Objective To investigate the levels of Nod-like receptor protein 3 (NLRP3) in primary cortical neurons with necroptosis.Methods Primary cortical neurons of SD rats were cultured for 12 days, before pretreated with Q-VD-Oph for 30min followed by oxygen-glucose deprivation to induce necroptosis.① The cells were divided into the following groups: a normal control group, an oxygen-glucose deprivation group (oxygen-glucose deprivation for 2 h followed by reperfusion for 12 h), and treatment groups (oxygen-glucose deprivation for 2 h followed by reperfusion for 3, 6, 12 and 24 h, and all of them were treated with Q-VD-Oph) Their levels of NLRP3 and lactate dehydrogenase (LDH) were examined.② The cells were divided into the following groups: a control group, an oxygen-glucose deprivation group (oxygen-glucose deprivation for 2 h followed by reperfusion for 12 h), and treatment groups (oxygen-glucose deprivation for 0.5, 1, 1.5, 2, and 3 h followed by reperfusion for 12 h, and all of them were treated with Q-VD-Oph).Their levels of NLRP3 and LDH were examined.③ The cells were subjected to oxygen-glucose deprivation for 2 h followed by reperfusion for 12 h (all of them were treated with Q-VD-Oph) before determination of their levels of NLRP3 by immunofluorescence.Results After oxygen-glucose deprivation for 2 h, the level of NLRP3 was increased as the time of reperfusion extended, which then reached the highest at 12 h and was remarkably decreased at 24 h (P<0.05).The level of LDH reached the highest at 12 h.After oxygen-glucose deprivation for different periods of time followed by reperfusion for 12 h, the level of NLRP3 reached the highest when oxygen-glucose deprivation for 2 h, which then was remarkably decreased at 3 h (P<0.05).The level of LDH reached the highest when oxygen-glucose deprivation for 2 h.The immunofluorescence intensity was the highest when the cells were oxygen-glucose deprived for 2 h followed by reperfusion for 12 h.Conclusions NLRP3 is present in primary cortical neuron with necroptosis.The degree of cell injury is gradually increased ass the level of NLRP3 increases.%目的 探讨Nod样受体蛋白3 (NLRP3)在大鼠原代皮质神经元坏死样凋亡中的表达情况.方法 SD大鼠原代皮质神经元细胞培养12天,Q-VD-Oph预处理30 min后缺糖缺氧刺激诱导坏死样凋亡.①将细胞分为正常对照组、缺糖缺氧组(缺糖缺氧2 h,复氧12 h)、实验组(缺糖缺氧2 h,复氧3、6、12、24 h,均用Q-VD-Oph处理),Western blot检测NLRP3蛋白表达,生化分析仪检测乳酸脱氢酶(LDH)水平;②将细胞分为正常对照组、缺糖缺氧组(缺糖缺氧2 h,复氧12 h)、实验组(缺糖缺氧0.5、1、1.5、2、3 h,复氧12 h,均用Q-VD-Oph处理),再次进行NLRP3及LDH测定;③细胞缺糖缺氧2 h、复氧12 h(用Q-VD-Oph处理)后免疫荧光观察NLRP3的表达.结果 缺糖缺氧2 h后,随着复氧时间的延长,NLRP3在12 h表达量最高,24 h表达量较12 h下降(P<0.05),LDH在12 h的表达量最高.缺糖缺氧时间不同,各组均复氧12 h时,NLRP3在缺糖缺氧 2 h表达量最高,3 h蛋白表达量较2 h下降(P<0.05),LDH在2 h的表达量最高.在缺糖缺氧2 h、复氧12 h,细胞免疫荧光强度最高.结论 NLRP3在原代皮质神经元细胞坏死样凋亡中有表达,且随着NLRP3表达量升高,细胞受损程度相应增高.
展开▼
