NLRP3炎症小体在神经病理性痛大鼠脊髓小胶质细胞激活中的作用

Role of NLRP3 inflammasomes in activation of microglia in rats with neuropathic pain

目的评价NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体在神经病理性痛大鼠脊髓小胶质细胞激活中的作用。方法健康成年雄性SD大鼠66只,体重180~220 g,采用随机数字表法分为3组(n=22):假手术组(S组)、神经病理性痛组(NP组)和神经病理性痛+MCC950组(NP+M组)。采用结扎坐骨神经的方法制备大鼠神经病理性痛模型。NP+M组于结扎坐骨神经后腹腔注射NLRP3炎症小体抑制剂MCC95010 mg/kg,1次/d,连续7 d。取10只大鼠,分别于造模前和造模后1、2、3、7、14 d时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。于造模后7和14 d时,处死6只大鼠,取脊髓组织,采用Western blot法测定Iba-1表达水平,采用免疫荧光法检测Iba-1和NLRP3的共表达情况。于造模后7 d时,取脊髓组织,采用Western blot法检测NLRP3、凋亡相关斑点样蛋白(ASC)、caspase-1、IL-1β和IL-18表达水平。结果与S组相比,NP组和NP+M组造模后各时点MWT降低,TWL缩短,脊髓NLRP3、ASC、caspase-1,IL-1β、IL-18和Iba-1表达上调(P<0.05);与NP组相比,NP+M组造模后各时点MWT升高,TWL延长,脊髓NLRP3、ASC、caspase-1、IL-1β、IL-18和Iba-1表达下调(P<0.05)。脊髓Iba-1和NLRP3存在共表达。结论NLRP3炎症小体可调控脊髓小胶质细胞激活,参与了大鼠神经病理性痛的过程。

Objective To evaluate the role of NLRP3 inflammasome in the activation of microglia in rat with neuropathic pain.Methods Sixty-six healthy adult male Sprague-Dawley rats,weighing 180-220 g,were divided into 3 groups(n=22 each)using a random number table method:sham operation group(group S),neuropathic pain group(group NP)and neuropathic pain plus MCC950 group(group NP+M).Neuropathic pain was induced by ligation of the sciatic nerve in the rats anesthetized with chloral hydrate.NLRP3 inflammasome inhibitor MCC95010 mg/kg was intraperitoneally injected once a day for 7 consecutive days starting from the end of ligating the sciatic nerve in group NP+M.Ten rats were selected,and the mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured before establishing the model and at 1,2,3,7 and 14 days after establishing the model.Six rats were sacrificed at 7 and 14 days after establishing the model,and spinal cord tissues were obtained for determination of the expression of Iba1(by Western blot)and co-expression of Iba-1 and NLRP3(by immunofluorescence).Spinal cord tissues were obtained at 7 days after establishing the model for determination of the expression of apoptosis-associated speck-like protein containing a CARD(ASC),caspase-1,interleukin-1beta(IL-1β)and IL-18 by Western blot.Results Compared with group S,the MWT was significantly decreased,and TWL was shortened at each time point after establishing the model,and the expression of NLRP3,ASC,caspase-1,IL-1β,IL-18 and Iba-1 was up-regulated in NP and NP+M groups(P<0.05).Compared with group NP,the MWT was significantly increased,and TWL was prolong at each time point after establishing the model,and the expression of NLRP3,ASC,caspase-1,IL-1β,IL-18 and Iba-1 was down-regulated in group NP+M(P<0.05).Co-expression of Iba-1 and NLRP3 was found.Conclusion NLRP3 inflammasomes can regulate the activation of microglia and is involved in neuropathic pain in rats.