脊髓富亮氨酸重复激酶2在大鼠神经病理性痛中的作用

Role of spinal Leucine-rich Repeat Kinase 2 in neuropathic pain in rats

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-791512.5、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。

ObjectiveTo evaluate the role of spinal Leucine-rich Repeat Kinase 2(LRRK2)in neuropathic pain in rats.MethodsFifty SPF healthy male Sprague-Dawley rats,aged 6-7 weeks,weighing 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-791512.5,25.0 and 50.0 mg/kg were intraperitoneally 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 staining),contents of interleukin-1β(IL-1β),monocyte chemotactic protein-1(MCP-1)and IL-18(by enzyme-linked immunosorbent assay),positive expression of phosphorylated LRRK2(p-LRRK2)(by immunofluorescence staining),and expression of LRRK2,IL-1β,MCP-1 and IL-18(by immunoblotting).The ratio of p-LRRK2/LRRK2 was calculated.ResultsCompared with C group,the MWT was significantly decreased,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).ConclusionsLRRK2 in the spinal cord may be involved in the pathophysiological mechanism of neuropathic pain by activating microglia and inducing inflammatory responses in rats.