Carvacryl Acetate Alleviates Cerebral Ischemia Reperfusion-induced Oxidative Stress Injury Via Nrf2 Signalling Molecule

Objective:Cerebral ischemia reperfusion model in SD（Sprague Dawley）rat was established to clarify the correlation between the protective effect of carvacryl acetate on cerebral ischemia reperfusion-induced oxidative stress injury and Nrf2 signalling molecule at the animal level.At the same time,the oxidative stress injury model was established by H₂O₂ on PC12 cells to investigate the relationship between the protective effect of carvacryl acetate on oxidative stress injury and Nrf2 signalling molecule at the cellular level.Finally,we construted PC12 cells with down-regulated Nrf2 gene and established the oxidative stress injury model to further confirm that the mechanism of carvacryl acetate in alleviating oxidative stress injury caused by cerebral ischemia reperfusion was mediated by Nrf2 signalling molecule.Methods:（1）Establishment of cerebral ischemia reperfusion model in SD rats:healthy male SD rats were randomly divided into sham operation group,MCAO model group,edaravone administration group and carvacryl acetate administration group.Cerebral ischemia reperfusion model was constructed in the MCAO model group and administrated groups by the suture method.The administratrated groups were given intraperitoneal injection of edaravone（5 mg/kg）and carvacryl acetate（30,75 and 120mg/kg）at 0 h and 12 h after reperfusion,while the sham operation group and the MCAO model group were received isopykinic normal saline.Construction of oxidative stress model of PC12 cells:PC12 cells were added H₂O₂（100、200、400、600、800μM）with concentration gradient to explore the optimal concentration to induce injury.PC12cells were treated with various edaravone（25、50、100、200、400μM）and carvacryl acetate(2×10^-4、2×10^-3、0.02、0.2、2 m M)at the optimal concentration of to H₂O₂explore the optimal administrated concentration.Nrf2 gene down-regulated（KD cells）and empty carrier PC12 cell line（NC cells）were constructed,the same methods were used to damage and administrate the NC/KD cells.（2）Determination of pathological indicators:neurobehavioral scores of Longa were used to evaluate rats after 24 h of cerebral ischemia reperfusion.The rats from each group were perfused transcardially with normal saline,the lesions of ischemic infarction in the cerebral tissues were determined by TTC staining,and the damage of nerve cells in the cerebral tissues was determined by HE staining and Nissl staining.The apoptosis of PC12 cells and NC/KD cells werw determined by Hoechst 33342 staining and TUNEL staining after injury and administration.（3）Determination of oxidative stress level:the contents of ROS,MDA and the activity of SOD in the cerebral tissues,PC12 cells and NC/KD cells were detected.（4）Determination of Nrf2 expression:the expression of Nrf2 in cerebral tissues was evaluated by immunohistochemistry and Western blot,and the expression of Nrf2 in PC12 cells was analyzed by Western blot.Results:（1）After 24 h of cerebral ischemia-reperfusion,SD rats showed severe behavioral dysfunction,a large range of pale infarct lesions appeared in the brain tissue,and nerve cells were arranged sparsely and the number significantly reduced.In addition,the increase of ROS and MDA content and the decrease of SOD activity in the ischemic cerebral tissues indicated that serious oxidative stress damage existed in the ischemic cerebral tissues.However,the administration of carvacryl acetate could significantly improve cerebral tissue lesions and reduce the level of oxidative stress.（2）After the injury induced by H₂O₂,the cell viability of PC12 cells significantly decreased and the apoptosis rate increased.the increase of intracellular ROS and MDA content and the decrease of SOD activity indicated that high level of oxidative stress in cells,and the administration of carvacryl acetate significantly improved cell viability,inhibited apoptosis and mitigated oxidative stress damage.（3）The immunohistochemistry of Nrf2 suggested that carvacryl acetate increased the expression level of Nrf2 in cerebral tissues.Combined with Westren blot analysis of Nrf2 in cerebral tissues and PC12 cells,we found that carvacryl acetate could increase the expression of Nrf2 to promote the activation of related signalling pathway.（4）After NC/KD cells were injured by the same concentration of H₂O₂,KD cells suffered more severe injury and the level of oxidative stress was higher than NC cells,while the protective effect of carvacryl acetate impaired on KD cells,which further revealed that the protective effect of carvacryl acetate was related to Nrf2 signalling molecule.Conclusions:（1）After cerebral ischemia reperfusion injury,the neurobehavioral function of SD rats significantly decreased,and severe pathological changes occurred in brain tissue accompanied by high level of oxidative stress.（3）Carvacryl acetate significantly reduced oxidative stress levels in vivo and in vitro and provided protective effects.（4）After the administration of carvacryl acetate,the expression of Nrf2 in brain tissue and PC12 cells increased significantly,while carvacryl acetate had no significant protective effect on KD cells after H₂O₂-induced injury.The results of this study suggested that the role of carvacryl acetate in alleviating cerebral ischemia-reperfusion induced oxidative stress is related to Nrf2 signalling molecule and carvacryl acetate had a potential effect in activating Nrf2 signalling pathway.