anti-inflammatory...