氧化应激通过TDP-43激活mtDNA-cGAS/STING通路诱发神经元损伤和小鼠痛觉敏化  

Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway

作　　者：李丽 黄鹏辉 崔剑 LI Li;HUANG Penghui;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2036-2045,共10页Journal of Army Medical University

基　　金：重庆市自然科学基金面上项目(CSTB2023NSCQ-MS×0683)。

摘　　要：目的探索TAR DNA结合蛋白43(transactive response DNA binding protein 43,TDP-43)在氧化应激诱导的小鼠神经元(neuro-2a,N2a)细胞损伤及小鼠痛觉敏化中的作用及机制。方法①为评估最佳诱导浓度,不同浓度的H_(2)O_(2)处理N2a细胞分为4组:对照组、200μmol/L H_(2)O_(2)组、400μmol/L H_(2)O_(2)组和800μmol/L H_(2)O_(2)组。②为评估最佳诱导时间,400μmol/L H_(2)O_(2)处理N2a细胞分为4组:对照组、6 h H_(2)O_(2)组、12 h H_(2)O_(2)组和24 h H_(2)O_(2)组。③为验证线粒体DNA(mitochondria DNA,mtDNA)释放途径,使用环孢素(cyclosporin,CsA)抑制线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)分为3组:对照组、24 h H_(2)O_(2)组和24 h H_(2)O_(2)+CsA组。④为验证TDP-43介导的细胞损伤机制,siRNA抑制TDP-43后分为3组:对照组、24 h H_(2)O_(2)组、24 h H_(2)O_(2)+siTDP-43组。⑤采用CCK-8检测细胞活性,EdU检测细胞增殖,Western blot检测TDP-43、神经元标志物(neuronal nuclei,NeuN)、环状GMP-AMP合酶(cylic GMP-AMP synthase,cGAS)和干扰素基因刺激因子(stimulator of interferon,STING)表达,qPCR检测mtDNA,免疫染色观察细胞内TDP-43表达变化,Calcein AM染色评估mPTP开放。⑥为验证TDP-43在神经病理性疼痛(neuropathic pain,NP)中的作用,将24只6~8周健康SPF级雄性C57BL/6J小鼠(体质量25~30 g)使用随机数字表法分为3组:对照组、慢性压迫性损伤(chronic constriction injury,CCI)组、CCI+siTDP-43组,术前1 d和术后7、14、21 d进行鞘内注射siTDP-43;术前1 d和术后1、3、5、7、14、21 d通过von Frey纤维丝和热辐射法测定小鼠机械痛阈值和热痛阈值,免疫荧光检测术后21 d腰段(L5-L6)脊髓背角中TDP-43与NeuN的变化。结果氧化应激刺激诱导N2a中TDP-43蛋白表达增加,刺激mtDNA通过mPTP释放,上调cGAS、STING的表达,影响N2a的细胞活性(P<0.05);CsA抑制mPTP通道的开放并减少mtDNA释放(P<0.05);下调TDP-43的表达后可显著降低mtDNA的释放,抑制cGASObjectiveTo investigate the role and possible mechanisms of transactive response DNA binding protein 43(TDP-43)in mediating neuronal injury induced by oxidative stress in mouse neuro-2a(N2a)cells and mouse pain sensitization.Methods①To evaluate the optimal induction concentration,N2a cells were treated with different concentrations of H_(2)O_(2),and the cells were divided into control group,200,400 and 800μmol/L H_(2)O_(2) groups.②To assess the optimal induction duration,N2a cells were treated with 400μmol/L H_(2)O_(2),and the cells were divided into control group,and the cell groups treated for 6,12 and 24 h,respectively.③To validate the mitochondrial DNA(mtDNA)release pathway,cyclosporin A(CsA)was used to inhibit the mitochondrial permeability transition pore(mPTP),and the cells were divided into control group,24 h H_(2)O_(2) group and 24 h H_(2)O_(2)+CsA group.④To validate TDP-43-mediated cellular damage,the cells were divided into control group,24 h H_(2)O_(2) group and 24 h H_(2)O_(2)+siTDP-43 group.⑤Cell viability was assessed using CCK-8 assay,while cell proliferation was determined using EdU assay.Western blot analysis was employed to examine the expression levels of TDP-43,neuronal nuclei(NeuN),cyclic GMP-AMP synthase(cGAS),and stimulator of interferon genes(STING).qPCR was utilized to measure the release of mtDNA.Immunostaining was conducted to observe intracellular expression of TDP-43,and Calcein AM staining was employed to evaluate mPTP opening status.⑥To elucidate the role of TDP-43 in neuropathic pain(NP),24 healthy SPF male C57BL/6J mice(6~8 weeks old,25~30 g)were randomly divided into control group,chronic constriction injury(CCI)group,and CCI+siTDP-43 group.In 1 d before and 7,14 and 21 d after surgery,intrathecal injections of siTDP-43 were administered.Mechanical and thermal pain thresholds of the mice were assessed using von Frey filaments and radiant heat,respectively,on 1 preoperatively and 1,3,5,7,14 and 21 d postoperatively.Immunofluorescence assay was conducted on 21 d post

关 键 词：神经病理性疼痛 TDP-43 cGAS/STING通路 MTDNA 氧化应激 

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