Neuroprotective Mechanism Of Vitexin Regulating Epac Pathway In Mitigating Chronic Hypoperfusion Cerebral Ischemia Injury In Rats

Objective:To study the role of Epac signaling pathway in chronic hypoperfusion cerebral ischemia injury in rats,and to explore the protective mechanism of vitexin on chronic hypoperfusion cerebral ischemia injury,and in order to develop vitexin as a safe and effective drug for the prevention and treatment of cerebral ischemia.Methods:1)Sixty male Sprague-Dawley rats were randomly divided into sham group,2VO group,2VO+Vitexin（2.5mg/kg）group,2VO+Vitexin（5mg/kg）group,2VO+Vitexin（10 mg/kg）and 2VO+breviscapine（5 mg/kg）group.Rats chronic cerebral ischemia models were establishment by permanently ligating the bilateral common carotid arteries of the rats.The learning and memory ability of rats were detected by Morris water maze test.The pathological analysis of brain tissue was detected by HE and Tunel staining respectively.Western blot was used to detect Epac1,Epac2,Rap1,P62,ERK1/2,NLRP3,Caspase1,IL-1?,IL-6,Cleaved caspase3 and p-ERK proteins expression in rat hippocampus.2)Establishment of oxygen glucose deprivation model:First,HT22 cells were seeded in a cμlture flask.When the cell density reached about80%,the medium was changed to an anaerobic and sugar-free medium,and then the flask was placed in a three-gas incubator at 1%O₂,5%CO₂.Five hours later,the anaerobic sugar-free medium was replaced with normal medium and placed in a common incubator for 24 h.The experiment was divided into three groups:control group,OGD group and OGD+Vitexin group.The activity of HT22 cells was detected by MTT assay.The damage of HT22 cells was detected by LDH kit.IL-6 release from HT22 cells detected by ELISA kit,ROS kit for detecting reactive oxygen species.The expression of Epac1,Epac2,NLRP3,Caspase1,Cleaved Caspase3 and IL-6 protein in HT22 cells was detected by western blot.Results:1)The MWM results showed that Compared with the sham group,the swimming speed of the model group did not change significantly,but the latency,swimming distance,target quadrant staying time and the number of crossing target quadrants were significantly reduced in the model group;The swimming speed of the rats in the high-dose group and the positive drug control group could not be changed,but it reduced the latency and swimming distance,increased the time of the target quadrant staying in the rat and the number of crossing the target quadrant,indicating that the vitexin was large.The spatial learning and memory skills of the rats have been significantly improved.2)The results of HE staining showed that compared with the sham group,the hippocampus and cortical neurons in the model group were significantly reduced,the intercellular space was enlarged,the arrangement was disordered,and the nucleus pyknosis was deeply stained.Compared with the model group,the vitexin medium and high dose groups and the positive drug control group significantly alleviated the pathological damage of hippocampus and cortex in rats.In the TUNEL staining results,the number of cells stained positive for the hippocampus and cortex of the model group was significantly increased compared with the sham group.Compared with the model group,the number of cells positive for TUNEL staining was significantly decreased in the hippocampus and cortex of vitexin,high dose group and positive control group,suggesting that vitexin protects neuronal damage after chronic hypoperfusion cerebral ischemia.3)Western blot results showed that the protein expression of Epac1,Epac2,Rap1,P62 and p-ERK/ERK1/2 was significantly decreased in the model group compared with the sham operation group,NLRP3,Caspase1,IL-1?,IL-6,Cleaved caspase-3 protein expression was significantly elevated.Compared with the model group,the vitexin medium,high dose group and positive control group can up-regulate Epac1,Epac2,Rap1,P62,p-ERK protein expression,down-regulate NLRP3,Caspase1,IL-1?,IL-6,Cleaved caspase3 protein expression.4)In the results of cell experiments,after hypoxia-reoxygenation of HT22 cells,cell viability decreased,cell damage increased,ROS production increased,IL-6 release increased,Epac1,Epac2protein expression decreased,NLRP3,Caspase1,Increased expression of Cleaved Caspase3 and IL-6 proteins.Compared with the OGD group,the vitexin group can enhance cell viability,reduce cell damage,reduce the release of inflammatory factors IL-6 and reactive oxygen species ROS,promote Epac activation,up-regulate Epac1,Epac2 protein expression,and down-regulate NLRP3,Caspase1,Cleaved Caspase3,IL-6 protein expression,reduce inflammation,inhibit apoptosis,and protect neuronal function.Conclusion:Vitexin can improve the learning and memory ability of rats with chronic hypoperfusion cerebral ischemia,protect damaged neuronal cells,reduce oxidative stress,inflammatory reaction and apoptosis caused by cerebral ischemia,and its mechanism may be related to regulation of EPAC and downstream related proteins are closely related,suggesting that EPAC may be an important molecular mechanism for vitexin to improve the occurrence of chronic cerebral ischemic injury,and provide a new experimental basis for vitexin treatment of neurological diseases caused by chronic hypoperfusion cerebral ischemia.