| Objective: To investigate the mechanism of Rac1 and Nox1 involved in ischemia-reperfusion injury through oxidative stress in the middle cerebral artery ischemia-reperfusion(MCAO / R)injury in rats.Methods: In this study,70 male SD rats were randomly divided into sham operation group(n=5)and MCAO/R group(n=65).MCAO/R group was treated with modified Zea-Longa method to establish rat model of middle cerebral artery ischemia-reperfusion injury.The neurological behavior of rats was evaluated by Longa score,and the neurological deficits were observed.After reperfusion about 3h,6h,12 h,24h,2d,3d,5d,7d,the rats were decapitated separately,then brain tissue was taken out to undergo TTC staining with the concentration of 0.2%.The percentage of the volume of cerebral infarction were measured in different time groups,and the stability of MCAO/R injury model was observed.After the model was stable,the rats were sacrificed at 3h,6h,12 h,24h,2d,3d,5d,7d after reperfusion.The morphological changes of cerebral ischemia-reperfusion injury were observed by HE staining.The expression of Rac1 and Nox1 was detected by immunohistochemistry.The expression of Rac1 and Nox1 protein in sham operation group and MCAO / R group was detected by Western-Blot technique.The expression of Rac1 and Nox1 protein was detected by immunohistochemistry and Western-Blot.Statistical analysis of the test results,to compare the expression of Rac1 and Nox1 protein in the brain tissue of each group and find the regularity of the expression.Results: 1.TTC staining showed that the percentage of cerebral infarct volume in rats at different time has a certain regularity.The percentage of infarct volume increased with the prolongation of reperfusion time(12h~3d)(P<0.05),and then decreased after reperfusion 5d and 7d(P<0.05).2.Compared with sham group,the results of HE staining showed that the neurons had acute swelling of nerve cells at 3h and 6h after reperfusion,and the neurons of 12h~3d had different degrees of damage,necrosis,disintegration and disappearance,red blood cells scattered in the perivascular brain tissue,after 5 days of reperfusion,brain infarction area can be observed the reduce of neurons number,astrocytes hyperplasia,and the vascular wall infiltrated of inflammatory cells,after 7 days of reperfusion,the capillary endothelial cells proliferated and the newborn capillaries were formed.3.Immunohistochemical results showed that,the expression of Rac1 protein in the nucleus of nerve cells was strongly positive after ischemia-reperfusion injury,Nox1 protein was mainly expressed in neurons and vascular endothelial cells.Compared with sham operation group,the positive expression of Rac1 protein in the hippocampus was appeared at 3h after reperfusion and reached the peak at 24 h after reperfusion(P<0.05),then the expression of Rac1 decreased gradually with the prolongation of reperfusion time(P<0.05).The positive expression of Rac1 protein in cortical was increased at 6h after reperfusion,and decreased at 2d after reperfusion(P <0.05).The positive expression of Nox1 protein in hippocampus was significantly lower than that in cortex.The positive expression of Nox1 protein in hippocampus was increased gradually at 24 hours after reperfusion,and then decreased gradually after reperfusion for 24 h.The positive expression of Nox1 protein was increased at 6h after reperfusion,and gradually decreased after 6h reperfusion,then the expression of Nox1 protein increased gradually after reperfusion for 5~7d(P<0.05).The results showed that the expression of Rac1 and Nox1 protein was specific in space,and the positive expression Rac1 protein in the hippocampus was earlier than that in the Nox1 protein,and the positive expression of Rac1 protein in hippocampus was earlier than that of Nox1 protein.4.The results of Western-Blot showed that Rac1 protein in MCAO/R group was up-regulated than sham group,and the expression of Rac1 protein in hippocampus and cortex increased gradually with the prolongation of reperfusion time,then decreased gradually after the peak,but the expression of Rac1 protein in the cortex was later than that in the hippocampus.Compared with sham group,the expression of Nox1 protein in hippocampus was significantly increased at 24 h after reperfusion,the difference of the expression level in the remaining time group was not obvious,but the expression of Nox1 protein still higher than that in sham group.The expression of Nox1 protein in the cortical region increased after 6 hours of reperfusion,and then decreased to the normal level after reperfusion for 6 hours,and the expression of Nox1 protein in the cortical part increased gradually after 5 ~ 7 days of reperfusion(P<0.05).Conclusions: 1.According to the percentage of cerebral infarct volume with TTC staining,the stability of MCAO / R model was determined.The percentage of cerebral infarct volume may rise first and then decrease with the prolongation of reperfusion time?2.Compared with sham group the histological changes were mainly included neurons injury,apoptosis,necrosis and edema after cerebral ischemia-reperfusion injury in rats.3.Compared with the sham operation group,Rac1 protein was mainly expressed in the cell nucleus of the neuronal cells after ischemia-reperfusion injury,Nox1 protein was mainly expressed in neurons and vascular endothelial cells,the expression of Rac1 and Nox1 protein was specific in space.With the prolongation of reperfusion time,the expression of Nox1 protein increased with the increase of Rac1 protein expression,further confirmed that Rac1 may be one of the key factors to regulate the expression of Nox1 protein by regulating Nox1-induced oxygen free radicals involved in ischemia-reperfusion injury.4.The expression of Nox1 protein in the early stage of reperfusion was increased first and then decreased,and then increased in the later stage of reperfusion,the trend of expression of Nox1 protein after reperfusion suggests that Nox1 protein is not only involved in the process of brain injury in the early stage of reperfusion,but may also be involved in the process of the brain injury repairing in the later stage of ischemia-reperfusion injury.To study the development and progression of ischemic reperfusion injury in stroke,it is very important to study the theory of ischemia-reperfusion injury and the development of clinical therapeutic drugs. |
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