NLRP-1,Caspase-1 Expression After Focal Ischemic/reperfusion Injury In Rats

NLRP-1,Caspase-1 expression after focal ischemic/reperfusion injury in ratsBACKROUNDSCerebral ischemia/reperfusion not only can save the brink of the death of nerve cells but also can aggravate ischemic cell damage called reperfusion injury. IL-1β(interleukin-1β) is the most important cytokines in the vary reasons causing of cerebral ischemia-reperfusion injury. While the cysteine protease-1 (caspase-1) activation is the important reason that leads to excessive release of IL-1β. Caspase-1 as an important inflammatory signal transduction material whose activation depends on its upstream factors. Recent studies have found that NLRP-1 is an important factor to make the procaspase-1 into an active caspase-1. So there is inflammsome constitutes of NLRP-1 (NOD-like receptor-1,to activate procaspase-1), caspase-1 which activated pro-IL-1βto become IL-1βinvolved in inflammatory response. This study will observe the changes of the key molecular NLRP-1 and caspase-1 expression and the inflammatory changes in penumbra at different time points using rats with focal cerebral ischemia/reperfusion model.Objectives To explore the fuction of NLRP-1 and caspase-1 in cerebral ischemia-reperfusion injury and to provide a theoretical basis for experimental study in cerebrovascular disease through the detection of NLRP-1 and caspase-1 mRNA and protein expression and their association with inflammation index changes and infarct volume of rats after cerebral ischemia-reperfusion at different time points.Methods144 adult male SD (sprague-Dawley) rats were included as research subjects. They were randomly divided into Middle Cerebral Artery Occlusion(MCAO) groups and sham operation group and nomal grope with 18rats in each grope. The model of focal cerebral ischemic in rats was established by MCAO using suture occlusion method. Repefusion was accomplished by withdrawing the suture at 2h. Reperfusion groups were subdivided into 1h,3h,6h,12h,24h and 48h according to reperfusion time. The experiment was finished as following steps:total RNA from ischemia brain tissues of peripheral zone in 6rats in each group was isolated, and then the changes of gene expression for NLRP-1and caspase-1 expression were detected by RT-PCR and their protein expression were detected by westernbloting. For HE staining 6rats in each grope were perfused with 4% paraformaldehyde solution under anesthetized and brain sections were randomly harvested to detect the inflammatory response. Another 6rats were used to detect the infart volume by TTC staining. All the results were thansformed to datas through image analysis system which were expressed as (x±s). The values were analyzed by ANOVA-SNK-q of SPSS.16 software and pearson correlations was used for naalysis of correlations. p<0.05 was considered statistieally significant.Results1.79.6%rats were induced ischemic neurological deficits. In model animals,5rats died in anesthesia,8rats died of subarachnoid hemorrhage,6rats died of cerebral edema,15rats unexplained deaths, but 3rats success but not observed neurologic deficits symptoms.2. Infarct volume increased since rat6h (p<0.001) and reach a peak at 24h(p<0.001).3. There were littlein flammatory cells in normal and sham-operated rats. The inflammatory gradually increased with the advance of time after cerebral ischemic-reperfusion and reach a peak at 48h after reperfusion (p<0.001).4. The expression of casapse-1 mRNA increased since 6h (p<0.001) after ischemia-reperfusion and peaked at 24h after reperfusion (P<0.05). The expression of casapse-1 45kd increased since 6h (p＜0.001) after ischemia-reperfusion and peaked at 24h after reperfusion(P<0.001). The expression of casapse-1 20kd was detected at 1h after ischemic-reperfusion and peaked at 24h after reperfusion (P＜0.001). 5. The expression of NLRP-1 had no significant change from 1h to 48h after reperfusion (p>0.05).6. There is significant relation beteen caspase-1 expression and inflammation index (r=0.829,p<0.01) and beteen NLRP-1 expression and caspase-1 expression (r=0.346, p<0.05).There is no relation beteen NLRP-1 and inflammation index(r=0.160, p>0.05).Conclusions1. There was increase of caspase-1 mRNA and protain level which may be involved in the ischemia-reperfusion neuronal injury.2. There was no significant change of NLRP-1mRNA and protain in ischemic/reperfusion rat model, and it may be indirectly involved in the ischemia-reperfusion neuronal injury.