Effects Of Etanercept On The Experssion Of MMP-9、Claudin-5after Cerebral Ischemic Reperfusion In Rats

Objective:This study was designed to observe the effect of the recombinant human tumor necrosis factor-a receptor Ⅱ:IgG Fc fusion protein for injection (Etanercept) in neurological deficit score, brain water content and the expression of Matrix metalloproteinases-9(MMP-9)/Claudin-5/Tumor necrosis factor-a (TNF-a) in cerebral cortex of cerebral ischemic-reperfusion model rats.To investigate the protective effects of TNF-alpha antagonist in the early stage of cerebral ischemic-reperfusion injury and its possible mechanisms, and supply experimental basis for the clinical treatment of cerebral ischemia and reperfusion injury.Methods:The middle cerebral artery occlusion(MCAO)models were established by using the intraluminal suture occlusion method in Sprague-Dawley rats. Cleaning adult male Sprague-Dawley rats weighing250-300g were randomly divided into normal group(n=10), sham-operated group(n=30), ischemia/reperfusion model group(n=30) and Etanercept treatment group(ETA group). The treatment group was further divided into the following groups according to different doses of Etanercept:Low-dose group (n=30):ETA0.2mg/kg;Medium-dose group (n=30):ETA0.4mg/kg;High dose group (n=30):ETA0.8mg/kg.The treatment group was treated with different doses of ETA intervention (on the right lateral ventricle injection), sham-operated group and ischemia/reperfusion model group were received the same dose of sterile saline intervention, Rats (except the normal group)were randomly divided into three sub-groups as follows:1d、2d and5d groups, according to the killing time after ischemia/reperfusion. The changement of neurological deficits were observed dynamically. Pathologic changes were evaluated by Hematoxylin.Edema was measured as water content of brain hemispheres according to the wet-dry weight method, the expressions of MMP-9/Claudin-5/TNF-a were detected by immunohistochemistry, and the datas were analyzed with the help of statistical software SPSS17.0Result:1.The success rate and the mortality rat of SD rats ischemia-reperfusion model is83.77%and5.76%.2.The effect of Etanercept on the change of neurological deficit score after ischemia-reperfusion:The normal control group and sham-operated group had no significant neurological deficits. The ischemia-reperfusion model group showed more severe neurological deficits than ETA treatment group(P<0.05) at the same time points.Camparing witn the ischemia-reperfusion model group, the ETA treatment group showed less neurological deficits at the same time points,indicating ETA may improve the symptoms of neurological deficits.3.The impact of ETA on the brain edema after ischemia-reperfusion:the wet and dry weight method showed that the brain edema of rats significantly increase in the1d, reach a peak at2d, and decline after5d,but still higher than the sham-operated group.ETA can significantly reduce the water content of the rats brain tiussue,which showed obviouely in middle-dose and hight-dose group(P<0.05).There is no significant difference in middle-dose group and hight-dose group.4. The effect of ETA on the histopathological morphological changes after ischemia-reperfusion:There is no significant morphological changes in the cerebral cortex tissue in normal-groups and the operated-group.The gliosis cells、the interstitial edeam and the necrosis of nerve cells is observed in ischemia/reperfusion group. ETA can reduce the inflammatory response of the ischemic brain tissue and the cerebral edeam, which showed obviouely in middle-dose and hight-dose group(P<0.05).There is no significant difference between middle-dose group and high-dose group.5.The effect of ETA on the expressions of MMP-9after ischemia-reperfusion:There is no MMP-9expressions in normal group.The expressions of MMP-9in the sham-operated group were rare observed and in the ischemic part of ischemia/reperfusion group the expressions of MMP-9began to increase at the time point of Id and reached the peak at the time point of2d,and started to decrease in5d after reperfusion.The expressions of MMP-9in the treatment groups is lower than the ischemia/reperfusion group(P<0.05).In ETA groups at the same time points,the expressions of MMP-9is least in middle-dose and high-dose group.The differences has statistical significant(P<0.05). There is no significant difference between middle-dose group and high-dose group.6.The effect of ETA on the expressions of Claudin-5after ischemia-reperfusion:In ischemia/reperfusion group the expressions of Claudin-5reduced significantly at the time point of1d and reached the lowest at the time point of2d,and increased in5d.ETN significantly increased the Claudin-5protein expression levels in each theatment group,which is obvious in middle-dose group and high-dose group(P<0.05). There is no significant difference between middle-dose group and high-dose group.7.The effect of ETA on the expressions of TNF-a after ischemia-reperfusion:The ischemic part of ischemia/reperfusion group the expressions of TNF-a reached the peak at the time point of1d,and started to decrease in2d and5d after reperfusion.The expressions of TNF-a in the treatment groups is lower than the ischemia/reperfusion group(P<0.05).In ETA groups at the same time points,the expressions of TNF-a is least in middle-dose and high-dose group.The differences has statistical significant(P<0.05). There is no significant difference between middle-dose group and high-dose group.Conclusions:1.ETN may play a neuroprotective role in focal cerebral ischemia-reperfusion injury in rats and the effects were dose-related.2.One of the mechanism of the ETN on neural protection might be related with down-regulation of MMP-9and up-regulation of Claudin-5expression to maintain the structral integrality of blood-brain barrier.