PTPIP51调节的线粒体相关内质网膜结构改变在七氟烷致大鼠海马神经元程序性坏死中的作用:离体实验
Role of PTPIP51-regulated mitochondria-associated endoplasmic reticulum membranes in sevoflu-rane-induced necroptosis in hippocampal neurons of rats:an in vitro experiment
张琦 1刘延琴 1齐林 1王俊霞 1巨英超 2石磊1
作者信息
- 1. 河北省儿童医院麻醉科,石家庄 050030
- 2. 河北医科大学第四医院动物实验中心,石家庄 050011
- 折叠
摘要
目的 采用离体实验评价蛋白酪氨酸磷酸酶相互作用蛋白51(PTPIP51)调节的线粒体相关内质网膜(MAMs)结构改变在七氟烷致大鼠海马神经元程序性坏死中的作用.方法 原代培养SD大鼠胎鼠的海马神经元,培养7 d时,以5×105个/ml细胞密度接种于培养孔(100 μl/孔)或培养瓶(3 ml/瓶)中,采用随机数字表法分为4组(n=19):对照组(C组)、七氟烷组(Sev组)、L氟烷+siRNA-PTPIP51 转染组(Sev+siPTPIP51 组)和七氟烷+无义 siRNA 转染组(Sev+siNC 组).将 Sev 组、Sev+siPTPIP51组和Sev+siNC组神经元置于含2％七氟烷的培养箱中37 ℃培养5 h.收集神经元,采用MTT法检测存活率,采用流式细胞术检测胞浆游离钙离子浓度([Ca2+]i)及程序性坏死率,采用Western blot法检测PTPIP51、受体相互作用蛋白激酶1(RIPK1)、RIPK3和磷酸化人混合系列蛋白激酶样结构域(p-MLKL)的表达,透射电镜下观察并记录MAMs长度、内质网周长和线粒体周长.结果 与C组比较,Sev组神经元活力下降,[Ca2+]i、程序性坏死率升高,PTPIP51、RIPK1、RIPK3和p-MLKL表达上调,MAMs长度/内质网周长比值和MAMs长度/线粒体周长比值升高(P＜0.05).与Sev组比较,Sev+siPTPIP51组神经元活力升高,[Ca2+]i和程序性坏死率降低,PTPIP51、RIPK1、RIPK3和p-MLKL表达下调,MAMs长度/内质网周长比值和MAMs长度/线粒体周长比值降低(P＜0.05),Sev+siNC组上述指标差异无统计学意义(P＞0.05).结论 PTPIP51表达上调介导MAMs的结构改变参与了七氟烷诱发海马神经元程序性坏死的过程.
Abstract
Objective To evaluate the role of mitochondria-associated endoplasmic reticulum mem-branes(MAMs)regulated by protein tyrosine phosphatase interacting protein 51(PTPIP51)in sevoflurane-induced necroptosis in hippocampal neurons of rats using the in vitro experiment.Methods Primary cul-tured hippocampal neurons from fetal rats of Sprague-Dawley rats were inoculated in culture wells(100μl/well)or culture flasks(3 ml/bottle)at a density of 5× 105 cells/ml at 7 days of culture and divided into 4 groups(n=19 each)using a random number table method:control group(C group),sevoflurane group(Sev group),sevoflurane+siRNA-PTPIP51 transfection group(Sev+siPTPIP51 group),and sevoflurane+nonsense siRNA transfection group(Sev+siNC group).The neurons were placed in a culture incubator con-taining 2％sevoflurane and incubated at 37 ℃ for 5 h in Sev,Sev+siPTPIP51 and Sev+siNC groups.Then neurons were collected for determination of the cell survival rate(by MTT method),cytoplasmic calcium concentration([Ca2+]i)and necroptosis rate(by flow cytometry),expression of PTPIP51,receptor-inter-acting protein kinase 1(RIPK1),RIPK3,and phosphorylated mixed lineage kinase domain-like protein(p-MLKL)(by Western blot)and for microscopic examination of the partial length,endoplasmic reticulum circumference,and mitochondrial circumference of MAMs(with a transmission electron microscope).Re-sults Compared with group C,the activity of neurons was significantly decreased,the[Ca2+]i and necrop-tosis rate were increased,the expression of PTPIP51,RIPK1,RIPK3 and p-MLKL was up-regulated,and the ratio of partial length of MAMs to endoplasmic reticulum perimeter and partial length of MAMs to mito-chondrial perimeter were increased in group Sev(P＜0.05).Compared with group Sev,the activity of neu-rons was significantly increased,the[Ca2+]i and necroptosis rate were decreased,the expression of PTPIP51,RIPK1,RIPK3 and p-MLKL was down-regulated,and the ratio of partial length of MAMs to en-doplasmic reticulum perimeter and partial length of MAMs to mitochondrial perimeter were decreased in group Sev+siPTPIP51(P＜0.05),and no statistically significant changes were found in the above parameters in group Sev+siNC(P＞0.05).Conclusions Up-regulation of PTPIP51 expression mediates structural changes in MAMs and is involved in the process of sevoflurane-induced necroptosis in hippocampal neurons of rats.
关键词
七氟醚/程序性坏死/神经元/蛋白质酪氨酸磷酸酶类/内质网/线粒体/偶联Key words
Sevoflurane/Necroptosis/Neurons/Protein tyrosine phosphatases/Endoplas-mic reticulum/Mitochondria/Coupling引用本文复制引用
基金项目
国家自然科学基金(82301382)
河北省自然科学基金(H2022316001)
河北省政府资助临床医学优秀人才项目(ZF2023126)
出版年
2024
NETL
NSTL
万方数据