灸法预处理脑缺血再灌注模型大鼠自噬及NLRP3炎症小体的表达

Effect of moxibustion pretreatment on autophagy and NLRP3 inflammasome expression in cerebral ischemia-reperfusion model rats

背景:灸法作为中医学疗法之一在防治脑卒中方面显示出明显的临床疗效。目的:探讨灸法预处理对脑缺血再灌注模型大鼠自噬及NLRP3炎症小体表达的影响。方法:采用随机数字表法将36只SD大鼠分为假手术组、模型组、灸法预处理组,每组12只。灸法预处理组在造模前给予百会、大椎、足三里麦粒灸3壮/穴,1次/d,共治疗7 d。灸法预处理组末次艾灸30 min后,模型组和灸法预处理组大鼠采用大脑中动脉线栓法制备大脑中动脉阻塞模型,脑缺血处理2 h后拔除中动脉线栓进行再灌注;假手术组仅给予颈总动脉、颈内动脉、颈外动脉剥离,而并不插入线栓。再灌注12 h后,对3组大鼠进行进行神经功能缺损评分,采用TTC染色法计算大鼠脑梗死体积,Western-blot法检测大脑皮质缺血区中自噬相关蛋白Beclin1、p62及NLRP3炎症小体的蛋白表达。结果与结论:①模型组大鼠神经功能缺损评分高于假手术组(P<0.01),灸法预处理组大鼠神经功能缺损评分低于模型组(P<0.01);②TTC染色显示,假手术组大鼠脑组织未见明显梗死灶,模型组、灸法预处理组大鼠右侧脑组织可见明显缺血灶,其中灸法预处理组大鼠右侧脑梗死体积小于模型组(P<0.01);③Western-blot检测显示,与假手术组比较,模型组、灸法预处理组Beclin1、NLRP3蛋白表达升高(P<0.01),p62蛋白表达降低(P<0.01);与模型组比较,灸法预处理组Beclin1蛋白表达升高(P<0.05),p62及NLRP3蛋白表达降低(P<0.01);④结果表明,灸法预处理可显著改善缺血再灌注后大鼠的神经功能,其机制可能与激活自噬抑制炎症因子表达有关。

BACKGROUND:Moxibustion as one of the Chinese medicine therapies has shown significant clinical efficacy in the prevention and treatment of stroke.OBJECTIVE:To explore the effect of moxibustion pretreatment on autophagy and NLRP3 inflammasome expression in rats with cerebral ischemia reperfusion.METHODS:Thirty-six Sprague-Dawley rats were randomly divided into sham operation group,model group,and moxibustion pretreatment group,with 12 rats in each group.The moxibustion pretreatment group was given moxibustion at Baihui,Dazhui,and Zusanli before modeling.Each acupoint was given moxibustion for 3 strengths,once a day for 7 days.In the model and moxibustion pretreatment groups,the rat model of middle cerebral artery occlusion was made by suturing of the middle cerebral artery 30 minutes after the last moxibustion.After 2 hours of cerebral ischemia,the middle artery suture was removed and the rats were reperfused for 12 hours.In the sham operation group,only the common carotid artery,internal carotid artery,and external carotid artery were dissected without suturing the middle cerebral artery.The neurological deficit score was assessed,the cerebral infarct volume was calculated using 2,3,5-triphenyltetrazolium chloride staining,and the expression levels of autophagy-related proteins Beclin1,p62 and NLRP3 inflammasome in cerebral cortex ischemic area were detected using western blot assay.RESULTS AND CONCLUSION:The neurological deficit score in the model group was significantly higher than that in the sham operation group(P<0.01),and the neurological deficit score of the moxibustion pretreatment group was significantly lower than that of the model group(P<0.01).2,3,5-Triphenyltetrazolium chloride staining results showed that there were no obvious infarcts in the brain tissue of rats in the sham operation group,and obvious ischemic lesions were detected in the right brain tissue of rats in the model and moxibustion pretreatment groups.The infarct volume in the moxibustion pretreatment group was significantly less than that in the model group(P<0.01).Western blot results showed that compared with the sham operation group,the protein expression of Beclin1 and NLRP3 was significantly increased in the model and moxibustion pretreatment groups(P<0.01),while the expression of p62 protein decreased(P<0.01).Compared with the model group,the expression of Beclin1 protein was significantly increased(P<0.05),and the expression of p62 and NLRP3 proteins was significantly decreased in the moxibustion pretreatment group(P<0.01).To conclude,moxibustion pretreatment can significantly improve the neurological function of rats after cerebral ischemia-reperfusion,and its mechanism may be related to the activation of autophagy and inhibition of the expression of inflammatory factors.