电针预处理减轻大鼠脑缺血再灌注损伤的机制:大麻素1型受体与NLRP3炎性小体的关系

Mechanism underlying reduction of cerebral ischemia-reperfusion injury by electroacupuncture preconditioning in rats:relationship between cannabinoid type 1 receptor and NLRP3 inflammasome

目的评价电针预处理减轻大鼠脑缺血再灌注损伤时大麻素1型受体(CB1R)与NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎性小体的关系。方法SPF级健康雄性SD大鼠40只,7~9周龄,体质量250~280 g,按照随机数字表法分为5组(n=8):假手术组(Sham组)、脑缺血再灌注组(I/R组)、电针预处理组(EA组)、CB1R拮抗剂AM251+电针预处理组(AM251+EA组)和CB1R激动剂WIN 55,212-2组(WIN组)。采用大脑中动脉栓塞法制备大鼠脑缺血再灌注损伤模型。EA组行电针预处理(取百会穴,电刺激参数为疏密波,频率2/15 Hz,强度1 mA,持续电刺激30 min,1次/d,连续电刺激5 d),末次电刺激后24 h制备脑缺血再灌注损伤模型。AM251+EA组每次电刺激前30 min腹腔注射CB1R拮抗剂AM2511 mg/kg,其余操作同EA组。WIN组腹腔注射CB1R激动剂WIN 55,212-21.5 mg/kg,连续注射5 d,末次注射后24 h时制备脑缺血再灌注损伤模型。于再灌注3 d时,进行神经行为学评分,随后处死大鼠,TTC法确定脑梗死体积百分比,提取缺血半暗带组织,采用Western blot法测定NLRP3、caspase-1及IL-1β表达。结果与Sham组相比,I/R组脑梗死体积百分比升高,神经行为学评分降低,缺血脑组织NLRP3、caspase-1和IL-1β表达上调(P<0.05);与I/R组相比,EA组和WIN组脑梗死体积百分比降低,神经行为学评分升高,缺血脑组织NLRP3、caspase-1和IL-1β表达下调(P<0.05);与EA组相比,AM251+EA组脑梗死体积百分比升高,神经行为学评分降低,缺血脑组织NLRP3、caspase-1和IL-1β表达上调(P<0.05)。结论电针预处理可能通过激活CB1R,进而抑制NLRP3炎性小体活化,减轻大鼠脑缺血再灌注损伤。

Objective To evaluate the relationship between cannabinoid receptor 1(CB1R)and the NOD-like receptor thermal protein domain-associated protein 3(NLRP3)during the reduction of cerebral ischemia-reperfusion(I/R)injury by electroacupuncture(EA)preconditioning in rats.Methods Forty SPF healthy male Sprague-Dawley rats,aged 7-9 weeks,weighing 250-280 g,were divided into 5 groups(n=8 each)according to the random number table method:sham operation group(Sham group),cerebral I/R group(I/R group),EA preconditioning group(EA group),CB1R antagonist AM251+EA preconditioning group(AM251+EA group),and CB1R agonist WIN 55,212-2 group(WIN group).Cerebral I/R was induced by middle cerebral artery occlusion(MCAO)in anesthetized animals.In EA group,EA preconditioning was performed,and the acupoint Baihui(GV20)was stimulated for 30 min with disperse-dense waves,the intensity of 1 mA and frequency of 2/15 Hz once a day for 5 consecutive days,and the model of cerebral I/R injury was developed at 24 h after the last EA.In AM251+EA group,CB1R antagonist AM2511 mg/kg was intraperitoneally injected at 30 min before each stimulation,and the remaining operations were the same as those previously described in EA group.CB1R agonist WIN 55,212-21.5 mg/kg was intraperitoneally injected for 5 consecutive days,and the model of cerebral I/R injury was prepared at 24 h after the last injection in WIN group.Neurological behavior was assessed and scored at 3 days of reperfusion.Then the rats were sacrificed,and brains were removed,and the infarct volume was measured by TTC staining,and the tissues in the ischemic penumbra were extracted for determination of the expression of NLRP3,caspase-1 and interleukin-1bata(IL-1β)by Western blot.Results Compared with Sham group,the percentage of cerebral infarct volume was significantly increased,the neurobehavioral score was decreased,and the expression of NLRP3,caspase-1 and IL-1βwas up-regulated in I/R group(P<0.05).Compared with I/R group,the percentage of cerebral infarct volume was significantly decreased,the neurobehavioral score was increased,and the expression of NLRP3,caspase-1 and IL-1βwas down-regulated in EA and WIN groups(P<0.05).Compared with EA group,the percentage of cerebral infarct volume was significantly increased,the neurobehavioral score was decreased,and the expression of NLRP3,caspase-1 and IL-1βwas up-regulated in AM251+EA group(P<0.05).Conclusions EA preconditioning may inhibit the activation of NLRP3 inflammasomes by activating CB1R,thus alleviating cerebral I/R injury in rats.