miR-124通过调控早期生长反应因子1对紫杉醇致神经病理性疼痛的机制研究  

Effect of miR-124 on neuropathic pain induced by paclitaxel via regulating early growth response1 EGR1

作　　者：王文康 李恒昌[2] 李湘莲[1] 冯杜杭 张汉裕 张庆文 朱瑞燕 侯春玲 WANG Wen-kang;LI Heng-chang;LI Xiang-lian;FENG Du-hang;ZHANG Han-yu;ZHANG Qing-wen;ZHU Rui-yan;HOU Chun-ling(Department of Anesthesiology,the Second People’s Hospital of Baiyun District,Guangzhou,Guangdong 510450,China;不详)

机构地区：[1]广州市白云区第二人民医院麻醉科,广东510450 [2]广州市第一人民医院麻醉科

出　　处：《环境与健康杂志》2024年第9期768-776,F0003,共10页Journal of Environment and Health

基　　金：广州市卫生健康科技一般引导项目(20231A010009)。

摘　　要：目的探讨微小RNA-124(microRNA-124,miR-124)的表达对紫杉醇诱导的神经病理性疼痛(neuropathic pain,NPP)的影响机制,以及其对早期生长反应因子1(early growth response 1,EGR1)的调控机制。方法将P12细胞分别暴露于0μmol/L(对照)、0.5μmol/L、1μmol/L、2μmol/L的紫杉醇溶液培养48 h;通过实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qRT-PCR)PCR实验、双荧光素酶实验以及蛋白质免疫共沉淀实验检测miR-124和EGR1的mRNA的表达以及两者之间的关系。将50只8~10周龄SD雄性大鼠分别在第1天、第3天、第5天、第7天腹腔注射2 mg/kg的紫杉醇,构建大鼠NPP模型;将大鼠随机分为假手术组、模型组、对照(agomiR-124-NC+pcDNA3.1-EGR1-NC)组、激动剂(agomiR-124)组、过表达(pcDNA3.1-EGR1)组,每组10只;其中,假手术组大鼠在造模过程中腹腔注射等剂量的生理盐水;在造模成功后,对照组大鼠尾静脉注射agomiR-124-NC+pcDNA3.1-EGR1-NC;激动剂组大鼠尾静脉注射agomiR-124+pcDNA3.1-EGR1;过表达组大鼠尾静脉注射agomiR-124+pcDNA3.1-EGR1,每日注射一次,连续注射14 d。测定大鼠的械缩足反射阈值(mechanical withdrawal threshold,MWT)和热缩足潜伏期(thermal withdrawal latency,TWL);末端脱氧核苷酸转移酶(TdT)介导的脱氧尿苷三磷酸(dUTP)缺口末端标记(TdT-mediated dUTPnick end labelling,TUNEL)染色检测脊髓组织中神经元的细胞凋亡率;采用酶联免疫吸附法检测各组大鼠血清中白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、IL-4和IL-10的水平;免疫荧光检测脊髓组织中小胶质细胞的表型转换;采用Western blot法检测各组脊髓组织中EGR1蛋白的表达。结果在紫杉醇诱导的P12细胞损伤模型中,miR-124的表达明显降低(均P<0.05),EGR1的mRNA的表达明显升高(均P<0.05),miR-124靶向调控EGR1的表达;动物实验中,与假手术组相比,模型组、对照组、激动剂组、过表达组大鼠的MWT和TWL、脊髓组织中Objective To understand the effect of the expression of microRNA-124(miR-124)on paclitaxel-induced neuropathic pain(NPP)and the regulating mechanism on early growth response 1(EGR1).Methods The P12 cells were exposed to paclitaxel at the doses of 0μmol/L,0.5μmol/L,1μmol/L and 2μmol/L respectively,for 48 h.Injury model of P12 cells induced by paclitaxel was established real-time quantitative polymerase chain reaction(qRT-PCR)and double luciferase test were used to detect the expression of mRNA of miR-124 and the mRNA expression of EGR1 and the relationship between them were detected though PCR test,double luciferase test and protein co-immunoprecipitation test.Totally 50 SD male rats aged 8 to 10 weeks were intraperitoneally injected 2 mg/kg of paclitaxel on day 1,day 3,day 5 and day 7,respectively,to establish the NPP models.The rats were divided into sham operation group(Sham),model group(Model),agomiR-124-NC+pcDNA3.1-EGR1-NC group(Control),agonist group(agomiR-124 group)and over expression group(pcDNA3.1-EGR1),with 10 rats in each group.The rats in the sham operation group were intraperitoneally injected with equal dose of normal saline during the modeling process.After successful modeling,the rats in the control group were injected with agomiR-124-NC+pcDNA3.1-EGR1-NC through tail vein.the rats in agonist group were injected with agomiR-124.The rats in the over expression group were injected with agomiR-124+pcDNA3.1-EGR1 through the tail vein once a day for 14 days..The mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were detected.TdT-mediated dUTPnick end labelling(TUNEL)staining was used to detect the apoptosis rate of neurons in spinal cord tissue.enzyme Enzyme linked immunosorbent assay was used to detect the levels of interleukin-1β(IL-1β),IL-6,IL-4 and IL-10 in serum of rats in each group.Immunofluorescence was used to detect the phenotypic transition of microglia in spinal cord tissue.Immunohistochemistry was used to detect the expression of B cell lymphoma/leukemia-2(Bcl-2)a

关 键 词：微小RNA-124 早期生长反应因子1 紫杉醇 神经病理性疼痛 小胶质细胞的表型转换 

分 类 号：R994.6[医药卫生—毒理学]