NOD样受体蛋白3炎性体在大鼠脑缺血再灌注损伤中的作用

NLRP3 inflammasome deficiency reduces rats brain damage after cerebral ischemia reperfusion

目的 探讨NOD样受体蛋白3（NLRP3）炎性体在大鼠正常脑组织中的表达及在脑缺血-再灌注损伤中的作用.方法 先取健康成年雄性SD大鼠40只,按随机数字表法随机分为假手术组,再灌注12、24、48 h组,每组10只.采用大脑中动脉栓塞法构建脑缺血（2 h）模型.免疫荧光双染色法检测正常脑组织NLRP3炎性体的表达分布,Western blot法、实时定量PCR法检测NLRP3炎性体mRNA、蛋白质的表达.再选取40只健康成年雄性SD大鼠,随机分为假手术对照组、再灌注对照组、假手术加药组、再灌注加药组,每组10只.两加药组于术前30 min经腹腔注射500 mg/kg格列苯脲,两对照组给予等量生理盐水,建模成功后24h,对各组行颅脑MRI检查和伊文思蓝染色,观察脑组织的损伤及血-脑屏障通透性的改变.结果 健康大鼠脑组织中,NLRP3炎性体仅表达于小胶质细胞和血管内皮细胞,神经元和星形胶质细胞中未见表达.再灌注后12、24、48 h,NLRP3的mRNA和蛋白表达均明显高于假手术组（P＜0.01）,且在24h达高峰.颅脑MRI显示,再灌注加药组损伤面积明显小于再灌注对照组[（21.7±4.2）％对比（36.0±4.7）％,P＜0.01].伊文思蓝染色显示,再灌注加药组伊文思蓝含量低于再灌注对照组[（18.7±3.8）μg/g对比（32.1±5.1）μg/g,P＜0.05].结论 NLRP3炎性体仅表达于大鼠脑组织的小胶质细胞及微血管内皮细胞中,其可能通过损伤血-脑屏障参与脑缺血-再灌注损伤.

Objective To observe the expression and distribution of NLRP3 in rats brain and to explore the significant correlations between NLRP3 and cerebral ischemia/reperfusion （IR） injuries.Methods FortyMale SD rats were randomly divided into the Sham group,ischemia/reperfusion 12h,24h,48h group.The ischemia model was induced by occlusion of the middle cerebral artery （MCAO） with a microfilament in rats.The expression and distribution of NLRP3 in rats brain was explored by immunofluorescence staining.After reperfusion injury,we examined the levels of protein and mRNA by Western blot and real-time PCR,respectively.Another 40 Male SD rats were randomly divided into the shamcontrol group,IR-control group,sham-glyburine group,IR-glyburine group.Glyburine used as inhibitor of NLRP3 was given to the rats in glyburine group before MCAO model.In order to analyze infarct and assess function of blood-brain barrier （BBB）,magnetic resonance imaging （MRI） and Evans blue stain were performed.Results NLRP3 was mainly expressed in microglia and endothelial cells by immunofluorescence staining.By real-time PCR and Western blot analyses,NLRP3 expression was markedly enhanced in the ischemic cerebral hemisphere with a peak expression of NLRP3 at 24 hours after reperfusion（P 〈 0.05）.In MRI the area of treatment/reperfusion group was significantly smaller than control/reperfusion group [（21.7±4.2）% vs.（36.0 ±4.7）%,（P 〈 0.05）.Our results also indicated that the content of Evans blue of treatment/reperfusion？ group was significantly lower than control/reperfusion group [（18.7 ± 3.8） μg/g vs.（32.1 ± 5.1） μg/g] by Evans blue stain （P 〈 0.05）.Conclusions NLRP3 was mainly expressed in microglia and endothelial cells.NLRP3 contributes to the development of cerebral ischemia/ reperfusion injury and ischemia-induced BBB disruption.