Study On The Relationship Between The Clinical Effect Of Tongdu Tiaoshen Acupuncture On Ischemic Stroke And The Regulation Of MicroRNA

1 Objectives1.1 MiRNA microarray was used to screen and analyze the differentially expressed miRNAs between patients with ischemic stroke and healthy people,and RT q PCR was used to verify and analyze the differentially expressed miRNAs,so as to explore the miRNAs that may be related to ischemic stroke.1.2 Through clinical research,observe and compare the difference of clinical efficacy and circulating miRNA of ischemic stroke patients before and after Tongdu Tiaoshen acupuncture,and explore the correlation between the clinical efficacy of Tongdu Tiaoshen acupuncture and the regulation of miRNA expression.2 Methods2.1 MiRNA microarray was used to detect circulating miRNA in serum of 3patients with ischemic stroke and 3 healthy subjects respectively,and the differentially expressed miRNA was screened,and the results were verified by RT q PCR;go significant enrichment analysis was performed based on gene ontology database,and the related target gene functional class was preliminarily identified;KEGG pathway was used as the unit for path analysis The biological pathway of related target genes was preliminarily identified by significance enrichment analysis.2.2 60 patients with ischemic stroke were randomly divided into Tongdu Tiaoshen group(30 cases)and non meridian non acupoint group(30 cases).On the basis of routine rehabilitation treatment,Tongdu Tiaoshen group was treated with acupuncture at Baihui,Jingjiaji,Fengfu,yamen,Dazhui,Shenzhu,Shendao,Zhiyang,Mingmen and Yaoyangguan.In the non meridian non acupoint group,the acupoints were selected by opening 1 inch beside the acupoints in the Tongdu Tiaoshen group.If the opening 1inch beside the acupoints was still the meridian acupoints,the opening 1 inch beside the acupoints was selected.If the opening 1 inch was less than 1 inch,0.5 inch was selected.The patients in the two groups received acupuncture 6 times a week,one day off,and continued acupuncture for 2 weeks / course of treatment.A total of 2 courses of treatment were observed.The scores of NIHSS and ADL before treatment(0 week)and after treatment(4 week)in the two groups were observed and recorded;the expression of miRNA in three serum circulation of the two groups before and after treatment was detected by RT-q PCR,and the correlation between the clinical efficacy of Tongdu Tiaoshen acupuncture and the regulation of miRNA expression was discussed by SPSS correlation analysis.3 Results3.1 Differential expression of circulating miRNAs in serum of patients with ischemic stroke and healthy subjects results: compared with healthy subjects,there were 55 miRNAs(fold Among them,there were 25 up-regulated miRNAs and 30 down regulated miRNAs;the target genes of 25 up-regulated miRNAs were mainly enriched in positive regulation of cell process,positive regulation of cell metabolism,morphogenesis of anatomical structure,synapses,cell junctions,cytoplasm and cells The 30 down regulated miRNA target genes are mainly enriched in the regulation of transcription by RNA polymerase II,the positive regulation of macromolecular metabolism and the regulation of RNA metabolism in biological processes,and the intracellular,cellular junction and nuclear chromatin in cell components The 25up-regulated miRNA target genes were significantly enriched in cell senescence,PI3K-Akt signaling pathway,Notch signaling pathway,TGF-β signaling pathway,MAPK signaling pathway,and the other 30 up-regulated miRNA target genes were significantly enriched in cell senescence,PI3K-Akt signaling pathway,Notch signaling pathway,TGF-β signaling pathway,MAPK signaling pathway,etc The down regulated miRNA target genes were mainly enriched in FOXO signaling pathway,lifespan regulation pathway,cell senescence,PI3 K Akt signaling pathway,aldosterone synthesis and secretion signaling pathway(P≤0.05).The 4 miRNAs with the most significant difference and stable expression were selected: hsa-miRNA-520c-3p,hsa-miRNA-6827-5p,hsa-miRNA-4772-5p andhsa-miRNA-552-3p for RT-q PCR verification,RT-q PCR results showed that the expression of hsa-mir-520c-3p in is patients was significantly increased(P<0.05),and the expression of hsa-mir-4772-5p and hsa-mir-552-3p were significantly decreased(P<0.05).The expression trend was consistent with the results of gene chip.3.2 Observation on clinical efficacy of acupuncture intervention in patients with ischemic stroke: there was no significant difference in demographic data(gender,age,education level),course of disease,stroke location and past history(hypertension,diabetes,hyperlipidemia)between the two groups(P>0.05).Before treatment,there was no statistically significant difference between the two groups(P=0.198037(29)0.05),and there was comparability between the two groups;before and after treatment,there was significant difference between the two groups(P(27)0.05),suggesting that both treatment methods can reduce NIHSS score;after treatment,the NIHSS score difference between the two groups was compared,and compared with non meridian and non acupoint group,the NIHSS score difference of Tongdu Tiaoshen group was higher(P=0.000113(27)0.05),with significant difference,suggesting that the NIHSS score of Tongdu Tiaoshen group decreased more significantly.There was no significant difference between the two groups before treatment(P=0.524337(29)0.05),and there was comparability between the two groups;there was significant difference between the two groups before and after treatment(P(27)0.01),which indicated that the two treatment methods could increase ADL score;the difference of ADL score between the two groups before and after treatment was compared between the two groups,and the difference of ADL score between the Tongdu Tiaoshen group before and after treatment was higher(P=0.001149(27)05),with significant difference,suggesting that the ADL score of Tongdu Tiaoshen group increased more significantly.3.3 Regulation of acupuncture on circulating miRNA level in serum of patients with ischemic stroke: before treatment,there was no significant difference in miRNA expression between Tongdu Tiaoshen group and non meridian non acupoint group,which was comparable;after treatment,compared with before treatment,the expression of hsa-mir-520c-3p and hsa-mir-6827-5p in Tongdu Tiaoshen group and non meridian non acupoint group decreased,with significant difference;hsa-mir-4772-5p The expression of hsa-mir-552-3p and hsa-mir-552-3p increased significantly.After treatment,the expressions of hsa-mir-520c-3p and hsa-mir-6827-5p in Tongdu Tiaoshen group were significantly lower than those in non meridian and non acupoint group,while the expressions of hsa-mir-4772-5p and hsa-mir-552-3p in Tongdu Tiaoshen group were significantly higher than those in non meridian and non acupoint group.3.4 Correlation analysis between the clinical efficacy of acupuncture intervention on patients with ischemic stroke and the level of circulating miRNA: after treatment,the correlation analysis between the clinical efficacy indexes and miRNA expression in Tongdu Tiaoshen group was conducted one by one.The results showed that hsa-mir-520c-3p was positively correlated with NIHSS(P(27)0.05),while hsa-mir-520c-3p was negatively correlated with ADL(P(27)0.05)Hsa-mir-552-3p was negatively correlated with NIHSS(P(27)0.05);hsa-mir-552-3p was positively correlated with ADL(P(27)0.05);after treatment,there was no significant difference between hsa-mir-520c-3p,hsa-mir-4772-5p and hsa-mir-552-3p in clinical effect indexes and miRNA expression(P(29)0.05).4 Conclusions4.1 There are abnormally expressed miRNAs in the peripheral blood of patients with ischemic stroke.These abnormally expressed miRNAs may be related to the PI3 K Akt signaling pathway,Notch signaling pathway and TGF-β signaling pathway related to the regulation of cell aging,apoptosis,autophagy and injury,which are accompanied by the occurrence,development and rehabilitation of ischemic stroke.4.2 Tongdu Tiaoshen acupuncture can significantly improve the NIHSS and ADL scores of is patients,alleviate the neurological deficit of is patients,and improve the quality of life of is patients,which may be related to down regulating the expression of hsa-mir-520c-3p and up regulating the expression of hsa-mir-552-3p.