To Investigate The Effect Of Acupuncture-rehabilitation Therapy On Rat Intestinal Flora And IL-23/IL-17 Pathway Related Protein After Cerebral Ischemia

Objective:To investigate the effect of Acupuncture-rehabilitation therapy on rat intestinal flora and IL-23/IL-17 pathway related protein after cerebral ischemia.Method:Methods:The permanent middle cerebral artery occlusion model was established.Rats were randomly divided into 5 groups,model group,sham group,rehabilitation group,acupuncture group and Acupuncture-rehabilitatio-n group.Model group and sham operation group without any treatment.Acupuncture group was treated by scalp acupuncture.Rehabilitation group received treadmill training,Acupuncture rehabilitation group was treatedby scalp acupuncture combined with rehabilitation.Neurological deficit score(m NSS)was used to observe the neurological deficit of rats in each group;the expression of IL-23,IL-17 protein in rat serum was detected by enzyme-linked immunosorbent assay(ELISA);the composition of intestinal flora was analyzed by high-throughput 16 S r DNA V4 sequencing;and the correlation between neurological deficit score and intestinal flora was analyzed by Spearman correlation analysis.Western Blot method was used to detect the expression levels of IL-23,IL-17,IL-6 and TNF-alpha protein in cerebral cortex of rats with ischemic penumbra,and enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of IL-23,IL-17,IL-6 and TNF-alpha protein in intestinal tissue of rats.Result:1.Neurologic impairment assessment:At 3d post operation,compared withmodel group,the neurologic impairment score in acupuncture group,rehabilitation group and acupuncture-rehabilitation group were reduced,there isno statistical significance(P>0.05).At 7d,14 d post operation,compared withmodel group,the neurologic impairment scoresin acupuncture group,rehabilitation group and acupuncture-rehabilitation group werereduced(P<0.05),the scores in Acupuncture-rehabilitation group is more obvious than acupuncture group and rehabilitation group(P<0.05).2.Changes of intestinal microbial community in rats:(1)Sequencing data results: 15 fecal samples of rats were collected for sequencing analysis.One of them failed to amplify by PCR.Finally,14 samples were sequenced according to the requirements.A total of 567,648high-quality sequences were obtained,of which 49,663 were the most,at least32,953 were the least.On average,40,546.28 sequences were obtained for each sample,of which 99.9% were valid.The length between 400bp-435 bp.(2)Alpha diversity:1)Rarefaction curve: The experimental results are sufficient to reflect the diversity of current samples,and can meet the requirements of subsequent bioinformatics analysis.2)Species accumulation curves: when the number of samples is small,the curve shows a rapid upward trend with the addition of new samples;when the number of samples is large,the curve begins to flatten.The results showed that the sample size is enough to reflect the richness.3)Rank abundance curve: the species richness of model group was lower;the species richness and evenness of other groups were better,and the abundance grade curve of acupuncture-rehabilitation group was closest to that of sham-operation group.4)Alpha diversity index: compared with sham-operated group,Alpha diversity index of other groups decreased significantly(P<0.05).Compared with the model group,the diversity index of Alpha in the rehabilitation group,the acupuncture group and the acupuncture-rehabilitation group increased significantly(P<0.05);compared with the rehabilitation group and the acupuncture-rehabilitation group,the diversity index of Alpha in the acupuncture-rehabilitation group increased significantly(P<0.05).(3)Taxonomic analysis of intestinal flora of rats in each group:1)Phylum level: There were structural differences at gate level between model group and sham-operated group.The levels of microflora in rat intestinal feces were Firmicutes(60.1% vs 56.9%),Bacteroidetes(21.3% vs37.2%),Proteobacteria(8.6% vs 1.3%),Spirochaetes(8.5% vs 0.1%).)The species and abundance of microorganisms at different levels in acupuncture-rehabilitation group,rehabilitation group and acupuncture group were as follows:(57.6% vs 61.0% vs 67.5%),Bacteroides:(32.8% vs 29.8%vs 23.0%),Proteus(4.8% vs 6.8% vs 5.8%)and Spirochaeta(3.5% vs 0.2% vs1.7%).2)Class level: Clostridia and Bacteroidia were relatively abundant in fecal samples of sham-operated group(43.5% vs 43.2%),Delta Proteobacteria was 11.3%,Gamma Proteobacteria and Fusobacteriia were less than 0.1%,while Clostridia and Bacteroidia were predominant in model group.(33.5% vs30.3%)followed by Fusobacterium,Bacillus and Bacilli(15.6% vs 13.7% vs0.3%).The acupuncture-rehabilitation group,rehabilitation group,acupuncture group in the Oomycetes composition of Clostridia levels(34.7%vs 39.2% vs 20.6%),Bacteroides classes(42.5% vs 35.3% vs 30.4%),?-Proteobacteria(0.1% vs 16.3% vs 18.8%),Fusobacterium(0.1% vs 4.5% vs4.0%).3.The correlation between neurological deficit score and intestinal flora in rats:The correlation analysis between the relative abundance of intestinal flora at the phylum level and the score of neurological deficit in rats showed that at the gate level,there was a positive correlation between the Proteobacteria and the score of neurological deficit in rats(P<0.01),while there was no significant correlation between the abundance of other intestinal flora and the score of neurological deficit in rats(P>0.05).At the class level,Fusobacteriia and Bacilli were positively correlated with the neurological deficit score of rats(P<0.05),Bacteroidia was negatively correlated with the neurological deficit score of rats(P<0.01),and other intestinal bacteria were not significantly correlated with the neurological deficit score of rats(P>0.05).4.Serum levels of IL-23 and IL-17:14th day after operation,the contents of IL-23 and IL-17 protein in the acupuncture group,rehabilitation group and acupuncture-rehabilitation group were significantly lower than those in the model group(P<0.05);compared with the acupuncture group and rehabilitation group,the contents of IL-23 and IL-17 protein in the acupuncture-rehabilitation group were significantly lower(P<0.05).5.Changes of IL-23,IL-17,IL-6 and TNF-alpha proteins in brain tissue of ischemic penumbra:14 days after operation,the expression of IL-23,IL-17,IL-6,TNF-alpha protein in acupuncture group,rehabilitation group and acupuncturerehabilitation group was significantly lower than that in model group(P<0.05);compared with acupuncture group and rehabilitation group,the expression of IL-23,IL-17,IL-6,TNF-alpha protein in acupuncture-rehabilitation group was significantly lower(P<0.05).6.Changes of IL-23,IL-17,IL-6 and TNF-alpha proteins in intestinal tissues of rats:14 days after operation,the expression of IL-23,IL-17,IL-6,TNF-alpha protein in acupuncture group,rehabilitation group and acupuncturerehabilitation group was significantly lower than that in model group(P<0.05);compared with acupuncture group and rehabilitation group,the expression of IL-23,IL-17,IL-6,TNF-alpha protein in acupuncture-rehabilitation group was significantly lower(P<0.05).Conclusion:1.Acupuncture-rehabilitation therapy can improve the degree of neurological deficit in rats with cerebral ischemia.2.Acupuncture-rehabilitation therapy can play a therapeutic role by improve-ng the quantity and composition of intestinal flora after cerebral ischemia,reducing opportunistic pathogenic bacteria,increasing resident bacteria or beneficial bacteria,and restoring the diversity of intestinal flora.3.Acupuncture-rehabilitation therapy can antagonize inflammatory response after cerebral ischemia by inhibiting the expression of IL-23,IL-17,IL-6 and TNF-alpha proteins in brain and intestinal tissues of ischemic penumbra.