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 sevoflurane-induced necroptosis in hippocampal neurons of rats:an in vitro experiment

作　　者：张琦 刘延琴 齐林[1] 王俊霞[1] 巨英超[2] 石磊[1] Zhang Qi;Liu Yanqin;Qi Lin;Wang Junxia;Ju Yingchao;Shi Lei(Department of Anesthesiology,Hebei Children′s Hospital,Shijiazhuang 050030,China;Animal Experiment Center of the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,China)

机构地区：[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

出　　处：《中华麻醉学杂志》2024年第7期806-810,共5页Chinese Journal of Anesthesiology

基　　金：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)。

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5×10^(5)个/ml细胞密度接种于培养孔(100μl/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)、七氟烷+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([Ca^(2+)]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,[Ca^(2+)]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,[Ca^(2+)]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。Objective To evaluate the role of mitochondria-associated endoplasmic reticulum membranes(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 cultured 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 5×10^(5) 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 containing 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([Ca^(2+)]i)and necroptosis rate(by flow cytometry),expression of PTPIP51,receptor-interacting 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).Results Compared with group C,the activity of neurons was significantly decreased,the[Ca^(2+)]i and necroptosis 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 mitochondrial perimeter were increased in group Sev(P<0.05).Compared with group Sev,the activity of neurons was significantly increased,the[Ca^(2+)]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 endoplasmic reticulum perimeter and partial length of MAMs to mitochond

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 

分 类 号：R614[医药卫生—麻醉学]