| Experiment 1 The Effect of Electroacupuncture Preconditioning on the Structure of Gut Microbiota in Rats with Cerebral Ischemia/Reperfusion InjuryObjective:To observe the effects of electroacupuncture preconditioning on neurological function and gut microbiota in rats with cerebral ischemia/reperfusion injury.Method:36 SPF male Sprague-Dawley rats were randomly divided into three groups:sham group,model group,and electroacupuncture preconditioning group,with 12 rats in each group.The electroacupuncture preconditioning group was given electroacupuncture preconditioning at Baihui(DU20)and Zusanli(ST36)for 2 weeks,while the other two groups were only given grasping under the same conditions.A rat middle cerebral artery ischemia/reperfusion model was prepared,and laser doppler flowmetry was used to detect the cerebral blood flow changes on the ischemic side.After 2 hours of ischemia,the fishing line was pulled out to restore reperfusion.After reperfusion for 12 hours,1 day,3 days,7 days,the modified Neurological Severity Scores was used to evaluate the nerve function,the grasping strength of rat forelimbs was measured with the grip tester for rats and mice,and the survival rate and weight were counted and recorded;after reperfusion for 7 d,TTC staining was used to measure cerebral infarction volume,HE staining was used to observe the pathological damage of the ischemic cortex and colon tissue,and high-throughput sequencing of 16S rDNA V3-V4 region was used to analyze the diversity of gut microbiota.Result:1.The modified Neurological Severity Scores:after reperfusion for 12 h,1 d,3 d,7 d,compared with the sham group,the modified Neurological Severity Scores of rats in the model group was increased significantly(P<0.05);compared with the model group,the modified Neurological Severity Scores of rats in the electroacupuncture preconditioning group was significantly decreased(P<0.05).2.Forelimb grasping strength test:after reperfusion for 12 h,1 d,3 d,7 d,compared with the sham group,the forelimb grasping strength of rats in the model group was significantly decreased(P<0.05);compared with the model group,the forelimb grasping strength of rats in the electroacupuncture preconditioning group was increased significantly(P<0.05).3.Survival rate:after reperfusion for 7 days,compared with the sham group,the survival rate of rats in the model group(46.15%)was significantly decreased(P<0.05);compared with the model group,the survival rate of rats in the electroacupuncture preconditioning group(80.00%)was increased significantly(P<0.05).4.TTC staining:after reperfusion for 7 d,compared with the sham group,the cerebral infarction volume of rats in the model group was significantly increased(P<0.05);compared with the model group,the cerebral infarction volume of rats in the electroacupuncture preconditioning group was significantly decreased(P<0.05).5.HE staining:after reperfusion for 7 d,compared with the sham group,the pathological damage degree of ischemic cortex and colon tissue of rats in the model group was significantly worse;compared with the model group,the pathological damage degree of ischemic cortex and colon tissue of rats in the electroacupuncture preconditioning group was significantly reduced.6.Sequencing of 16S rDNA V3-V4 region:At the phylum level,compared with the sham group,the relative abundances of Proteobacteria,TM7 and Tenericutes in the cecum content of rats in the model group were increased significantly(P<0.05);and compared with the model group,the relative abundances of Proteobacteria,TM7 and Tenericutes in the cecal content of rats in the electroacupuncture preconditioning group were significantly decreased(P<0.05).At the family level,compared with the sham group,the relative abundance of Lachnospiraceae and Bifidobacteriaceae in the cecal content of rats in the model group were significantly decreased(P<0.05),and the relative abundance of Leuconostocaceae and Enterobacteriaceae were increased significantly(P<0.05);compared with the model group,the relative abundance of Leuconostocaceae and Enterobacteriaceae in the cecal content of rats in the electroacupuncture pretreatment group were significantly decreased(P<0.05).At the genus level,compared with the sham group,the relative abundances of Lactobacillus,Coprococcus,Bifidobacteriunz,Blautia and Prevotella in the cecum content of rats in the model group were significantly decreased(P<0.05),and the relative abundance of Clostridiaceae Clostridium and[Ruminococcus]were significantly increased(P<0.05);compared with the model group,the relative abundance of Lactobacillus,Blautia and Prevotella in the cecal content of rats in the electroacupuncture pretreatment group were significantly increased(P<0.05),and the relative abundance of Clostridiaceae_Clostridium and[Ruminococcus]were significantly decreased(P<0.05).The correlation analysis between gut microbiota and mNSS score showed that at the phylum level,the relative abundance of Proteobacteria,TM7 and Tenericutes was positively correlated with the mNSS score(P<0.05);at the family level,the relative abundance of Leuconostocaceae and Enterobacteriaceae was positively correlated with the mNSS score({<0.05);at genus level,the relative abundance of Clostridiaceae Clostridium and[Ruminococcus]was positively correlated with the mNSS score(P<0.05),while the relative abundance of Lactobacillus,Blautia and Prevotella was negatively correlated with the mNSS score(P<0.05).Conclusion:1.Electroacupuncture preconditioning can down-regulate the relative abundance of Proteobacteria,TM7,Tenericutes,Leuconostocaceae,Enterobacteriaceae,Clostridiaceae Clostridium and[Ruminococcus],and up-regulate the relative abundance of Lactobacillus,Blautia and Prevotella in the cecum content of rats with cerebral ischemia/reperfusion,reduce the pathological damage degree of the ischemic cortex and colon tissue,the volume of cerebral infarction,the mortality rate,and Neurological Severity.Experiment 2 Effect of Electroacupuncture Preconditioning on Th17/Treg Immune Balance in Rats with Cerebral Ischemia/ReperfusionObjective:To observe the effects of electroacupuncture preconditioning on Th17/Treg immune imbalance and the expression of related inflammatory factors in rats with cerebral ischemia/reperfusion.Method:36 SPF male Sprague-Dawley rats were randomly divided into three groups:sham group,model group,and electroacupuncture preconditioning group,with 12 rats in each group.The electroacupuncture preconditioning group was given electroacupuncture preconditioning at Baihui(DU20)and Zusanli(ST36)for 2 weeks,while the other two groups were only given grasping under the same conditions.A rat middle cerebral artery ischemia/reperfusion model was prepared,and laser doppler flowmetry was used to detect the cerebral blood flow changes on the ischemic side.After 2 hours of ischemia,the fishing line was pulled out to restore reperfusion.After reperfusion for 7 days,immunohistochemistry was used to observe the expression of IL-17A and IL-10 protein in ischemic cortex and colon tissue,Western blotting was used to detect the expression of IL-17A and IL-10 protein in ischemic cortex and colon tissue and the expression of ROR?t and Foxp3 protein in the ischemic cortex,spleen and thymus tissue,and Elisa was used to determine the serum IL-17A,IL-23,IL-10,and TGF-?1 levels in the abdominal aorta of rats.Result:1.Immunohistochemistry:after reperfusion for 7 days,compared with the sham group,the mean optical density value of IL-17A and IL-10 protein in the ischemic cortex and colon tissue of rats in the model group significantly increased(P<0.05);compared with the model group,the mean optical density value of IL-17A protein in the ischemic cortex and colon tissue of rats in the electroacupuncture preconditioning group significantly decreased,the mean optical density value of IL-10 protein significantly increased(P<0.05).2.IL-17A and IL-10 protein expression:after reperfusion for 7 days,compared with the sham group,the IL-17A and IL-10 protein expression in the ischemic cortex and colon tissue of rats in the model group were significantly increased(P<0.05);compared with the model group,the expression of IL-17A protein in the ischemic cortex and colon tissue of rats in the electroacupuncture preconditioning group was significantly decreased,and the expression of IL-10 protein was significantly increased(P<0.05).3.The expression of ROR?t and Foxp3 protein:after reperfusion for 7 days,compared with the sham group,the ROR?t and Foxp3 protein expression in the ischemic cortex,spleen and thymus tissue of rats in the model group were significantly increased(P<0.05);compared with the model group,the expression of ROR?t protein in the ischemic cortex,spleen and thymus tissue of rats in the electroacupuncture preconditioning group was significantly decreased,and the expression of Foxp3 protein was significantly increased(P<0.05).4.Serum Elisa:after reperfusion for 7 days,compared with the sham group,the content of serum inflammatory factors IL-17A,IL-23,IL-10,and TGF-?1 in the abdominal aorta of rats in the model group were significantly increased(P<0.05);compared with the model group,the content of serum proinflammatory factors IL-17A and IL-23 in the abdominal aorta of rats in the electroacupuncture preconditioning group were significantly decreased,and the content of anti-inflammatory factors IL-10 and TGF-?1 were significantly increased(P<0.05).5.Correlation analysis of ROR?t and Foxp3 protein with mNSS score:after reperfusion for 7 days,ROR?t and Foxp3 protein expression of ischemic cortex was positively correlated with the mNSS score(P<0.05);under the intervention of electroacupuncture,the ROR?t protein expression of ischemic cortex was decreased was positively correlated with the mNSS score(P<0.05),and the expression of Foxp3 protein was negatively correlated with the mNSS score(P<0.05).Conclusion:1.Electroacupuncture preconditioning can down-regulate the expression of IL-17A,up-regulate the expression of IL-10 in ischemic cortex and colon tissue,reduce the content of peripheral IL-17A,IL-23,increase the content of IL-10,TGF-?1,and reduce intestinal-central inflammation damage after cerebral ischemia;2.Electroacupuncture preconditioning can down-regulate the expression of ROR?t,up-regulate the expression of Foxp3 in the ischemic cortex,spleen and thymus tissue,regulate Th17/Treg immune balance,and play brain-gut protection.Experiment 3 Research on the Immune Mechanism of Electroacupuncture Preconditioning Inducing Cerebral Ischemic Tolerance through Gut MicrobiotaObjective:Observe the effects of microbiota clearance and microbiota transplantation(microbiota of electroacupuncture preconditioning intervention for 2 weeks)on the neurological function and the expression of related inflammatory factors in rats with cerebral ischemia/reperfusion.Method:60 SPF male Sprague-Dawley rats were randomly divided into 5 groups:sham group,model group,ABX-Pre+model+FMT group,ABX-Pre+model+PBS group,ABX+model group,with 12 rats in each group.Throughout the experiment,both the sham group and the model group were given sterile drinking water.Before the operation,the ABX-Pre+model+FMT group and ABX-Pre+model+PBS group were given antibiotic drinking water for 2 weeks and were given sterile water after modeling.Before the operation,the ABX+model group was given antibiotic drinking water for 2 weeks and continued to be given antibiotic drinking water after modeling.A rat middle cerebral artery ischemia/reperfusion model was prepared,and laser doppler flowmetry was used to detect the cerebral blood flow changes on the ischemic side.After 2 hours of ischemia,the fishing line was pulled out to restore reperfusion.After reperfusion for 12 hours,1 day,3 days,7 days,the modified Neurological Severity Scores was used to evaluate the nerve function and the grasping strength of rat forelimbs was measured with the grip tester for rats and mice;after reperfusion for 7 d,HE staining was used to observe the pathological damage of the ischemic cortex,immunohistochemistry was used to observe the expression of IL-17A and IL-10 protein in ischemic cortex,and Elisa was used to determine the serum IL-17A,IL-23,IL-10,and TGF-?1 levels in the abdominal aorta of rats.Result:1.The modified Neurological Severity Scores:after reperfusion for 12 h,1 d,3 d,7 d,compared with the sham group,the modified Neurological Severity Scores of rats in the model group was increased significantly(P<0.05);compared with the model group,the modified Neurological Severity Scores of rats in the ABX-Pre+model+FMT group was significantly decreased(P<0.05),and the modified Neurological Severity Scores of rats in the ABX-Pre+model+PBS group was significantly increased(P<0.05);compared with the ABX-Pre+model+PBS group,the modified Neurological Severity Scores of rats in the ABX+model group increased significantly(P<0.05).2.Forelimb grasping strength test:after reperfusion for 12 h,1 d,3 d,7 d,compared with the sham group,the forelimb grasping strength of rats in the model group was significantly decreased(P<0.05);compared with the model group,the forelimb grasping strength of rats in the ABX-Pre+model+FMT group was significantly increased(P<0.05),and the forelimb grasping strength of rats in the ABX-Pre+model+PBS group was significantly decreased(P<0.05);compared with the ABX-Pre+model+PBS group,the forelimb grasping strength of rats in the ABX+model group was significantly decreased(P<0.05).3.HE staining:after reperfusion for 7 d,compared with the sham group,the pathological damage degree of ischemic cortex and colon tissue of rats in the model group was significantly worse;compared with the model group,the pathological damage degree of ischemic cortex and colon tissue of rats in the ABX-Pre+model+FMT group was significantly reduced,the pathological damage degree of ischemic cortex and colon tissue of rats in the ABX-Pre+model+PBS group was significantly worse;compared with the ABX-Pre+model+PBS group,the pathological damage degree of ischemic cortex and colon tissue of rats in the ABX+model group was significantly worse.4.Immunohistochemistry:after reperfusion for 7 days,compared with the sham group,the mean optical density value of IL-17A and IL-10 protein in the ischemic cortex of rats in the model group were significantly increased(P<0.05);compared with the model group,the mean optical density value of IL-17A protein in the ischemic cortex of rats in the ABX-Pre+model+FMT group was significantly decreased,the mean optical density value of IL-10 protein was significantly increased(P<0.05),the mean optical density value of IL-17A protein in the ischemic cortex of rats in the ABX-Pre+model+PBS group was significantly increased,the mean optical density value of IL-10 protein was significantly decreased(P<0.05);compared with the ABX-Pre+model+PBS group,the mean optical density value of IL-17A protein in the ischemic cortex of rats in the ABX+model group was significantly increased,the mean optical density value of IL-10 protein was significantly decreased(P<0.05).5.Serum Elisa:after reperfusion for 7 days,compared with the sham group,the content of serum inflammatory factors IL-17A,IL-23,IL-10,and TGF-?1 in the abdominal aorta of rats in the model group were significantly increased(P<0.05);compared with the model group,the content of serum proinflammatory factors IL-17A and IL-23 in the abdominal aorta of rats in the ABX-Pre+model+FMT group were significantly decreased,and the content of anti-inflammatory factors IL-10 and TGF-?1 were significantly increased(P<0.05),the content of serum proinflammatory factors IL-17A and IL-23 in the abdominal aorta of rats in the ABX-Pre+model+PBS group were significantly increased,and the content of anti-inflammatory factors IL-10 and TGF-?1 were significantly decreased(P<0.05);compared with the ABX-Pre+model+PBS group,the content of serum proinflammatory factors IL-17A and IL-23 in the abdominal aorta of rats in the ABX-Pre+model group were significantly increased,and the content of anti-inflammatory factors IL-10 and TGF-?1 were significantly decreased(P<0.05).Conclusion:Electroacupuncture preconditioning can reduce the pathological damage degree of the ischemic cortex and colon tissue and the intestinal-central inflammation injury after cerebral ischemia and improve neurobehavioral function through the gut microbiota. |
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