Research On The Mechanism Of Candesartan On Oxidative Stress In Rats With Cerebral Ischemia Reperfusion

Objective: The experiment is to observe the effect of candesartan on serum and brain tissue SOD,MDA,CAT,GSH-Px,NO and NOS in cerebral ischemia-reperfusion rats,and to observe the effects of candesartan on8-OHDG and 4-HNE on brain tissue,Cortex NADPH oxidase p22-phox,p47 phox,gp91-phox,and to explore its cerebral ischemia protective effect and its mechanism.Methods: 144 SD rats were randomly divided into normal group(sham operation group),cerebral ischemia-reperfusion model group,low dose group of candesartan(0.2mg / kg),and high-dose group of candesartan(1mg/kg).Each group was 32,and each group was randomly divided into 24 h subgroup,72 h subgroup,with 18 rats in each group.Normal group and model group were given saline(0.9% NS)1ml / d by gavage,low dose group of candesartan was given candesartan 0.2mg /(kg.d)by gavage,and high-dose group of candesartan was given candesartan 1mg /(kg.d)by gavage.Two weeks later,bilateral common carotid artery occlusion was used to clamp the common carotid artery.After 2 hours of ischemia,the artery clamp was removed to achieve reperfusion injury.Rat models of cerebral ischemia-reperfusion were prepared.According to the different time points of experimental design,6 rats were decapitated at 24 h or 72 h reperfusion for TTC staining and determination of the volume of cerebral infarction;Another 6 ratsin each group were fasted overnight,weighed and anesthetized with 3% sodium pentobarbital(0.15 ml / 100g).Abdominal aorta blood was collected and centrifuged at low temperature to take serum,sacrificed and brain tissue was taken 10% brain homogenate,the supernatant was centrifuged at low temperature.The levels of SOD,MDA,CAT,GSH-Px in serum and brain homogenate were detected by enzyme-linked immunosorbent assay(ELISA).NO and NOS levels in serum and brain tissue were measured by nitrate reductase method.The expression of NADPH oxidase subunits p22-phox,p47 phox,gp91-phox in brain tissues were detected by RT-PCR.The remaining rats in each group were anesthetized and fixed with 4% paraformaldehyde perfusion.The brain tissue was sectioned and decapitated.HE staining was used to observe the pathological changes and neuronal apoptosis in hippocampal CA1 area under light microscope.The expression of 8-OHdG and4-HNE were observed by immunohistochemistry.Results:1.TTC staining results showed that the normal group TTC staining have no white and no cerebral infarction area.Compared with the model group,24-h low dose group of candesartan,72-h low dose group of candesartan,24-h high dose group of candesartan and 72-h high dose group of candesartan were significantly reduced the infarct size.The difference was statistically significant(P<0.05);Compared with 24-h high dose group of candesartan and 72-h high dose group of candesartan,24-h low dose group of candesartan and 72-h low dose group of candesartan had slightly larger cerebral infarction area,but the differencewas not statistically significant(P> 0.05).The results of determination of SOD,MDA,CAT,GSH-Px,NO and NOS in serum and brain tissue of rats showed that compared with the normal group,the levels of SOD,CAT and GSH-Px in the serum and brain tissue of the model group were significantly decreased and the difference was statistically significant(P< 0.05).Compared with the model group,24-h low dose group of candesartan,72-h low dose group of candesartan and 24-h high dose group of candesartan,72-h high dose group of candesartan in serum and brain tissue of SOD,CAT and GSH-Px levels were significantly increased,and the difference was statistically significant(P< 0.05);Compared with the normal group,the levels of MDA,NO and NOS in the serum and brain tissues of the model group were significantly increased,and the difference was statistically significant(P< 0.05).Compared with the model group,the levels of serum and brain MDA,NO and NOS decreased significantly in 24-h low dose group of candesartan,72-h low dose group of candesartan,24-h high dose group of candesartan and 72-h high dose group of candesartan,with significant difference(P< 0.05).The results of HE staining in the CA1 region of the hippocampus showed that the normal tissue staining was uniform and the nuclear structure was clear in the normal group.In the model group,the cell density was reduced,the structure was disordered,the shape of the cells was irregular,and the nuclei were deformed,fixed,and even dissolved.24-h low dose group of candesartan,72-h low dose group of candesartan,24-h high dose group of candesartan,72-h high dose group of candesartan had sparse tissuestructure,and a small amount of nuclei appeared deformable and pyknosis.The results of immunohistochemistry showed that the number of 8-OHdG and4-HNE positive cells in the model group increased significantly compared with the normal group,and the difference was statistically significant(P< 0.05).Compared with model group,24-h low dose group of candesartan,72-h low dose group of candesartan,24-h high dose group of candesartan,72-h high dose group of candesartan had significantly decreased 8-OHdG and 4-HNE immunoreactive cells,and the difference was statistically significant(P <0.05).2.RT-PCR test showed that compared with the normal group,the expression of p22-phox,p47-phox and gp91-phox increased significantly in the model group,and the difference was statistically significant(P< 0.05).Compared with the model group,the expression of p22-phox,p47 phox and gp91-phox in 24-h low dose group of candesartan,72-h low dose group of candesartan,24-h high dose group of candesartan,and 72-h high dose group of candesartan were significantly decreased,and the difference was statistically significant(P < 0.05).Conclusion: Candesartan can significantly reduce the volume of cerebral infarction in rats,brain tissue and serum MDA,NO,NOS,8-OHdG and 4-HNE levels of cerebral ischemia,increased serum and brain tissue of SOD,CAT,GSH-Px,levels reduce the apoptosis of hippocampal neurons,suggesting that candesartan on cerebral ischemia reperfusion in rats with cerebral protective effect of antioxidant.Candesartan can obviously reduce the volume of cerebral infarction,the level of SOD,CAT,GSH-Px,8-OHdGand 4-HNE,and it can increase the level of MDA,NO,NOS in serum and brain tissue to reduce apoptosis of hippocampal neurons,suggesting that candesartan has protective effect on cerebral ischemia and reperfusion in rats.Candesartan can significantly reduce the cerebral cortex NADPH oxidase p22-phox,p47 phox,gp91-phox expression in cerebral ischemia-reperfusion rats,suggesting that this may be one of its mechanisms of cerebral ischemia protective effect.