miRNA-149改善坐骨神经慢性压迫损伤大鼠神经病理性疼痛的机制研究  

Mechanism of miRNA-149 in improving neuropathic pain in rats with chronic compression injury of the sciatic nerve

作　　者：余艳丽 程小宁[2] 薛静[3] YU Yan-li;CHENG Xiao-ning;XUE Jing(Department of Blood Transfusion,No.215 Hospital of Shaanxi Nuclear Industry,Xianyang 712000,Shaanxi,CHINA;Department of Pediatric Cardiology and Nephrology,Second Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,Xianyang 712021,Shaanxi,CHINA;Department of Laboratory Medicine,Second Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,Xianyang 712021,Shaanxi,CHINA)

机构地区：[1]陕西省核工业二一五医院输血科,陕西咸阳712000 [2]陕西中医药大学第二附属医院儿童心肾病科,陕西咸阳712021 [3]陕西中医药大学第二附属医院检验科,陕西咸阳712021

出　　处：《海南医学》2023年第20期2898-2905,共8页Hainan Medical Journal

摘　　要：目的 探究miR-149对大鼠坐骨神经慢性压迫损伤(CCI)引起的神经病理性疼痛(NP)的影响及分子机制。方法 将72只SD大鼠随机分为假手术组(sham)、CCI组、CCI+miR-149 agomir阴性对照(CCI+agomir-NC)组、CCI+miR-149 agomir组、CCI+miR-149 agomir+pc-NC组、CCI+miR-149 agomir+pc-CaMKⅡγ组,每组12只。采用CCI诱导建立NP大鼠模型,于建模前及建模后的第3天、第7天、第11天、第14天利用Von Frey检测大鼠的机械痛敏,热辐射法检测大鼠的热痛敏;建模后第14天用RT-qPCR法检测大鼠背根神经节(DRG)中miR-149和钙调蛋白依赖性蛋白激酶Ⅱγ (CaMKⅡγ) mRNA表达;蛋白免疫印迹法(Western blot)检测DRG中CaMKⅡγ蛋白水平;尼氏(Nissl)染色观察DRG神经元尼氏小体变化;免疫荧光法检测脊髓神经元中CaMKⅡγ表达情况;酶联免疫吸附法(ELISA)检测DRG中肿瘤坏死因子α (TNF-α)、白介素1β (IL-1β)和干扰素-γ (IFN-γ)含量;双荧光素酶报告基因实验验证miR-149与CaMKⅡγ的靶向关系。结果 与sham组比较,CCI组大鼠机械痛阈值和热痛阈、DRG中miR-149表达下降,DRG中CaMKⅡγ的mRNA和蛋白水平、CaMKⅡγ阳性表达神经元数量、TNF-α、IL-1β和IFN-γ水平升高,且DRG中尼氏小体减少,差异均有统计学意义(P<0.05)。过表达miR-149可改善CCI大鼠机械痛阈和热痛阈,降低DRG中CaMKⅡγ的mRNA和蛋白水平、CaMKⅡγ阳性表达神经元数量以及TNF-α、IL-1β、IFN-γ水平,尼氏小体明显增多,差异均有统计学意义(P<0.05)。与CCI+miR-149 agomir+pc-NC组比较,CCI+miR-149 agomir+pc-CaMKⅡγ组机械痛阈值和热痛阈下降,DRG中CaMKⅡγ的m RNA和蛋白水平、CaMKⅡγ阳性表达神经元数量、TNF-α、IL-1β和IFN-γ水平升高,尼氏小体减少,差异均有统计学意义(P<0.05)。双荧光素酶结果显示,过表达miR-149可降低CaMKⅡγ-WT的荧光素酶活性。结论 过表达miR-149可能通过靶向抑制CaMKⅡγ表达,缓解CCI大鼠的神经病理性疼痛。Objective To explore the effect and molecular mechanism of miR-149 on neuropathic pain(NP)caused by chronic compression injury(CCI)of the sciatic nerve in rat.Methods Seventy-two SD rats were randomly divided into sham group(sham),CCI group,CCI+miR-149 agomir negative control(CCI+agomir-NC)group,CCI+miR-149 agomir group,and CCI+miR-149 agomir+pc-NC group,CCI+miR-149 agomir+pc-CaMKⅡγgroup,with 12 rats in each group.The model of NP rats was induced by chronic compression injury(CCI)of sciatic nerve.Before modeling and on the 3rd,7th,11th,and 14th day after modeling,Von Frey was used to detect the mechanical hyperalgesia in rats,and the thermal hyperalgesia of rats was detected by thermal radiation method.RT-qPCR was used to detect the ex-pression of miR-149 and calmodulin-dependent kinaseⅡγ(CaMKⅡγ)mRNA in dorsal root ganglion(DRG)of rats on the 14th day after modeling.The expression of CaMKⅡγin DRG was detected by Western blot.Nissl staining was used to observe the changes of NissL bodies in DRG neurons.The expression of CaMKⅡγin spinal cord neurons was detect-ed by immunofluorescence.The levels of tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β)and interferon-γ(IFN-γ)in DRG were detected by enzyme-linked immunosorbent assay(ELISA).The targeting relationship between miR-149 and CaMKⅡγwas verified by dual-luciferase reporter gene assay.Results Compared with sham group,the mechanical pain threshold,thermal pain threshold,and the expression of miR-149 in DRG were decreased in CCI group,the mRNA and protein levels of CaMKⅡγ,the number of CaMKⅡγpositive expression neurons,and the levels of TNF-α,IL-1β,and IFN-γin DRG were increased,and the number of Nissl bodies in DRG decreased,with statistically significant differences(P<0.05).Overexpression of miR-149 could significantly improve the mechanical pain threshold and thermal pain threshold in CCI rats,reduce the mRNA and protein levels of CaMKⅡ,the number of CaMKⅡγposi-tive expression neurons,and the levels of TNF-α,IL-1β,and IFN

关 键 词：SD大鼠 神经病理性疼痛 miR-149 钙调蛋白依赖性蛋白激酶Ⅱγ 神经炎症 病理性疼痛 作用机制 

分 类 号：R-332[医药卫生]