摘要
目的 评价脊髓富亮氨酸重复激酶2(LRRK2)在大鼠神经病理性痛中的作用.方法 SPF级健康雄性SD大鼠50只,6～7周龄,体质量210～245 g,采用随机数字表法分为5组(n=10):对照组(C组)、神经病理性痛组(NP组)、GNE-7915低剂量组、GNE-7915中剂量组和GNE-7915高剂量组.采用选择性坐骨神经损伤术建立神经病理性痛模型.建模后7 d时,GNE-7915低、中、高剂量组分别腹腔注射LRRK2抑制剂GNE-7915 12.5、25.0和50.0 mg/kg.于建模前、建模后7 d、注射抑制剂后4 h时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL).痛阈测定结束后处死大鼠,取脊髓组织,采用免疫荧光染色法检测离子钙结合衔接分子1(Iba-1)阳性表达,采用ELISA法检测IL-1β、单核细胞趋化蛋白-1(MCP-1)和IL-18的含量,免疫荧光染色法检测磷酸化LRRK2(p-LRRK2)阳性表达,采用免疫印迹法检测LRRK2、IL-1β、MCP-1和IL-18的表达,计算p-LRRK2/LRRK2比值.结果 与C组相比,NP组MWT和TWL降低,脊髓组织Iba-1与p-LRRK2阳性细胞百分比、IL-1β、MCP-1与IL-18含量、p-LRRK2/LRRK2比值升高,IL-1β、MCP-1和IL-18表达上调(P＜0.05);与NP组相比,GNE-7915低、中、高剂量组MWT和TWL升高,脊髓组织Iba-1与p-LRRK2阳性细胞百分比、IL-1β、MCP-1 与 IL-18 含量、p-LRRK2/LRRK2 比值降低,IL-1β、MCP-1 和 IL-18 表达下调(P＜0.05).结论 脊髓LRRK2可能通过激活小胶质细胞,诱发炎症反应,参与大鼠神经病理性痛的病理生理机制.
Abstract
Objective To evaluate the role of spinal Leucine-rich Repeat Kinase 2(LRRK2)in neuropathic pain in rats.Methods Fifty SPF healthy male Sprague-Dawley rats,aged 6-7 weeks,weig-hing 210-245 g,were divided into 5 groups(n=10 each)using a random number table method:control group(C group),neuropathic pain group(NP group),low dose GNE-7915 group(low-dose GNE-7915 group),medium-dose GNE-7915 group(medium-dose GNE-7915 group),and high-dose GNE-7915 group(high-dose GNE-7915 group).Neuropathic pain was induced by the spared nerve injury in anesthetized rats.At 7 days after developing the model,LRRK2 inhibitor GNE-7915 12.5,25.0 and 50.0 mg/kg were intrap-eritoneally injected in low-,medium-and high-dose GNE-7915 groups,respectively.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured before developing the model,at 7 days after developing the model,and at 4 h after injecting the inhibitor.After measurement of the pain threshold,the rats were sacrificed and the spinal cord tissues were taken for determination of the positive expression of ionized calcium-binding adapter molecule 1(Iba-1)(by immunofluorescence stai-ning),contents of interleukin-1β(IL-1β),monocyte chemotactic protein-1(MCP-1)and IL-18(by en-zyme-linked immunosorbent assay),positive expression of phosphorylated LRRK2(p-LRRK2)(by immu-nofluorescence staining),and expression of LRRK2,IL-1β,MCP-1 and IL-18(by immunoblotting).The ratio of p-LRRK2/LRRK2 was calculated.Results Compared with C group,the MWT was significantly de-creased,the TWL was shortened,the proportion of Iba-1 and p-LRRK2 positive cells in spinal cord tissues,contents of IL-1β,MCP-1 and IL-18,and p-LRRK2/LRRK2 ratio were increased,and the expression of IL-1β,MCP-1 and IL-18 proteins was up-regulated in NP group(P＜0.05).Compared with NP group,the MWT was significantly increased,the TWL was prolonged,the proportion of Iba-1 and p-LRRK2 positive cells in spinal cord tissues,contents of IL-1β,MCP-1 and IL-18,and p-LRRK2/LRRK2 ratio were decreased,and the expression of IL-1β,MCP-1 and IL-18 proteins was down-regulated in low-,medium-and high-dose GNE-7915 groups(P＜0.05).Conclusions LRRK2 in the spinal cord may be involved in the pathophysiological mechanism of neuropathic pain by activating microglia and inducing inflammatory responses in rats.
基金项目
湖北省卫生健康委科研资助(WJ2023M176)
湖南省人民医院医联体专项科研基金项目(2022YLT006)
NETL
NSTL
万方数据