脊髓背角LRRK2在大鼠DNP中的作用及其与NF-κB信号通路的关系  

Role of LRRK2 in spinal dorsal horn in DNP and relationship with NF-κB signaling pathway in rats

作　　者：徐艳艳 周俊[2] 郝泉水 Xu Yanyan;Zhou Jun;Hao Quanshui(Department of Anesthesiology,Wuhan No.1 Hospital,Wuhan 430022,China;Department of Anesthesiology,Huanggang Central Hospital of Yangtze University,Huanggang 438000,China)

机构地区：[1]武汉市第一医院麻醉科,武汉430022 [2]长江大学附属黄冈市中心医院麻醉科,黄冈438000

出　　处：《中华麻醉学杂志》2025年第3期341-347,共7页Chinese Journal of Anesthesiology

摘　　要：目的:评价脊髓背角富含亮氨酸重复激酶2(LRRK2)在大鼠糖尿病神经病理性痛(DNP)中的作用,及其机制是否与NF-κB信号通路有关。方法:SPF级健康雄性SD大鼠,8周龄,体质量205~238 g,采用高糖高脂饲料喂养联合腹腔注射链脲佐菌素的方法制备2型糖尿病大鼠模型。2型糖尿病大鼠机械缩足反应阈(MWT)和热缩足潜伏期(TWL)低于基础值85%以下则为DNP造模成功。取DNP造模成功的大鼠36只,采用随机数字表法分为3组(n=12):DNP组、MLi-2(LRRK2抑制剂)组和MLi-2+佛波醇-12-豆蔻酰-13乙酸酯(PMA,NF-κB激活剂)组。MLi-2组于大鼠L 5-6间隙鞘内注射MLi-21 mg/kg。MLi-2+PMA组鞘内注射MLi-21 mg/kg,1 h后鞘内注射PMA 20μg/10μl。DNP组鞘内注射等量5%二甲基亚砜。随机取12只正常大鼠作为对照组(C组),采用普通饲料进行喂养,腹腔注射生理盐水2 ml,鞘内注射等量5%二甲基亚砜。鞘内注射每日1次,连续注射7 d。于鞘内注射第1、3、5和7天时测定MWT和TWL,使用神经电生理学方法检测大鼠运动神经传导速度(MNCV)。取大鼠L 4-6段脊髓组织,HE染色观察病理学结果,免疫荧光染色观察脊髓背角LRRK2与小胶质细胞活化标志物离子化钙结合适配分子-1(Iba-1)共定位情况。取L 4-6段脊髓背角组织,采用酶联免疫吸附法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)水平,蛋白免疫印迹法检测LRRK2、Iba-1、核因子-κB(NF-κB)p65和磷酸化NF-κB p65(p-NF-κB p65)表达水平。结果:与C组比较,DNP组MWT降低,TWL缩短,MNCV降低,脊髓背角LRRK2与Iba-1共表达细胞百分比升高,TNF-α、IL-1β和IL-6含量升高,LRRK2和Iba-1表达上调,p-NF-κB p65/NF-κB p65比值升高(P<0.05),脊髓背角细胞核皱缩,出现炎性细胞浸润;与DNP组比较,MLi-2组MWT升高,TWL延长,MNCV升高,脊髓背角LRRK2与Iba-1共表达细胞百分比降低,TNF-α、IL-1β和IL-6含量降低,LRRK2和Iba-1表达下调,p-NF-κB p65/NF-κB p65比�Objective:To evaluate the role of leucine-rich repeat kinase 2(LRRK2)in the spinal dorsal horn in diabetic neuropathic pain(DNP)and to determine whether the mechanism involved nuclear factor kappa B(NF-κB)signaling pathway in rats.Methods:SPF healthy male Sprague-Dawley rats,aged 8 weeks,weighing 205-238 g,were fed a high-sugar and high-fat diet and received intraperitoneal injection of streptozotocin to induce a type 2 diabetes mellitus model.Successful DNP model was defined as the mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)being below 85%of the baseline in type 2 diabetes mellitus rats.Thirty-six rats with DNP were allocated into 3 groups(n=12 each)using a random number table method:DNP group,MLi-2(LRRK2 inhibitor)group,and MLi-2+phorbol 12-myristate 13-acetate(PMA)(NF-κB activator)group.MLi-21 mg/kg was intrathecally injected at L 5-6 in MLi-2 group.In MLi-2+PMA group,MLi-21 mg/kg was intrathecally injected,and 1 h later PMA 20μg/10μl was intrathecally injected.In group DNP,the equal volume of 5%dimethyl sulfoxide was intrathecally injected.Twelve healthy rats were randomly selected and served as control group(C group).The animals were fed a standard diet,normal saline 2 ml was intraperitoneally injected,and the equal volume of 5%dimethyl sulfoxide was intrathecally injected in group C.Intrathecal injection was performed once a day for 7 consecutive days.The MWT and TWL were measured at 1,3,5 and 7 days after intrathecal injection,and the motor nerve conduction velocity(MNCV)was measured using neuroelectrophysiological techniques.The L 4-6 segments of the spinal cord were collected,and histopathological changes were observed through HE staining.The co-localization of leucine-rich repeat kinase 2(LRRK2)in the spinal dorsal horn with the microglial activation marker ionized calcium-binding adaptor molecule-1(Iba-1)was observed by immunofluorescence staining.The L 4-6 segments of the spinal dorsal horn were harvested for determination of the level of tumor necrosis factor-a

关 键 词：富含亮氨酸重复丝氨酸-苏氨酸蛋白激酶2 糖尿病神经病变 小神经胶质细胞 炎症 NF-ΚB 脊髓背角 

分 类 号：R587.2[医药卫生—内分泌]