摘要
目的:观察“醒脑开窍”针刺法对脑缺血再灌注损伤(CIRI)大鼠缺血侧皮质区N6-甲基腺苷(m6A)甲基化水平及关键甲基转移酶、去甲基化酶表达的影响,探讨针刺改善CIRI的作用机制。方法:从68只SPF级雄性SD大鼠中随机选取15只作为假手术组,其余大鼠采用线栓法制备大脑中动脉闭塞模型,缺血2 h后再灌注实现CIRI,剔除造模失败及中途死亡大鼠,将剩余的45只大鼠随机分为模型组、针刺组、非穴位针刺组,每组15只。针刺组予“醒脑开窍”针刺法干预,穴取双侧“内关”及“水沟”;非穴位针刺组于双侧腋中线下3 mm处及尾骨尖旁开3 mm处3个非穴位点行针刺干预。两组均针刺1次,留针30 min。造模前及缺血2 h后采用激光散斑血流仪监测大鼠脑血流灌注量;于缺血2 h后、再灌注后24 h评定大鼠神经行为学评分;采用氯化三苯基四氮唑(TTC)染色法测定大鼠脑梗死体积;Dot blot法检测大鼠缺血侧皮质区m6A甲基化水平;Western blot法及实时荧光定量PCR法检测大鼠缺血侧皮质区关键去甲基化酶脂肪量和肥胖相关蛋白(FTO)、AlkB同源蛋白5(ALKBH5)及关键甲基转移酶甲基转移酶样3(METTL3)、甲基转移酶样14(METTL14)、辅助蛋白Wilms肿瘤1相关蛋白(WTAP)蛋白及mRNA表达。结果:缺血2 h后大鼠脑血流灌注量降低>70%。与假手术组比较,模型组大鼠神经行为学评分、脑梗死体积百分比升高(P<0.01),缺血侧皮质区m6A甲基化水平升高(P<0.01),FTO蛋白及mRNA表达降低(P<0.01),ALKBH5、METTL3、METTL14蛋白及mRNA表达升高(P<0.01,P<0.05);与模型组和非穴位针刺组比较,针刺组大鼠神经行为学评分、脑梗死体积百分比降低(P<0.01),缺血侧皮质区m6A甲基化水平降低(P<0.01,P<0.05),FTO蛋白及mRNA表达升高(P<0.01)。结论:“醒脑开窍”针刺法可对脑缺血再灌注损伤大鼠发挥脑保护作用,其作用机制可能与上调FTO表达,进而调控m6A甲基化修饰有关。
Objective To observe the effects of Xingnao Kaiqiao acupuncture technique(for regaining consciousness and opening orifice)on methylation of N6-methyladenosine(m6A),and key methyltransferases and demethylases,so as to clarify the mechanism of acupuncture on cerebral ischemia-reperfusion injury(CIRI).Methods Of 68 male Sprague-Dawley rats of SPF grade,15 rats were randomly selected as a sham-operation group,and the remaining rats were subjected to the model of middle cerebral artery occlusion using the suture ligation.CIRI was induced by ischemia for 2 h followed by reperfusion.Rats that failed to modeling or died were excluded.The rest 45 rats were randomly divided into three groups,i.e.model group,acupuncture group,and non-point acupuncture group,with 15 rats in each group.The rats in the acupuncture group were treated with acupuncture at bilateral"Neiguan"(PC6)and"Shuigou"(GV26).In the non-point acupuncture group,acupuncture was delivered at three non-points,located 3 mm below the bilateral midaxillary line and 3 mm lateral to the tip of the coccyx.One intervention was operated in these two acupuncture groups and the needles were retained for 30 min.Before modeling start and 2 h after ischemia,a laser speckle flowmeter was used to monitor the cerebral blood perfusion.In 2 h of ischemia and 24 h of reperfusion,the neurological behavioral score was evaluated.The volume of rat cerebral infarction was determined by triphenyltetrazolium chloride(TTC)staining,and the level of m6A methylation in ischemic cortical area was detected by Dot blot,and the protein and mRNA expression of the demethylase i.e.fat mass and obesity-associated protein(FTO),AlkB homolog 5(ALKBH5)and key methyltransferases,i.e.methyltransferase-like 3(METTL3),methyltransferase-like 14(METTL14),and Wilms'tumor 1-associated protein(WTAP)in ischemic cortical area were detected by Western blot and real-time PCR.Results Cerebral blood perfusion decreased by>70%after 2 h ischemia.Compared with the sham-operation group,the neurobehavioral score and the percentage of cerebral infarction volume increased in the model group(P<0.01);the level of m6A methylation in the ischemic cortical area increased(P<0.01),the protein and mRNA expression of FTO decreased(P<0.01),and that of ALKBH5,METTL3,and METTL14 increased(P<0.01,P<0.05)in the model group.When compared with the model group and the non-point acupuncture group,the acupuncture group showed a decrease in the neurobehavioral score and the percentage of cerebral infarction volume(P<0.01),the level of m6A methylation in the ischemic cortical area was reduced(P<0.01,P<0.05),and the protein and mRNA expression of FTO was elevated(P<0.01).Conclusion Xingnao Kaiqiao acupuncture technique presents its protective effect on the brain in the rats with CIRI,which is related to up-regulating the expression of FTO and modulating m6A methylation.
作者
刘欣雨
张新昌
黄正
刘倩倩
赵艺
陆天靓
张智慧
倪光夏
LIU Xinyu;ZHANG Xinchang;HUANG Zheng;LIU Qianqian;ZHAO Yi;LU Tianliang;ZHANG Zhihui;NI Guangxia(College of Acupuncture-Moxibustion and Tuina,College of Health and Rehabilitation,Nanjing 210023,Jiangsu Province,China;Key Laboratory of Acupuncture and Medicine of Ministry of Education,Nanjing University of Chinese Medicine,Nanjing 210023,Jiangsu Province,China)
出处
《中国针灸》
北大核心
2025年第5期670-677,共8页
Chinese Acupuncture & Moxibustion
基金
国家自然科学基金资助项目:82074525、82205238。
关键词
脑缺血再灌注损伤
针刺
m6A甲基化修饰
脂肪量和肥胖相关蛋白
Scerebral ischemia-reperfusion injury
acupuncture
m6A methylation
fat mass and obesity-associated protein(FTO)
