| Purpose:1.To retrospectively observe the clinical effect of Tongnaoyin(TNY)on patients with acute ischemic stroke(AIS)with syndrome of phlegm and blood stasis blocking collaterals.2,To evaluate the effect of TNY on angiogenesis in zebrafish vascular injury model.3.With the support of transcriptomics,the potential mechanism of TNY in promoting angiogenesis was explored by analyzing the differentially expressed genes sequenced from the Tongnaoyin intervened zebrafish vascular injury model.Method:Part I:Clinical retrospective study of TNY in treating AIS.Based on the stroke registration database of the inpatient electronic information system of the Affiliated Hospital of Nanjing University of Chinese Medicine,the patients who were discharged from hospital from January 1,2018 to December 31,2020,met the diagnostic criteria of modern medicine for AIS,TCM diagnostic criteria for stroke,and TCM syndrome differentiation belonged to phlegm and blood stasis blocking collaterals were included.The patients further screened according to the inclusion criteria and exclusion criteria.After screening,the patients who were only treated with modern medicine were set as the control group,while the patients treated with TNY combined with modern medicine were set as the treatment group.The national institute of health stroke scale(NIHSS),modified Rankin scale(mRS)and Barthel index score(BIS)were used to evaluate the efficacy.The proportion of patients with 90-day mRS score(1 was taken as the primary efficacy endpoint,and the secondary efficacy endpoints included 90-day BIS score,NIHSS score at discharge,and the proportion of patients with mRS score≤1 at discharge.The propensity score matching was applied to balance important patient characteristics between groups,and the primary and secondary effficacy endpoints were then analyzed.Part II:Based on transcriptomics,to explore the potential mechanism of TNY in treating zebrafish vascular injury model.Transgenic zebrafish expressing enhanced green fluorescent protein in endothelial cells were obtained from model animal Institute of Nanjing University,and zebrafish embryos were obtained by pairing.Twenty-three hour post-fertilization(hpf),zebrafish embryos were treated with VEGFR tyrosine kinase inhibitor ii(VRI)to establish a zebrafish vascular injury model.Rinse away VRI at 27 hpf,and use blank solvent or five different concentrations of TNY solution,including 1:10(1375 μg/ml),1:20(687.5 μg/ml),1:50(275μg/ml),1:100(137.5 μg/ml),1:200(68.75 μg/ml),continually to treat zebrafish embryos.Embryos without any treatment were used as blank control,and embryos pre-treated with VRI and then treated with blank solvent were used as negative control.At 48 hpf,the internodal blood vessels of embryos were observed,and the drug effect was evaluated by calculating the number of internodal blood vessels.Furthermore,the zebrafish embryos under the optimal angiogenesis promoting effect of TNY were collected for transcriptome analysis.RNA of zebrafish was extracted and a library was then constructed,which was sequenced by Illumina HiSeq2500 sequencer.The quality of RNA samples can be ensured by testing sample quality,controlling data quality,comparing sequences,and analyzing gene types,coverage,randomness,sequencing saturation and expression.Gene ontology(GO)and KEGG function enrichment analysis were carried out on the differential expressed genes between TNY group and blank solvent group after treatment for 21h,and the interaction network of differential expressed genes and proteins was constructed.In addition,the trend analysis was carried out on three samples with action time sequence treated by TNY for 0h,10h and 21h,and the expression patterns of the three groups of differential expressed genes were analyzed,and the function enrichment analysis was carried out on the gene set with increasing expression.Results:Part Ⅰ:649 patients were included in the treatment group and 153 patients in the control group.After propensity score matching,153 patients who received TNY combined with western medicine treatment were matched with 153 patients only treated with modern medicine.After matching,the baseline data of the two groups were comparable(P>0.05).The NIHSS score of the treatment group was significantly lower than that of the control group at discharge(P=0.031).At discharge,the proportion of patients with mRS score≤1 in the treatment group was higher than that in the control group,and there was no significant difference(P=0.248).The proportion of patients with mRS score≤in the 90-day treatment group was significantly higher than that in the control group(P=0.037).The proportion of patients with BIS scores ranging from 76 to 100 in the 90-day treatment group was significantly higher than that in the control group(P=0.012).Part Ⅱ:After incubation with VRI for 4 h,the development of blood vessels in the internode of zebrafish embryos was significantly damaged.After incubation for 21 hours,TNY reversed the vascular injury caused by VRI and promoted angiogenesis in a dose-dependent manner.TNY showed the most significant effect of promoting angiogenesis if the concentration was 1375μg/ml(1:10).The quality of mRNA extracted from zebrafish embryos met the requirements,and the quality of data obtained by sequencing was at a good level.After 21h treatment,there were 861 differential expressed genes obtained between TNY group and blank solvent group,of which 654 were up-regulated and 207 were down-regulated.The results of GO enrichment show that the mechanism of TNY was mainly related to regulating the activity of structural molecules,the binding of metal cations such as calcium ions,the activity of ion channels,energy metabolism,the activity of transcription factors,etc.It also regulates the cytoskeleton structure such as intermediate fibrils,troponin complex,contractile fibers,extracellular matrix and other structures,and participates in the development of various tissues and organs including sensory organs.The results of KEGG enrichment mainly involved regulation of steroid synthesis,pentose phosphate pathway,tyrosine metabolism,caffeine metabolism,interaction between extracellular matrix and receptor,glycolysis and gluconeogenesis,MAPK signaling pathway,cell adhesion molecule,purine metabolism,adhesion spot,tight junction and other metabolic pathways or signaling pathways.The importance of differentially expressed genes was comprehensively evaluated from three aspects:degree centrality,closeness centrality and betweenness centrality.A total of 5535 genes and 8 trends were obtained by trend analysis,among which the expression of 515 genes continued to rise.These genes were further analyzed by GO and KEGG enrichment and gene protein network interaction.The results were mainly related to signal transduction,cell structure,cell combination,biosynthesis,and metabolism of various substances.Conclusion:1.Compared with modern medicine treatment,TNY combined with modern medicine treatment can further improve the neurological deficit of patients with stroke with syndrome of the phlegm and blood stasis blocking collaterals when they are discharged from hospital,and increase their good prognosis in 90 days,and further improve their basic activities of daily life in 90 days.2.TNY reversed the vascular injury of zebrafish caused by VRI in a dose-dependent manner and significantly promoted the angiogenesis of zebrafish,with the best concentration of 1375μg/ml(1:10).3.TNY can promote angiogenesis by regulating the signal transduction between vascular endothelial extracellular matrix and its receptors,such as hyaluronic acid,collagen-integrin pathway,neuroprotein-VEGFA pathway;regulation of cytoskeleton structure,including microfilament and microtubule skeleton,and structures that related closely to cytoskeleton,such as focal adhesion,intercellular connection,cell adhesion,and molecular motor;regulating multiple metabolic pathways,including steroid synthesis,pentose phosphate pathway,tyrosine metabolism,caffeine metabolism,glycolysis/gluconeogenesis,purine metabolism,fructose and mannose metabolism,glyceride metabolism;regulating the signal transduction of MAPK signaling pathway,including Raf/MEK/ERK/c-fos pathway. |
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