受损神经来源的mtDNA通过GRP75增加线粒体内Ca2+水平诱导U87细胞凋亡和小鼠痛觉敏化  

Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca2+level through GRP75

作　　者：黄鹏辉 李丽 崔剑 HUANG Penghui;LI Li;CUI Jian(Department of Pain Medicine,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院疼痛科,重庆400038

出　　处：《陆军军医大学学报》2024年第18期2081-2091,共11页Journal of Army Medical University

基　　金：重庆市自然科学基金面上项目(CSTB2023NSCQ-MSX0683)。

摘　　要：目的探讨受损神经来源的线粒体DNA(mitochondrial DNA,mtDNA)对人脑星形胶质母细胞瘤细胞(human glioblastoma cell line U-87MG,U87)内GRP75蛋白表达、线粒体Ca^(2+)水平、细胞凋亡的影响,及其诱导小鼠痛觉敏化的机制。方法采用坐骨神经慢性缩窄(chronic constriction injury,CCI)建立小鼠神经病理性疼痛(neuropathic pain,NeP)模型,采用随机数字表法将20只C57BL/6雄性小鼠(6~8周龄,体质量20~30 g)分为4组:假手术组、CCI-7天组、CCI-14天组、CCI-21天组,行为学检测痛觉敏化情况,实时荧光定量PCR法(quantitative Real-time PCR,qPCR)检测小鼠脊髓中mtDNA含量变化。从H_(2)O_(2)处理的人神经母细胞瘤细胞(human neuroblastoma cell line SH-SY5Y,SH-SY5Y)提取mtDNA。采用浓度为0~1 ng/μL的mtDNA处理U87细胞。CCK-8检测细胞活力变化,根据结果选择适宜的实验浓度。将U87细胞分为5组:对照组、mtDNA组、mtDNA+GRP75抑制剂(MKT-0771μg/mL)组、mtDNA+钙螯合剂(BAPTA-AM 10μmol/L)组和mtDNA+MKT-077+BAPTA-AM组,mtDNA处理前2 h分别予以MKT-077、BAPTA-AM预处理。Western blot检测GRP75蛋白(glucose-regulated protein 75,GRP75)的表达,内质网-线粒体共定位染色观察内质网-线粒体连接;钙离子荧光探针检测线粒体Ca^(2+)水平;活性氧(reactive oxygen species,ROS)及线粒体膜电位检测评估线粒体氧化应激功能;PI荧光标记检测U87细胞凋亡率。结果与假手术组相比,CCI-21天组小鼠脊髓中用于检测mtDNA含量的细胞色素c氧化酶I(cytochrome c oxidase I,CO1)和NADH脱氢酶亚基1(nicotinamide adenine dinucleotide dehydrogenase 1,ND1)均升高,CO1:1.01±0.20 vs 2.22±0.26(P<0.05),ND1:1.00±0.12 vs 1.79±0.07(P<0.05)。CCK-8结果显示:mtDNA(1 ng/μL)可抑制U87细胞活力(P<0.01);0.2 ng/μL mtDNA可上调GRP75蛋白的表达(P<0.05),促进内质网-线粒体偶联,增加线粒体Ca^(2+)水平(109.4±62.6 vs 540.3±150.3,P<0.05),并诱使线粒体释放ROS增多(P<0.05)。MKT-077或/和BAPTA-AM处理后,线粒ObjectiveTo investigate the effects of damaged nerve-derived mitochondrial DNA(mtDNA)on GRP75 protein expression,mitochondrial Ca^(2+)level and apoptosis in human glioblastoma cell line U-87MG(U87)cells,and its mechanism of inducing pain sensitization in mice.MethodsChronic constriction injury(CCI)of the sciatic nerve was used to establish a mouse model of neuropathic pain(NeP).A total of 20 male C57BL/6 mice(6~8 weeks old,weighing 20~30 g)were randomly divided into sham group,and CCI-7,-14 and CCI-21 d groups.Paw withdrawal mechanical threshold(PWMT)was measured,and the changes in mtDNA content in the spinal cord were detected with quantitative real-time PCR(qPCR).After mtDNA was extracted from human neuroblastoma cells(SH-SY5Y)treated with H_(2)O_(2),U87 cells were treated with mtDNA at a dose of 0~1 ng/μL.CCK-8 assay was utilized to detect cell viability,and the appropriate concentration was selected according to the results.Then U87 cells were divided into:control group,mtDNA group,mtDNA+GRP75 inhibitor(MKT-0771μg/mL)group,mtDNA+calcium chelator(BAPTA-AM 10μmol/L)group,and mtDNA+MKT-077+BAPTA-AM group.MKT-077 and BAPTA-AM were pre-treated in 2 h before mtDNA treatment,respectively.Western blotting was employed to assess the expression of glucose-regulated protein 75(GRP75)protein,and endoplasmic reticulum-mitochondrial colocalization staining was conducted to observe the endoplasmic reticulum-mitochondrial junction.Calcium ion fluorescent probe was used to measure mitochondrial Ca^(2+)levels.The oxidative stress and function of mitochondria were evaluated by reactive oxygen species(ROS)and mitochondrial membrane potential.PI fluorescent labeling was employed to examine apoptotic rate of U87 cells.ResultsCompared with the sham group,the cytochrome C oxidase I(CO1)and nicotinamide adenine dinucleotide dehydrogenase 1(ND1),which were utilized to measure mtDNA content in the spinal cord of mice in the CCI-21 group,were increased(1.01±0.20 vs 2.22±0.26,P<0.05,1.00±0.12 vs 1.79±0.07,P<0.05).CCK-8 assay showe

关 键 词：神经病理性疼痛 MTDNA GRP75 钙 凋亡 

分 类 号：R322.85[医药卫生—人体解剖和组织胚胎学] R363.21[医药卫生—基础医学] R441.1