乳酸诱导的脊髓神经元线粒体分裂在小鼠糖尿病神经病理性痛中的作用  

Role of lactate-induced mitochondrial division of spinal cord neurons in diabetic neuropathic pain in mice

作　　者：潘文燕 贺俭[2] 王福宇 卢冠华 何万友[2] 张磊[2] 张创强 王汉兵 Pan Wenyan;He Jian;Wang Fuyu;Lu Guanhua;He Wanyou;Zhang Lei;Zhang Chuangqiang;Wang Hanbing(The First Clinical Medical College,Guangdong Medical University,Zhanjiang 524000,China;Department of Anaesthesiology,The First People's Hospital of Foshan,Foshan 528000,China)

机构地区：[1]广东医科大学第一临床医学院,湛江524000 [2]佛山市第一人民医院麻醉科,佛山528000

出　　处：《中华麻醉学杂志》2024年第5期603-608,共6页Chinese Journal of Anesthesiology

基　　金：广东省基础与应用基础研究基金(2021A1515111193,2022A1515140047);广东省医学科学技术研究基金(A2022105)。

摘　　要：目的评价乳酸诱导的脊髓神经元线粒体分裂在小鼠糖尿病神经病理性痛(DNP)中的作用。方法选择SPF级健康成年雄性C57BL/6小鼠36只,2月龄,体质量20~25 g,采用随机数字表法分为3组(n=12):对照组(C组)、DNP组和DNP+草氨酸钠组(OXA组)。采用腹腔注射链脲佐菌素130 mg/kg构建糖尿病模型。糖尿病建模成功后,OXA组腹腔注射草氨酸钠750 mg/kg,1次/d,持续4周,抑制乳酸生成;C组和DNP组于相同时点腹腔注射等容量生理盐水。于造模前、造模后1、2、3和4周时测定左侧后足机械缩足阈值(MWT)。最后一次行为学测试完成后,取血标本和L4-6段脊髓组织,采用比色法检测血清和脊髓组织乳酸水平,分别采用JC-1和DHE探针检测线粒体膜电位和ROS含量,采用Western blot法检测线粒体动力相关蛋白1(Drp1)和线粒体融合蛋白2(Mfn2)的表达,透射电镜观察线粒体的结构和数量,采用免疫荧光双标观察Drp1与神经元标志物NeuN共表达情况。结果与C组比较,DNP组和OXA组造模后MWT降低,血清和脊髓组织乳酸水平、脊髓ROS含量升高,线粒体膜电位下降,Drp1表达上调,Mfn2表达下调,线粒体数量增多,面积减小(P<0.05),Drp1和NeuN共表达增加;与DNP组比较,OXA组造模后MWT升高,血清和脊髓组织乳酸水平、脊髓ROS含量降低,线粒体膜电位升高,Drp1表达下调,Mfn2表达上调,线粒体数量减少,面积增大(P<0.05),Drp1和NeuN共表达减少。结论乳酸诱导的脊髓神经元线粒体过度分裂可导致线粒体功能障碍,可能参与小鼠DNP的维持机制。Objective To evaluate the role of lactate-induced mitochondrial division of spinal cord neurons in diabetic neuropathic pain(DNP)in mice.Methods Thirty-six SPF-grade healthy adult male C57BL/6 mice,aged 2 months,weighing 20-25 g,were divided into 3 groups(n=12 each)using a random number table method:control group(CON group),DNP group,and DNP+sodium oxalate group(OXA group).The diabetic model was established by intraperitoneal injection of streptozotocin 130 mg/kg.After the diabetic model was successfully established,sodium oxalate 750 mg/kg was intraperitoneally injected once a day for 4 consecutive weeks to inhibit lactate production in OXA group,while the equal volume of normal saline was given instead at the same time in C group and DNP group.The mechanical paw withdrawal threshold(MWT)of the left hindpaw was measured before developing the model and at 1,2,3 and 4 weeks after developing the model.After completing the last behavioral testing,the spinal cord tissue of the lumbar segment(L4-6)was taken for determination of the levels of lactate in serum and spinal cord tissues(by the colorimetric method),expression of the mitochondrial membrane potential,reactive oxygen species(ROS)content(using JC-1 or DHE probes),expression of mitochondrial dynamin-related protein 1(Drp1)and mitochondrial protein mitofusin 2(Mfn2)(by Western blot),and co-expression of Drp1 and neuronal neuronal marker neuronal nuclear protein(NeuN)(by immunofluorescence double labeling)and for examination of the structure and the number of mitochondria(with a transmission electron microscope).Results Compared with C group,the MWT was significantly decreased after developing the model,the levels of lactate in serum and spinal cord tissues and ROS content in the spinal cord were increased,the mitochondrial membrane potential was decreased,the Drp1 expression was up-regulated,the Mfn2 expression was down-regulated,the number of mitochondria was increased,the area was reduced(P<0.05),and the co-expression of Drp 1 and NeuN was increased in DNP grou

关 键 词：糖尿病神经病变 乳酸 线粒体 

分 类 号：R587.2[医药卫生—内分泌] R741[医药卫生—内科学]