头穴丛刺结合康复训练对脑缺血再灌注大鼠肠道菌群及CaM/CaMKⅡ信号通路的影响

Effects of scalp clustering acupuncture combined with rehabilitation training on gut microbiota and CaM/CaMK II signaling pathway in rats with cerebral ischemia-reperfusion

目的观察头穴丛刺结合康复训练对脑缺血再灌注大鼠肠道菌群及CaM/CaMKⅡ信号通路的影响。方法将大鼠随机分为假手术组(sham组)、脑缺血再灌注损伤模型组(CIRI组)、头穴丛刺组(SCA组)(头穴丛刺干预,1次/日,连续14 d)、康复训练组(RT组)(跑台训练,30 min/次/日,连续14 d)和头穴丛刺联合康复训练组(SCA+RT组)(头穴丛刺干预,同时进行跑台训练),每组12只。分别于造模成功后3、7、14 d对大鼠进行神经功能评分;干预14 d后采用TTC染色检测脑梗死面积,ELISA检测脑组织中COX2、NOS2水平,16S-rDNA测序检测结肠内容物肠道菌群变化,Western blot检测脑组织中CaM、CaMKⅡ蛋白表达情况。结果CIRI组大鼠神经功能评分,脑梗死面积,脑组织中COX2、NOS2含量,CaM、CaMKⅡ蛋白水平及OUT水平的Simpson指数均显著高于sham组,OUT水平的Shannon指数、Ace指数、Chao指数均显著低于sham组(均P<0.05);与CIRI组相比,SCA组、RT组和SCA+RT组大鼠神经功能评分,脑梗死面积,脑组织中COX2、NOS2含量,CaM、CaMKⅡ蛋白相对表达量,以及OUT水平的Simpson指数均显著降低,OUT水平的Shannon指数、Ace指数、Chao指数均显著升高(均P<0.05),且以SCA+RT组变化更显著。与sham组相比,CIRI组拟杆菌门、放线杆菌门、脱硫弧菌门、厚壁菌门、双歧杆菌属、norank-f-Muribaculaceae、杜氏杆菌属、拟普雷沃菌属、红蝽菌属_UCG-002、普雷沃菌属菌落相对丰度显著降低,变形菌门菌落、贺菌群属菌落对丰度显著升高(均P<0.05);与CIRI组相比,SCA组、RT组和SCA+RT组拟杆菌门、放线杆菌门、脱硫弧菌门、厚壁菌门、双歧杆菌属、norank-f-Muribaculaceae、杜氏杆菌属、拟普雷沃菌属、红蝽菌属_UCG-002、普雷沃菌属菌落相对丰度显著升高,变形菌门菌落、贺菌群属相对丰度显著降低(均P<0.05),且以SCA+RT组变化更显著。结论头穴丛刺结合康复训练可改善脑缺血再灌注损伤大鼠神经功能,调节肠道菌群失调,抑制CaM/CaMKⅡ信号通路的活化,从而对脑组织发挥保护作用。

Objective To investigate the effects of scalp cluster acupuncture on intestinal flora and CaM/CaMKⅡsignaling pathway in rats with cerebral ischemia-reperfusion injury.Methods Rats were randomly divided into a sham operation group(Sham group),a cerebral ischemia-reperfusion injury model group(CIRI group),a head acupoint cluster acupuncture group(SCA group)(head acupoint cluster acupuncture intervention,1 time/day,continuous for 14 days),a rehabilitation training group(RT group)(treadmill training,30 minutes/time/day,continuous for 14 days),and a head acupoint cluster acupuncture combined rehabilitation training group(SCA+RT group)(head acupoint cluster acupuncture intervention,simultaneous treadmill training),with 12 rats in each group.Neurological function scores were performed on rats 3,7,and 14 days after successful modeling;After 14 days of intervention,the area of cerebral infarction was detected using TTC staining,the levels of COX2 and NOS2 in brain tissue were detected using ELISA,changes in gut microbiota of colon contents were detected using 16S rDNA sequencing,and the expression of CaM and CaMK II proteins in brain tissue was detected using Western blot.Results The neurological function score,cerebral infarction area,COX2,NOS2 content in brain tissue,CaM,CaMK II protein levels,and the Simpson index of OUT levels in the CIRI group were significantly higher than those in the sham group.The Shannon index,Ace index,and Chao index of OUT levels were significantly lower than those in the sham group(all P<0.05);Compared with the CIRI group,the neurological function score,cerebral infarction area,COX2 and NOS2 content in brain tissue,relative expression of CaM and CaMK II proteins,and Simpson index of OUT level were significantly reduced in the SCA group,RT group,and SCA+RT group.The Shannon index,Ace index,and Chao index of OUT level were significantly increased(all P<0.05),and the changes were more significant in the SCA+RT group.Compared with the Sham group,the CIRI group includes Bacteroidetes,Actinobacteria,Desulfovibrio,Firmicutes,Bifidobacteria,norank-f-Muribaculaceae,Durobacillus,Prevotella,and Red Stigma.The relative abundance of UCG-002 and Prevotella colonies was significantly reduced,while the abundance of Proteobacteria and Shigella colonies was significantly increased(all P<0.05);Compared with the CIRI group,the SCA group,RT group,and SCA+RT group have Bacteroidetes,Actinobacteria,Desulfovibrio,Firmicutes,Bifidobacteria,norank-f-Muribaculaceae,Durobacillus,Prevotella,and Rhodotobacter genera_The relative abundance of UCG-002 and Prevotella colonies significantly increased,while the relative abundance of Proteobacteria colonies and Shigella groups significantly decreased(all P<0.05),and the changes were more significant in the SCA+RT group.Conclusion The combination of scalp acupuncture and rehabilitation training can improve the neural function of rats with cerebral ischemia-reperfusion injury,regulate intestinal microbiota imbalance,inhibit the activation of CaM/CaMK II signaling pathway,and thus exert a protective effect on brain tissue.