CAJ | 학술논문

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.
목적 탐토NOD양수체단백3(NLRP3)염성체재대서정상뇌조직중적표체급재뇌결혈-재관주손상중적작용.방법 선취건강성년웅성SD대서40지,안수궤수자표법수궤분위가수술조,재관주12、24、48 h조,매조10지.채용대뇌중동맥전새법구건뇌결혈(2 h)모형.면역형광쌍염색법검측정상뇌조직NLRP3염성체적표체분포,Western blot법、실시정량PCR법검측NLRP3염성체mRNA、단백질적표체.재선취40지건강성년웅성SD대서,수궤분위가수술대조조、재관주대조조、가수술가약조、재관주가약조,매조10지.량가약조우술전30 min경복강주사500 mg/kg격렬분뇨,량대조조급여등량생리염수,건모성공후24h,대각조행로뇌MRI검사화이문사람염색,관찰뇌조직적손상급혈-뇌병장통투성적개변.결과 건강대서뇌조직중,NLRP3염성체부표체우소효질세포화혈관내피세포,신경원화성형효질세포중미견표체.재관주후12、24、48 h,NLRP3적mRNA화단백표체균명현고우가수술조(P＜0.01),차재24h체고봉.로뇌MRI현시,재관주가약조손상면적명현소우재관주대조조[(21.7±4.2)％대비(36.0±4.7)％,P＜0.01].이문사람염색현시,재관주가약조이문사람함량저우재관주대조조[(18.7±3.8)μg/g대비(32.1±5.1)μg/g,P＜0.05].결론 NLRP3염성체부표체우대서뇌조직적소효질세포급미혈관내피세포중,기가능통과손상혈-뇌병장삼여뇌결혈-재관주손상.
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.