The Effect And Mechanism Of Intervention PI3k/Akt/mTOR Pathway Regulating Tregs/Th17 Imbalance From Immunologic Homeostasis On Ventricular Remodeling In Heart Failure

Objective:Heart failure is a complex clinical syndrome of insufficient cardiac output due to abnormal cardiac structure or function,which impairs the filling or ejection function of the heart.Clinically,the existing therapeutic drugs mainly work by inhibiting the activation of the neuroendocrine system(renin-angiotensin-aldosterone receptor antagonists)and improving metabolism(sodium-glucose symporter antagonists).Nevertheless,there are still some patients with heart failure whose prognosis cannot be improved.Therefore,finding new treatment methods is an urgent clinical problem to be solved.In recent years,studies have found that Tregs/Th17 balance disorder plays an important role in the formation and progression of heart failure.Maintaining the balance of Tregs/Th17 is beneficial for the body to maintain immune homeostasis,thereby improving myocardial remodeling and cardiac function.Therefore,regulating the balance of Tregs/Th17 may be an important direction to improve the prognosis of patients with heart failure.m TOR is an important link between cellular metabolism and immune function,and by increasing m TOR activation in the phosphatidylinositol 3-kinase-sirolimus target protein(PI3k/Akt/m TOR)pathway,it can increase Immune cell generation,neutralization of Tregs differentiation.Some studies speculate that the activation and inhibition of the PI3k/Akt/m TOR signaling pathway both affect the expression of Foxp3~+,thereby interfering with the activation of Tregs,thereby affecting the changes in the immune homeostasis of Tregs/Th17.However,until now,the reasons for the disturbance of Tregs/Th17 balance in chronic heart failure and the molecular mechanisms regulating Tregs/Th17 are still poorly understood.Therefore,this research will focus on three aspects:(1)To investigate whether Tregs/Th17 imbalance in peripheral blood of patients with chronic ejection fraction reduced heart failure.(2)To explore the potential molecular mechanisms or targets related to Tregs/Th17 in the process of chronic heart failure by constructing a heart failure dog model and performing full-length transcriptome sequencing.(3)Transcriptional analysis of post-MI heart failure rats based on public datasets to explore immune-related mechanisms in post-MI heart failure.(4)To explore the effect and mechanism of regulating PI3k/Akt/m TOR on myocardial Tregs/Th17 imbalance and ventricular remodeling,and provide new ideas and therapeutic targets for the treatment of heart failure.Methods:Part 1:Collect 80 patients with chronic ejection fraction reduced heart failure who were hospitalized in the Department of Pacing Electrophysiology in our hospital from December 2019 to December 2021,and 60 patients in the control group.Peripheral blood was detected by flow cytometry.Tregs cells and Th17 cells were extracted,and the data of cardiac ultrasonography and B-type natriuretic peptide at the time of enrollment were extracted.Part 2:control group(n=3)and heart failure group(HF,n=3)were used to full-length transcriptome sequencing.The heart failure model was constructed by rapid right ventricular pacing.The left ventricular ejection fraction(EF)and BNP were calculated by echocardiography in the postoperative week to determine the success of the modeling.After determining the indication of heart failure,the right ventricular myocardium group was subjected to full-length transcriptome sequencing(based on the nanopore platform),and the differentially expressed transcripts and differentially expressed genes were subjected to Gene Ontology(GO)and(KEGG)analysis.Alternative splicing analysis was performed using the AStalavista tool,the Animal TFDB 3.0 software was used to predict transcription factors,and the CNCI,CPC,CPAT,and Pfam databases were used to identify lnc RNAs.Finally,KEGG pathway analysis of alternative splicing,transcription factors and lnc RNAs was performed using Enrichr4 software.In addition,we also assessed candidate gene expression related to Th1,Th2,Th17 by quantitative real-time PCR(q RT-PCR).Part 3:In this study,the gene expression data of the left ventricle of GSE47495(Affymetrix Rat Gene 1.0 ST Array)rats were retrieved from GEO,a comprehensive database of gene expression,including 23 left ventricular samples and 5sham-operated samples.and 18 heart failure models.Differentially expressed genes(DEGs)between HF and sham-operated rats were identified using algorithms from the R language limma package linear fitting method,Bayesian analysis and t-test.Genes with P<0.05 were defined as differentially expressed genes.The differentially expressed genes were analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database.We performed ss GSEA analysis through R packages(GSVA,GSEABase and limma),Correlation of immunological characteristics and immune function with clinical symptoms for each sample in the study.And the WGCNA gene was screened and the weighted gene co-expression network was constructed.Select modules significantly related to Tregs and Th1,Th2,Th17 for functional enrichment analysis(GO/KEGG),and finally perform gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA)and pathway core.Correlation analysis between genes and immune cell scores.Part 4:24 healthy male SD rats were randomly divided into 6 control groups(n=6).The heart failure group(n=6),the rapamycin group(rapamycin+heart failure group,n=6),the methotrexate group(the methotrexate+heart failure group,n=6),The heart failure model after myocardial infarction was established by the method of ligation of the left anterior descending coronary artery.After 4 weeks of heart failure,the methotrexate group and the rapamycin group were given methotrexate and rapamycin for continuous administration,respectively.medicine for 4 weeks.After 4 weeks of treatment,echocardiography was used to evaluate the cardiac function of the rats;ELISA method was used to detect BNP,IL-1β,IL-6,TNF-αin serum;Masson staining was used to observe the pathological changes of rat myocardial tissue,and TUNEL method was used to detect Apoptosis of rat cardiomyocytes,flow cytometry was used to detect the number of Th17 and Tregs cells,and PCR was used to detect the expression of PI3k-Akt-m TOR signaling pathway in rat myocardial tissue.Results:Part 1:Compared with the normal control group,the proportion of Tregs in chronic heart failure patients with reduced ejection fraction was significantly decreased,Th17 cells were significantly increased,Th1 was significantly increased,and Th2 was significantly decreased.This change is important for immune activation in chronic heart failure.Part 2:Four weeks after the establishment of the rapid pacing heart failure model,the peripheral blood serum BNP and IL-17 of the dogs in the heart failure group were significantly decreased,and the ejection fraction was significantly lower than that in the control group.A total of 67,458 transcripts were identified by full-length transcriptome sequencing.785differential transcripts(DETs)were obtained from the HF group and the control group.Among them,DETs is enriched in immune response,especially in the differentiation of Th1,Th2 and Th17,and enriched 8 genes involved in the differentiation of Th1,Th2 and Th17 cells,among which,DLA-DMA,DLA-DQB1,DLA-DRA And HLA-DRB1 gene expression was up-regulated,FOS,JAG2 and JUN gene expression was down-regulated;From the analysis of differentially expressed transcripts and differentially expressed genes,the enrichment of many signaling pathways was found,such as TGF-βsignaling pathway,MAPK,Ras,PI3k/Akt and other signaling pathways.Alternative splicing analysis of sarcomere genes associated with the heart failure group revealed that five genes(TTN,TNNI2,TNNI3,MYBPC3,and FLNC)had alternative splicing events.KEGG enrichment analysis revealed that differentially spliced genes are enriched in aldosterone synthesis and secretion,mitophagy,signaling in adrenergic cardiomyocytes,hypertrophic cardiomyopathy,and dilated cardiomyopathy.And found 4892 transcription factors and406 lnc RNAs.Transcription factors with alternative splicing events are involved in myocardial fibrosis,hypertrophy,and Tregs stability.Part 3:Transcriptome analysis of post-MI rat heart failure identified a total of 1498 differential genes,of which 665 were up-regulated and 883 were down-regulated.GO enrichment analysis Differential genes were enriched in mitochondrial gene expression,energy metabolism,ribonucleotide metabolism process,etc.The differential gene KEGG was enriched in carbon metabolism,autophagy,MAPK signaling pathway,PI3k/Akt signaling pathway,m TOR signaling pathway,Th17 cell differentiation,Th1 and Th2 cell differentiation.Immune infiltration analysis Th1,Th17 increased significantly,while Th2,Tregs decreased significantly.The functional enrichment analysis of important modules,gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA)were both found to be related to immunity,TGF-βsignaling pathway,and PI3k/Akt/m TOR signaling pathway.The correlation analysis of genes differentially expressed and regulating Th17,Th1 and Th2 cell differentiation and ss GSEA immune score showed that RUNX1,HLA-DMA,CD4 had a high positive correlation with Tregs,Th1,and Th2,while RORC had a high positive correlation with Tregs,Th1,Th2 has a high negative correlation.Secondly,RUNX1,HLA-DMA,CD4have a high negative correlation with Th17,RORC,PPP3CA,JAG2 have a high positive correlation with Th17.Part 4:Compared with the control group,the expression of m TOR in the heart failure rats after myocardial infarction was significantly increased,Tregs were significantly decreased,and Th17 was significantly increased.Compared with the heart failure group,rapamycin and methotrexate inhibited m TOR m RNA expression,and compared with the heart failure group,rapamycin significantly reduced myocardial cell apoptosis,significantly improved cardiac function,significantly increased Tregs,Th17significantly reduced.Compared with the heart failure group,methotrexate significantly reduced myocardial apoptosis,significantly increased Tregs and significantly decreased Th17.Conclusion:1.The proportion of Th17 cells increased and the proportion of Tregs decreased in patients with heart failure with reduced ejection fraction.2.Full-length transcriptome sequencing enriched the immune-related processes involved in Th1,Th2 and Th17 cell differentiation.And the PI3k/Akt/m TOR,MAPK,Ras signaling pathways were significantly enriched.3.Transcriptome analysis of post-MI rats with heart failure based on public datasets Differential genes are enriched in Th1,Th2,Th17 differentiation,PI3k/Akt/m TOR signaling pathway.4.The m TOR signaling pathway is activated in heart failure rats after myocardial infarction,and rapamycin inhibits the m TOR signaling pathway,inhibits myocardial apoptosis,myocardial inflammation,up-regulates Tregs,and down-regulates Th17 cells,thereby reversing cardiac remodeling and delaying the progression of heart failure.Methotrexate up-regulates Tregs and down-regulates Th17cells by inhibiting m TOR signaling pathway.