| ObjectiveCerebral ischemia could not restore brain function after blood flow reperfusion for a certain period of time,but aggravate brain function injury,a process called Cerebral ischemia reperfusion injury(CIRI).There was a strong inflammatory response after cerebral ischemia.Intestinal symbiotic microorganisms could regulate ischemic brain injury.There was two-way communication between the brain and gut,known as the Microbiome-Gut-Brain Axis(MGBA).The Th17,γδT cells and Treg played an important role in the regulation of intestinal microbiota.Th17 and γδT cells released the pro-inflammatory factor IL-17,which aggravated the inflammatory reaction and pathological injury of ischemia reperfusion tissue.Treg cells release the anti-inflammatory factor IL-10,which was relatively increased in expression after cerebral ischemia and inhibits inflammatory response.Acupuncture was an important means of functional rehabilitation after cerebral infarction.Exosomes was an effective treatment method to study stroke.Based on brain-gut axis theory,this paper focuseed on the mechanism of electroacupuncture Baihui(GV20)and Zusanli(ST36)combined with iPSC-EVs in the treatment of cerebral ischemia reperfusion injury.MethodsHealthy male mice(C57BL/6,weight 21-23g,6-8 weeks of age)were randomly divided into 5 groups by random number table method:Sham operation(Sham)group,model(MCAO)group,electroacupuncture GV20 and ST36(EA)group,iPSC-EVs(EV)group and electroacupuncture GV20 and ST36 combined iPSC-EVs(EA+EV)group,with 10 mice per group,A middle cerebral artery occlusion(MCAO)model was established.In Sham group,only the right common carotid artery,internal carotid artery and external carotid artery were separated,and no thread plug was inserted.In the MCAO group,the right middle cerebral artery was blocked for 1h by thread embolism and reperfusion for 72h.In the EA group,acupuncture GV20 and the left ST36 after successful modeling and connected with pulse therapy instrument,continuous wave,frequency 2Hz,intensity 1mA,30min each time,once a day.In the EV group were injected with iPSC-EVs 20μg/mice once a day in the tail vein immediately after modeling.In the EA+EV group received electroacupuncture intervention and caudal intravenous injection of iPSC-EVs once a day after successful modeling.EA and iPSC-EVs treatments were performed four times at 0 h,24 h,48 h,and 70 h after reperfusion.Each group of mice underwent Clark score,open field test to evaluate nerve function injury.The infarct volume and apoptosis were evaluated by TTC(2,3,5-Triphenyltetrazolium chloride)and Nissl staining.HE staining showed pathological changes of intestinal tissue.The expressions of IL-10 and IL-17 in brain and intestinal tissues were detected by Western Blot.Intestinal contents were collected and 16S rRNA gene sequence were analyzed to reveal changes in intestinal flora.To verify the scientific hypothesis that electroacupuncture GV20 and ST36 combined with iPSC-EVs could regulate intestinal microorganisms to inhibit inflammation by interfering with the gut brain axis,thus playing a brain protective role,providing new research and experimental basis for the treatment of cerebral ischemia reperfusion injury.Results1.Effects of electroacupuncture with GV20 and ST36 combined with iPSC-EVs on the behavior of CIRI miceThe results of Clark neurological scoreshowed that the score of MCAO group was significantly increased(P<0.001).Compared with the MCAO group,the Clark score in the EA,EV,and EA+EV groups significantly decreased(P<0.001).However,there was no statistically significant difference between three treatment groups(P>0.05).The results of open field test showed that the movement distance and speed of mice in MCAO group were significantly lower than those in Sham group(P<0.001).Compared with MCAO group,the movement distance and speed of EA,EV and EA+EV groups were significantly increased(P<0.001),and there was difference between EA and EV groups compared with EA+EV group(P<0.05,P<0.01),EA+EV group was better than EA and EV groups.2.Effects of electroacupuncture with GV20 and ST36 combined with iPSC-EVs on cerebral infarction volume,neuronal injury and intestinal tissue in CIRI miceTTC staining results showed that the brain tissue of Sham group mice was normal without infarction,while MCAO group mice showed significant white infarction.Compared with the MCAO group,the cerebral infarction volume in the EA,EV,EA+EV groups was significantly reduced(P<0.01,P<0.01,P<0.001),there was no significant difference between the EA,EV,and EA+EV groups(P>0.05).Nissl staining showed that the structure of the cerebral cortex in Sham group mice was intacted,and the number of Nissl bodies was not significantly reduced or damaged,with regular arrangement.The cortical structure on the injured side of MCAO mice was incomplete,the number of Nissl bodies decreased or destroyed,and the arrangement was irregular(P<0.05).Compared with the MCAO group,EA,EV,and EA+EV groups significantly reduced neuronal damage(P<0.05).There was statistically significant difference between three treatment groups(P<0.05),the number of neuron damage in EA+EV group was significantly lower than that in EA and EV groups.HE staining showed that the colon tissue structure of the Sham group mice was intacted,and no obvious pathological changes such as without intestinal mucosal edema,villus injury,rupture and dissection,the decrease in the number of goblet cells.According to Chiu’s score,there were significant pathological changes(P<0.01)in the colon tissue in the MCAO group,including intestinal mucosal edema,integrity destruction,villus injury,rupture and dissection,and a decrease in the number of goblet cells.Compared with the MCAO group,the pathological changes of intestinal mucosal edema,intestinal villus injury,and colon tissue goblet cell failure in the EA,EV,and EA+EV treatment groups were significantly reduced(P<0.05,P<0.05,P<0.01).There was no difference between the three treatment groups(P>0.05).3.Effects of electroacupuncture with GV20 and ST36 combined with iPSC-EVs on inflammatory factors in brain and intestinal tissue of CIRI miceWestern blot results showed that,compared with Sham group,the expression of IL-10 in MCAO group was increased(P<0.05);compared with MCAO group,it was significantly higher in EA,EV and EA+EV groups(P<0.05,P<0.05,P<0.01),there was no difference between three treatment groups(P>0.05).Compared with Sham group,the expression of IL-17 in MCAO group was significantly increased(P<0.01),compared with MCAO group,EA,EV,and EA+EV groups were significantly decreased(P<0.05).There was no difference between three treatment groups(P>0.05).In colon tissue,compared with Sham group,the level of IL-10 in MCAO group increased(P<0.05);compared with MCAO group,the expression of IL-10 in EA,EV,and EA+EV groups were significantly increased(P<0.05).There was no difference between three treatment groups(P>0.05).Compared with Sham group,the expression of IL-17 in MCAO group was significantly increased(P<0.05);Compared with the MCAO group,the expression of IL-17 in EA,EV,and EA+EV groups were significantly decreased(P<0.01,P<0.05,P<0.01).There was no difference between the EA and EA+EV groups(P>0.05).Compared with the EA+EV group,the EA+EV group was superior to the EV group(P<0.05).4.Effect of electroacupuncture combined with GV20 and ST36 combined with iPSC-EVs on intestinal microorganisms of CIRI miceThe 16S test results showed that the number of taxonomic notes in the Sham group was the highest,that in the MCAO group was the lowest,and in the treatment groups that EV group>EA+EV group>EA group.ASV/OTU showed that the number of Sham group was 7403,MCAO group 972,EA group 1912,EV group 2729 and EA+EV group 2449.The number of species in Sham,MCAO,EA,EV and EA+EV groups was 393.The number of taxonomic units showed that the number of taxonomic units in MCAO group was the lowest,and the number of taxonomic units in Sham group was the highest.The EA group treatment significantly increased the number of taxonomic units in intestinal flora(P<0.05),mainly up-regulated the number of genera and species,which was more significant than EV group and EA+EV group(P<0.01).At phylum level,the proportion of Proteobacteria abundance,MCAO group>EV group>EA+EV group>EA group>Sham group and EA group was better than EV group and EA+EV group after treatment.Abundance ratio of Bacteroides EA+EV group>Sham group>MCAO group>EV group>EA group,EA+EV were superior to EA group and EV group.Firmicutes abundance ratio EA group>Sham group>EV group>EA+EV group>MCAO group,EA group was better than EV group and EA+EV group after treatment.Verrucomicrobacteria abundance ratio EA group>Sham group>MCAO group>EV group>EA+EV group,EA group was superior to EV group and EA+EV group after treatment.At species level,the proportion of Akkermansia abundance EA group>Sham group>MCAO group>EV group>EA+EV group,EA group was superior to EV group and EA+EV group(P<0.01,P<0.001).Alpha diversity results showed that,at the Rarefaction Curve,in EA group Faith’s PD value was most significant;in EV group,Chao1 and Observed Species were most significant;in EA+EV group Shannon,Simpson,and Pielou’s evenness values were most significant;there was almost no difference in Good’s coverage.There was a significant difference between the MCAO group and the Sham group in terms of Chaol,Observed Species,Shannon,Simpson,Faith’s PD,Pielou’s Evenness,and Good’s coverage(P<0.05),there was no significant difference between the MCAO group and the EA,EV,EA+EV groups.Beta diversity results showed that,in the Sham,MCAO,EA,EV and EA+EV groups,the weighted and unweighted unifrac(Stress<0.2,P<0.05).In the Sham and MCAO groups,the weighted unifrac and unweighted unifrac(Stress<0.2,P<0.05).In the MCAO,EA,EV,EA+EV groups,the weighted unifrac and unweighted unifrac(Stress<0.2),unweighted unifrac(P<0.05).In the MCAO and EA groups,the weighted unifrac(Stress<0.2),unweighted unifrac(P<0.05).In the MCAO and EV groups,the weighted and unweighted unifrac(Stress<0.2,P>0.05).In the MCAO and EA+EV groups,the weighted and unweighted unifrac(Stress<0.2,P>0.05).In the EA and EA+EV groups,the weighted unifrac and unweighted unifrac(Stress<0.2,P<0.05).In the EV and EA+EV groups,the weighted unifrac and unweighted unifrac(Stress<0.2,P>0.05).LEfSe results showed that the number of different species between Sham group and MCAO group that in Sham group(87)and in MCAO group(8).The number of different species among MCAO,EA,EV and EA+EV groups MCAO(5),EA(33),EV(1)and EA+EV(5);MCAO group compared with the treatment group in pairs,MCAO group(6)and EA group(57);MCAO group(9),EV group(11);MCAO group(19),EA+EV group(13).EA intervention was superior to EV group and EA+EV group,and EA+EV group was superior to EV group.The comparison of EA group,EV group and EA+EV group showed that EA group could improve the imbalance of intestinal microflora by regulating actinomyces,cyanobacteria,verrucobacteria,Firmicutes,and parasicutes,while EA+EV group could regulate intestinal microflora by Bacteroidetes.Abundance of six metabolic pathways pathway/group>1000 and the number of their secondary pathways:Biosynthesis(1),Degradation/Utilization/Assimilation(11),Generation of Precursor Metabolite and Energy(4),Glycan Pathways(2),Macromolecule Modification 1),Metabolic Clusters(2).Conclusion:1.The treatment of electroacupuncture GV20 and ST36,iPSC-EVs and the combination of both treatments could improve neurological function scores,alleviate neurological damage,reduce cerebral infarction volume,improve neuronal apoptosis,and intestinal tissue damage in CIRI mice.The protective effects of electroacupuncture GV20 and ST36 and iPSC-EVs on CIRI mice were equivalent,and the combined intervention was superior to the single treatmen in the distance,speed and number of neuron damage.2.The treatment of electroacupuncture GV20 and ST36,iPSC-EVs,and the combination of both treatments could reduce the protein expression level of IL-17 in brain and colon tissues of CIRI mice,increase the protein expression level of IL-10,inhibit inflammatory reactions,and had a protective effect on brain and colon tissues.Moreover,the electroacupuncture group alone and the iPSC-EVs group alone have similar protective effects on the brain and intestinal tissues of CIRI mice,and the expression of IL-17 in colon tissue the electroacupuncture combined with iPSC-EVs group was superior to single iPSC-EVs treatment in intestinal tissues.3.The treatment of electroacupuncture GV20 and ST36,iPSC-EVs,and the combination of both treatments could regulate the composition,diversity,and differential distribution of gut microbiota in CIRI mice,improve the gut environment,and inhibit inflammatory reactions.Electroacupuncture,iPSC-EVs and combined intervention increase the overall abundance of intestinal flora species and regulate microbial diversity more significantly.Electroregulation of beneficial flora was superior to exosome and combined intervention.Innovation:1.Innovation of intervention means:Used electroacupuncture GV20,ST36 and caudal vein injection of iPSC-EVs as new intervention means,compared the effect difference of electroacupuncture GV20,ST36 and iPSC-EVs and combined intervention.It provided a new experimental basis for the possibility of combined application of acupuncture and iPSC-EVs in ischemic stroke.2.Theoretical innovation:Based on the theory of brain-gut axis,this paper focused on the regulatory effects of electroacupuncture of GV20,ST36 and iPSC-EVs and combined intervention on the expression of IL-10 and IL-17 protein in the brain and intestinal tissues of CIRI mice,as well as the effect mechanisms of the composition and diversity of intestinal microflora and the different distribution of species in the intestine.It provided a new insight into the regulation of intestinal microbiota by acupuncture combined with iPSC-EVs in ischemic stroke.The literature search did not find the same report as this study. |
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