脊髓NLRP3炎症小体对神经病理性疼痛的影响及机制

发布时间：2018-11-06 14:57

【摘要】：目的:通过抑制NLRP3炎症小体,观察坐骨神经慢性压榨性损伤(chronic constriction injury,CCI)模型大鼠机械痛敏变化,探讨NLRP3炎症小体对神经病理性疼痛的影响及机制。方法:Sprague-Dawley雄性大鼠36只,8周龄,体重250~300 g,随机分为4组(n=9):假手术组(control组)、慢性坐骨神经压榨性损伤组(CCI组)、溶剂对照组(CCI-vehicle组)和抑制剂组(CCI-MCC950组)。分别于建模前1 d、建模后1、4和7 d测定机械缩足反应阈(MWT);CCI-vehicle组和CCI-MCC950组于建模后4d测定MWT后分别鞘内注射等容积生理盐水和MCC950溶液,每天1次,连续3天;于建模后7 d MWT测量后处死大鼠取L4-6脊髓组织,采用Western blot法测定脊髓组织NLRP3、ASC、caspase-1及IL-1β蛋白表达,采用实时荧光定量PCR法测定脊髓组织NLRP3、ASC、caspase-1及IL-1βm RNA表达,采用Td T介导d UTP末端标记法(Td T-mediated d UTP Nick-End Labeling,TUNEL)检测脊髓组织凋亡细胞,采用免疫荧光双标法观察脊髓组织NLRP3与GFAP共表达。结果:与control组比较,CCI组、CCI-vehicle组和CCI-MCC950组MWT显著降低(P0.05),NLRP3、ASC、caspase-1和IL-1β蛋白和m RNA表达上调(P0.05),凋亡阳性细胞数升高(P0.05);与CCI组、CCI-vehicle组比较,CCI-MCC950组经MCC950处理后MWT升高(P0.05),且脊髓组织NLRP3、ASC、caspase-1和IL-1β蛋白和m RNA表达下调(P0.05),凋亡阳性细胞数减少(P0.05)。免疫荧光双标结果显示:NLRP3蛋白主要表达于脊髓星形胶质细胞和神经元胞浆。结论:NLRP3炎症小体在神经病理性疼痛大鼠脊髓中活化,抑制该炎症小体能一定程度改善CCI模型大鼠机械痛敏;NLRP3炎症小体参与促进大鼠神经病理性疼痛的发展,其机制可能与脊髓星型胶质细胞活化导致免疫炎症反应和神经元凋亡有关。
[Abstract]:Aim: to investigate the effect of NLRP3 inflammatory body on neuropathic pain and its mechanism by observing the changes of mechanical pain sensitivity in rats with chronic sciatic nerve compression injury (chronic constriction injury,CCI) model by inhibiting the inflammatory body of NLRP3. Methods: Thirty-six male Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (control group) and chronic sciatic nerve compression injury group (CCI group). Solvent control group (CCI-vehicle group) and inhibitor group (CCI-MCC950 group). Mechanical foot contraction reaction threshold was measured 1 day before modeling, 1 day and 7 days after modeling. (MWT); CCI-vehicle group and CCI-MCC950 group were injected intrathecal normal saline and MCC950 solution respectively once a day for 3 days after MWT was measured 4 days after modeling. The L4-6 spinal cord tissue was measured by MWT 7 days after modeling. The expression of NLRP3,ASC,caspase-1 and IL-1 尾 protein in spinal cord tissue was measured by Western blot method, and NLRP3,ASC, in spinal cord tissue was measured by real-time fluorescence quantitative PCR method. The expression of caspase-1 and IL-1 尾 m RNA was detected by Td T mediated d UTP terminal labeling (Td T-mediated d UTP Nick-End Labeling,TUNEL), and the coexpression of NLRP3 and GFAP in spinal cord tissue was observed by immunofluorescence double labeling method. Results: compared with control group, MWT in CCI group, CCI-vehicle group and CCI-MCC950 group were significantly decreased (P0.05), the expression of NLRP3,ASC,caspase-1 and IL-1 尾 protein and m RNA were up-regulated (P0.05), and the number of apoptotic positive cells was increased (P0.05). Compared with CCI group and CCI-vehicle group, MWT in CCI-MCC950 group increased after MCC950 treatment (P0.05), and the expression of NLRP3,ASC,caspase-1 and IL-1 尾 protein and m RNA in spinal cord tissue decreased (P0.05), and the number of apoptotic positive cells decreased (P0.05). Immunofluorescence double labeling results showed that NLRP3 protein was mainly expressed in spinal astrocytes and neuron cytoplasm. Conclusion: NLRP3 inflammatory corpuscles are activated in the spinal cord of rats with neuropathic pain, and the inhibition of the inflammatory bodies can improve the mechanical pain sensitivity of CCI rats to some extent. The inflammatory bodies of NLRP3 are involved in promoting the development of neuropathic pain in rats. The mechanism may be related to the immune inflammation and neuronal apoptosis caused by the activation of astrocytes in spinal cord.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R402

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