| Objective:The study aimed to elucidate the protecting effects and mechanisms of piceatannol-3’-O-glucoside for the injection on the cerebral ischemia/reperfusion injury.Also,the study aimed to provide new experimental supports to the related clinical applications.Methods:1.Animal experiments(1)Using a simple random assorting method:ICR mice were grouped into five:sham operation group,tMCAO group,the low-concentration group of piceatannol-3’-O-glucoside for the injection(4mg/kg),the high-concentration group of piceatannol-3’O-glucoside for the injection(8mg/kg),and the edaravone injection group(4mg/kg).(2)In the sham operation group,only carotid artery was separated without middle artery embolization,and normal saline was given intravenously at the beginning of reperfusion.Except for the sham group,transient middle cerebral artery embolization model was prepared by suture embolization in other groups,the middle artery was embolized for 1 hour and then reperfused for 24 hours.The tail vein of tMCAO group was treated with normal saline at the beginning of reperfusion.The low-concentration group of piceatannol-3’-O-glucoside for the injection(4mg/kg),the high-concentration group of piceatannol-3’-O-glucoside for the injection(8mg/kg),and the edaravone injection group(4mg/kg)were treated with 4mg/kg of piceatannol-3’-O-glucoside for the injection,8mg/kg of piceatannol-3’O-glucoside for the injection and 4mg/kg of edaravone injection in the tail vein at the beginning of reperfusion,respectively.(3)Compare the survival rate within 24 hours of reperfusion after 1 hour of middle artery embolization in ICR mice;longa score method was used as the evaluation of nerve function damage in ICR mice;brain coefficient was used as brain tissue damage during cerebral ischemia reperfusion and cerebral edema evaluation of.Triphenyltetrazolium chloride staining method was used to detect the change of cerebral infarct area in ICR mice.2.Cell experiments(1)PC12 cells were cultured in vitro to establish a cobalt-dichloride induced oxygen-glycosylation/reoxygenation(OGD/R)model.MTS method was used to screen the optimal concentration of piceatannol-3’-O-glucoside for the injeetion(2)The PC12 cells were randomly assorted into groups of five:control group,OGD/R group,the group of low-concentration of piceatannol-3’-O-glucoside for the injection(200μM),the high-concentration group of piceatannol-3’-O-glucoside for the injection(1000μM),and the edaravone injection group(100μM).Except for.the control group,OGD/R method was used for modeling in other groups for 18h of oxygen-glucose deprivation and 6h of oxygen-glucose reoxygenation.After OGD/R group was deprived of oxygen and sugar for 18h,it was replaced with fresh high glucose DMEM medium and continued to be cultured for 6h.the group of low-concentration of piceatannol-3’-O-glucoside for the injection(200μM),the high-concentration group of piceatannol-3’-O-glucoside for the injection(1000μM),and the edaravone injection group(100μM)were deprived of oxygen and glucose for 18h,and then replaced with 200μM piceatannol-3’-O-glucoside high-glucose DMEM culture medium for the injection,1000μM piceatannol-3’-O-glucoside high-glucose DMEM culture medium for the injection and 100μM Edaravone high-glucose DMEM culture medium,respectively,and continued for 6h.(3)MTS method was employed to measure the cell viability of individual groups;the spectrophotometric method was used to measure the superoxide dismutase activity and lactic acid in PC12 cells after cobalt dichloride-induced oxygen glucose deprivation/reoxygenation of cobalt dichloride by piceatannol-3’-O-glucoside for the injection changes in the content of lactate dehydrogenase;fluorescent probe method to detect the effects of piceatannol-3’-O-glucoside for the injection on cobalt dichloride-induced oxygen glucose deprivation/reactive oxygen species in PC12 cells after reoxygenation.fluorescence intensity changes;western blot method was used to detect the changes of caspase-3,bcl-2,and bax protein expression in PC12 cells after cobalt dichloride-induced oxygen glucose deprivation/reoxygenation and reoxygenation of piceatannol-3’-O-glucoside for the injection.Results:1.Animal experiment:in comparison with the sham operation group,the rate of survival for mice in the tMCAO group was reduced,the brain coefficient increased,and infarct regions appeared in the brain of the mice.The model was successfully copied(P<0.05);as compared with the tMCAO group with the low as well as high concentration groups of piceatannol-3’-O-glucoside for the injection increased the survival rate of mice,decreased the cerebral coefficients,and minimized the area of cerebral infarction(P<0.05).2.Cell experiment(1)1000μM cobalt dichloride induced PC12 cells to be hypoglycemic and hypoxia for 18 hours/saccharose and reoxygenation for 6 hours,the cell viability was reduced to 63%,the model was successfully copied(P<0.01).(2)Piceatannol-3’-O-glucoside for the injection at 1200μM and 1400μM significantly reduced the viability of PC12 cells,suggesting that piceatannol-3’-O-glucoside for the injection at 1200μM and 1400μM had toxic effects on PC12 cells.In subsequent experiments,the maximum concentration of piceatannol-3’-O-glucoside for the injection was 1000μM.Compared with OGD/R group,the cell viability of the 200-1000μM piceatannol-3’-O-glucoside for the injection was significantly increased(P<0.01),and the protective effect was concentration-dependent.In the follow-up experiments,200μM piceatannol-3’-O-glucoside for the injection was selected as the low concentration group,and 1000μM piceatannol-3’-O-glucoside for the injection was selected as the high concentration group.(3)In comparison with the control group,the rate of cell survival of the OGD/R group was decreased,LDH release was increased,SOD activity was weakened,and ROS content was increased,the protein expression of bc1-2 in the OGD/R group was decreased,while that of caspase-3 together with bax was increased(P<0.05);as compared with the OGD/R group,the edaravone injection group,the low and high concentration groups of piceatannol-3’-O-glucoside for the injection,LDH release was decreased,SOD activity was increased,and ROS content was decreased,the protein expression of bcl-2 in the OGD/R group was increased,while that of caspase-3 together with bax was decreased(P<0.05);Conclusion(s):1.Injection with piceatannol-3’-O-glucoside can prolong the survival time of mice with cerebral ischemia/reperfusion injury,mitigate the area of cerebral infarction,mininize cerebral edema,and it has a protecting effect on cerebral ischemia/reperfusion injury in mice.2.Injection with piceatannol-3’-O-glucoside improve the survival rate of oxygen-glucose deprivation/reoxygenation model cells,reduces LDH release,reduces ROS content,enhances SOD activity,and downregulates caspase-3 and bax protein The expression of bcl-2 protein up-regulates the expression of bcl-2 protein,reduces oxidative stress damage and inhibits cell apoptosis.Injection with piceatannol-3’-O-glucoside has a protective effect on PC12 cell injury induced by cobalt dichloride. |
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