大鼠炎性痛形成时脊髓神经元P2X7受体与NLRP3/IL-1β信号通路的关系  

Relationship between P2X7 receptors and NLRP3/IL-1βpathway in spinal neurons in development of inflammatory pain in rats

作　　者：王丽蓉 朱进飘 方媛媛 钟琦[1] 张宗泽[1] 王焱林[1] 陈畅[1] Wang Lirong;Zhu Jinpiao;Fang Yuanyuan;Zhong Qi;Zhang Zongze;Wang Yanlin;Chen Chang(Department of Anesthesiology,Zhongnan Hospital of Wuhan University,Whuhan 430071,China)

机构地区：[1]武汉大学中南医院麻醉科,430071

出　　处：《中华麻醉学杂志》2020年第4期437-441,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81671060,81771160)。

摘　　要：目的评价大鼠炎性痛形成时脊髓神经元P2X7受体与NOD样受体热蛋白结构域相关蛋白3(NLRP3)/IL-1β信号通路的关系。方法SPF级健康成年雄性Wistar大鼠,体重180~220 g,取鞘内置管成功的大鼠40只,采用随机数字表法分为5组(n=8):对照组(CON组)、炎性痛组(IP组)、炎性痛+二甲基亚砜组(IP-DMSO组)、炎性痛+P2X7受体拮抗剂A740003组(IP-A组)和炎性痛+P2X7受体激动剂ATP组(IP-ATP组)。采用右后足踝关节腔内注射完全弗氏佐剂50μl的方法制备炎性痛模型,CON组于右后足踝关节腔内注射等容量生理盐水。于造模前1 d、造模后即刻、1、2和3 d时,IP-DMSO组鞘内注射1%二甲基亚砜10μl,IP-A组鞘内注射A7400030.1 nmol(溶于10μl二甲基亚砜中),IP-ATP组鞘内注射ATP 150 nmol(溶于10μl二甲基亚砜中)。于造模后3 d时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。采用ELISA法检测右后足踝关节组织前列腺素E2(PGE2)含量和脑脊液IL-1β浓度。然后处死大鼠,取L4-6脊髓组织,采用Western blot法检测NLRP3、casepase-1和IL-1β的表达,采用免疫荧光法检测P2X7与神经元特异性核蛋白(NeuN)、NLRP3与NeuN的共表达情况。结果与CON组比较,IP组关节组织PGE2含量升高,其余4组MWT降低,TWL缩短,脑脊液IL-1β浓度升高,脊髓NLRP3、caspase-1和IL-1β表达上调(P<0.05);与IP组比较,IP-A组MWT升高,TWL延长,脑脊液IL-1β浓度降低,脊髓NLRP3、caspase-1和IL-1β表达下调,IP-ATP组MWT降低,TWL缩短,脑脊液IL-1β浓度升高,脊髓NLRP3、caspase-1和IL-1β表达上调(P<0.05),IP-DMSO组上述各指标差异无统计学意义(P>0.05)。脊髓P2X7与NeuN存在共表达,NLRP3与NeuN存在共表达。结论脊髓神经元P2X7受体可通过激活NLRP3/IL-1β信号通路,参与了大鼠炎性痛的形成。Objective To evaluate the relationship between P2X7 receptors and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3)/interleukin-1beta(IL-1β)pathway in spinal neurons in the development of inflammatory pain(IP)in rats.Methods SPF healthy adult male Wistar rats,weighing 180-220 g,were used in this study.Forty rats in which intrathecal catheters were successfully implanted were divided into 5 groups(n=8 each)using a random number table method:control group(group CON),group IP,IP plus dimethyl sulfoxide(DMSO)group(group IP-DMSO),IP plus P2X7 receptor antagonist A740003 group(group IP-A)and IP plus P2X7 receptor agonist ATP group(group IP-ATP).Rats were anesthetized with pentobarbital sodium 40 mg/kg.IP was induced by injecting complete Freund′s adjuvant 50μl into the right ankle joint cavity,while group CON was injected with the equal volume of normal saline instead.On 1 day before establishing the model,immediately after establishing the model,and on 1,2 and 3 days after establishing the model,1%DMSO 10μl was intrathecally injected once a day in group IP-DMSO,A7400030.1 nmol(dissolved in DMSO 10μl)was intrathecally injected once a day in group IP-A,and ATP 150 nmol(dissolved in DMSO 10μl)was injected intrathecally once a day in group IP-ATP.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured on 3 days after establishing the model.Enzyme-linked immunosorbent assay was used to determine the prostaglandin E2(PGE2)concentrations in right ankle tissues and IL-1βconcentrations in cerebrospinal fluid(CSF).Then rats were sacrificed,and the lumber segments(L4-6)of the spinal cord were removed for determination of the expression of NLRP3,casepase-1,IL-1β(by Western blot)and co-expression of P2X7 receptors with neuron-specific nucleoprotein(NeuN)and NLRP3 and with NeuN(by immunofluorescence).Results Compared with group CON,PGE2 contents in ankle tissues were significantly increased in group IP,and the MWT was significantly decreased,th

关 键 词：炎症 慢性疼痛 受体 嘌呤能P2X7 NLR家族 热蛋白结构域包含蛋白3 白细胞介素1Β 

分 类 号：R402[医药卫生—临床医学]