脊髓线粒体自噬在姜黄素减轻小鼠糖尿病神经病理性痛中的作用  

Role of spinal cord mitochondrial autophagy in alleviation of diabetic neuropathic pain by curcumin in mice

作　　者：张创强 王汉兵[1] 陈碧芸 张磊[1] 黄慧慧[1] 李东林 贺俭[1] Zhang Chuangqiang;Wang Hanbing;Chen Biyun;Zhang Lei;Huang Huihui;Li Donglin;He Jian(Department of Anesthesiology,First People′s Hospital of Foshan,Foshan 528000,China;Department of Anesthesiology,Maternity and Infant Health Hospital of Foshan,Foshan 528000,China)

机构地区：[1]佛山市第一人民医院麻醉科,佛山528000 [2]佛山市妇幼保健院麻醉科,佛山528000

出　　处：《中华麻醉学杂志》2022年第9期1081-1085,共5页Chinese Journal of Anesthesiology

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

摘　　要：目的评价脊髓线粒体自噬在姜黄素减轻小鼠糖尿病神经病理性痛(DNP)中的作用。方法选择SPF级健康雄性C57BL/6小鼠,2月龄,体重20~25 g,采用腹腔注射链脲佐菌素130 mg/kg方法制备DNP模型。取DNP建模成功的小鼠36只,采用随机数字表法分成3组(n=12):DNP组、DNP+姜黄素组(DPR组)、DNP+姜黄素+环孢素A组(DRC组)。另取12只正常C57BL/6小鼠腹腔注射等容量生理盐水,为正常对照组(NC组)。DPR组给予姜黄素200 mg/kg灌胃,1次/d,持续7 d;DRC组每次姜黄素灌胃前,鞘内注射线粒体自噬抑制剂环孢素A 10 mg/kg,1次/d,持续7 d,NC组和DNP组于同时点灌胃等容量生理盐水,1次/d,持续7 d。于灌胃前、灌胃1、3、5、7 d时测定机械缩足反应阈(MWT)。最后1次行为学测试完成后,取L4-6段脊髓组织,采用JC-1法和DCFH-DA法分别联合流式细胞仪检测线粒体膜电位和ROS含量,采用Western blot法检测微管相关轻链蛋白3(LC3)、Beclin1和P62的表达水平,计算LC3-Ⅱ/Ⅰ比值,透射电镜检测线粒体自噬小体,采用免疫荧光双标技术观察LC3-Ⅱ与线粒体外膜转位酶复合体蛋白20(TOM20)共表达情况。结果与NC组比较,DNP组MWT和线粒体膜电位降低,ROS含量和LC3-Ⅱ/Ⅰ比值升高,Beclin1表达上调,P62表达下调(P<0.05),线粒体自噬小体形成增加,LC3-Ⅱ和TOM20共表达增加。与DNP组比较,DPR组MWT和线粒体膜电位升高,ROS含量降低,LC3-Ⅱ/Ⅰ比值升高,Beclin1表达上调,P62表达下调(P<0.05),线粒体自噬小体形成增加,LC3-Ⅱ和TOM20共表达增加。与DPR组比较,DRC组MWT和线粒体膜电位降低,ROS含量升高,LC3-Ⅱ/Ⅰ比值降低,Beclin1表达下调,P62表达上调(P<0.05),线粒体自噬小体形成减少,LC3-Ⅱ和TOM20共表达减少。结论姜黄素减轻小鼠DNP的机制可能与上调脊髓线粒体自噬水平,改善线粒体功能有关。Objective To evaluate the role of spinal cord mitochondrial autophagy in alleviation of diabetic neuropathic pain(DNP)by curcumin in mice.Methods SPF healthy male C57BL/6 mice,aged 2 months,weighing 20-25 g,in which DNP model was established by intraperitoneal injection of streptozotocin(STZ)130 mg/kg,were used in this study.A total of 36 mice with successfully established DNP model were divided into 3 groups(n=12 each)using the random number table method:DNP group,DNP+curcumin group(DPR group),and DNP+curcumin+cyclosporine A group(DRC group).Another 12 C57BL/6 mice were selected and served as normal control group(NC group),and the equal volume of normal saline was intraperitoneally injected.In group DPR,curcumin 200 mg/kg was administered by intragastric gavage,once a day,for 7 consecutive days.In group DRC,the mitochondrial autophagy inhibitor cyclosporine A 10 mg/kg was intrathecally injected once a day for 7 consecutive days before each administration of curcumin.The equal volume of normal saline was administered by intragastric gavage at the same time point,once a day,for 7 consecutive days in group NC and group DNP.The mechanical paw withdrawal threshold(MWT)was measured before intragastric gavage and at 1,3,5 and 7 days after intragastric gavage.After the last behavioral testing,the L4-6 spinal cord tissues were removed for determination of the mitochondrial membrane potential and ROS content(by JC-1 and DCFH-DA combined with flow cytometry),expression of microtubule-associated protein light chain 3(LC3),Beclin1 and P62(by Western blot),and mitochondrial autophagosomes(by transmission electron microscopy)and for microscopic examination of the co-expression of LC3-Ⅱwith mitochondrial translocase outer membrane protein 20(TOM20)(using immunofluorescence double-labeling technique).Results Compared with group NC,the MWT and mitochondrial membrane potential were significantly decreased,the ROS content and LC3-Ⅱ/Ⅰratio were increased,the expression of Beclin1 was up-regulated,the expression of P62 was down

关 键 词：糖尿病神经病变 姜黄素 线粒体 自噬 

分 类 号：R285.5[医药卫生—中药学]