Effects Of Epicatechin On Oxidative Stress And Neuronal Apoptosis After Cerebral Ischemia-reperfusion In Rats

Objective: Epicatechin(EC)is an active substance isolated from tea polyphenols,which has antioxidant activity and can easily penetrate the blood-brain barrier.After cerebral ischemia reperfusion injury(CIRI),a large amount of reactive oxygen species(ROS)accumulates,resulting in imbalance of oxidation and antioxidant system,and oxidative damage.At present,EC has been widely studied in anti-oxidation,anti-inflammatory,anti-tumor and other aspects,but the anti-oxidation mechanism in CIRI has not been deeply studied,and some studies suggest that EC may have potential application value in anti-apoptosis.Our previous studies have found that EC pretreatment can reduce the content of oxidized glutathione and increase the activity of SOD in H9C2 cells induced by high glucose and high fat,which can play a protective role on myocardial cells.At the same time,green tea polyphenols can increase the expression of endogenous antioxidant enzymes SOD1 and SOD2 in brain tissue in rat cardiac arrest cardiopulmonary resuscitation model.Based on the previous research of our group and others,we established the middle cerebral artery occlusion(MCAO)model by thread occlusion method.After 2 hours of ischemia and reperfusion,we administered EC at 0h,12 h and 24 h after reperfusion respectively to explore the protective effect of EC on the brain after CIRI from two aspects of oxidative stress and apoptosis.Methods: To combine the two methods 114 Rats were randomly divided into 6 groups 19 Rats were divided into sham operation group(sham group),model group(I/R group),EC5mg/kg group,EC10mg/kg group,EC20mg/kg group and edaravone(ED)3mg/kg group.After one week of adaptive feeding,except sham group,middle cerebral artery occlusion(MCAO)model was established in other groups by thread occlusion method,and reperfusion was performed after 2 hours of ischemia.At 0h,12 h and 24 h after reperfusion,EC groups were intraperitoneally injected with 5mg/kg,10mg/kg and 20mg/kg epicatechin;ED group was intraperitoneally injected with 3mg/kg edaravone;sham group and I/R group were injected with the same volume of normal saline.Longa and MNSs were used to evaluate the neurological deficit at 12 h and 24 h after reperfusion;Brain samples were taken at 36 h after reperfusion,and brain index was calculated by wet weighing to evaluate the degree of brain edema.And the infarct area of ischemic side was detected by triphenyltetrazolium chloride(TTC)staining to observe the infarct area.Hematoxylin and eosin(H&E)staining was used to observe the pathological damage of ischemic cerebral cortex.The apoptosis of neurons in the cortex was observed by TDT mediated d UTP nick end labeling(TUNEL).The content of malondialdehyde(MDA),SOD and GSH peroxidase(GPx)activity were measured by colorimetry to evaluate the antioxidant level of brain tissue in rats.Western blotting was used to detect the relative expression levels of nuclear factor E2 related factor 2,heme oxygenase 1,NAD(P)H:quinone oxidoreductase 1 and apoptosis related proteins Caspase-3,Bax and bcl-2.Results:(1)The score of nerve function defect,brain edema and infarction in rats.Compared with sham group,the scores of two kinds of nerve function in I/R group were significantly increased(P<0.05),the percentage of cerebral infarction area increased(P<0.05),and there was no significant difference in brain index between the groups.Compared with the I/R group,the scores of two kinds of nerve function decreased,the percentage of infarction area decreased(P<0.05),and the brain function scores of the two groups decreased at the two time points,and the percentage of infarction area decreased(P<0.05).There was no significant difference in index.(2)Pathological damage of cerebral cortex on the ischemic side of rats.The number of cells in sham group was more,arranged closely,the shape was round and clear,and the nucleolus was in the middle.Compared with sham group,the number of cells in I/R group decreased,the arrangement of cells was scattered,the morphology of cells was irregular,some cells had nuclear pyknosis and pathological damage;Compared with I/R group,the number of cells in EC group and ED3 mg/kg group was significantly increased,with regular morphology and obvious nucleocytoplasmic gap.(3)The contents of MDA,the activities of SOD and GPx in serum and ischemic cortex of rats,and the relative expression levels of Nrf2,HO-1 and NQO1.Compared with sham group,MDA content in serum and ischemic cerebral cortex increased,SOD activity decreased(P<0.05),GPx level decreased(P<0.05),Nrf2,NQO1 protein relative expression increased(P<0.05),HO-1 expression also increased,but there was no significant difference;Compared with I/R group,MDA content in serum and cerebral cortex of ischemic side decreased and SOD activity increased in EC10mg/kg,EC20mg/kg and ED3mg/kg groups,while GPx activity increased in EC20mg/kg and ED3mg/kg groups(P<0.05).The expression of Nrf2 protein was significantly increased in EC three dose groups and ED group(P<0.05).Except for EC5mg/kg group,the expression of NQO1 protein was significantly increased in other groups(P<0.05).Only the expression of HO-1 protein was significantly increased in EC20mg/kg group and ED3mg/kg group(P<0.05).(4)The apoptosis of cerebral cortex on the ischemic side of rats and the relative expression of Caspase-3,Bax and Bcl-2.Compared with sham group,the apoptosis index of neurons in I/R group was serious,apoptosis index increased,caspase-3 and Bax protein expression increased significantly,bcl-2 protein expression decreased(P<0.05);Compared with the I/R group,the apoptosis index,caspase-3 protein expression and bcl-2protein expression in the three dose EC groups and ED group were significantly decreased(P<0.05).Except for the EC5mg/kg group,the expression of Bax protein in the other groups was significantly decreased(P<0.05).Conclusion: EC may activate Nrf2/HO-1 signaling pathway,then reduce neuronal apoptosis and protect the brain after CIRI.