| Chronic Heart Failure(CHF)is a chronic inflammatory state characterized by elevated levels of circulating and myocardial pro-inflammatory cytokines promoting pathological left ventricular remodeling.Although preclinical studies have shown that cytokine antagonists have therapeutic effects in CHF,they have not shown positive efficacy in large-scale randomized controlled clinical trials.The failure of immunomodulation test shows that we don’t know enough about the inflammatory nature of heart failure.Specifically,because plasma/serum cytokine levels are largely determined by the combined effects of innate and adaptive immune system activation,they may not be specific indicators of the potential pathogenesis of CHF.Activated CD4+T cells have been shown to infiltrate the heart extensively and play a key role in promoting cardiac fibrosis,hypertrophy and remodeling in non-ischemic and pressure-overloaded CHF mouse models.However,whether and how CD4+T cells affect the pathophysiology of Ischemic Heart Failure(IHF)remains unclear.Traditional Chinese medicine(TCM)believes that qi deficiency is one of the pathological bases of IHF.Throughout the occurrence and development of the disease,the deficiency of heart qi leads to the stagnation of excess evil,and the accumulation of tangible evil in the body in turn affects the function of heart qi,so that the deficiency is more deficiency and the excess is more solid,forming a vicious circle,which is highly consistent with the process of immune activation.In the treatment of IHF,many doctors start from warming yang and promoting diuresis,promoting blood circulation and removing blood stasis,or nourishing heart yin,all of which are based on benefiting qi.Shenqi Compound Recipe is developed by the research group according to clinical experience,which has proved to have good efficacy at animal level.Through proteomics,it is preliminarily found that Shenqi Compound Recipe may play a protective role in heart by regulating the expression of immune-related proteins,but the specific mechanism is not clear.This study intends to further verify the curative effect of Shenqi Compound Recipe on IHF and the mechanism of regulating CD4+T lymphocyte subsets from multiple dimensions through clinical and animal levels,combined with network pharmacology technology.This study is divided into the following parts:Study 1 Clinical study on the treatment of Ischemic Heart Failure with Shenqi Compound Recipe and its effect on CD4+T lymphocyte subsetsObjective:To explore the therapeutic effect of Shenqi Compound Recipe on IHF patients and the regulation of CD4+T lymphocyte subsets based on clinical research,so as to provide scientific basis for Shenqi Compound Recipe in treating IHF.Methods:A case-control and self-control study was conducted,including IHF patients who met the standard of nano-excretion and comparable healthy subjects.Patients with IHF were treated with Shenqi Compound Recipe for 8 weeks on the basis of routine treatment,and the differences of clinical curative effect indexes were compared before and after treatment for 8 weeks.The percentage of Thl/Th2/Th17/Treg cell subsets in peripheral blood mononuclear cells and the contents of cytokines IL-2,IL-4,IL-6,IL-10,IL-17,TNF-α and IFN-y in serum were compared by flow cytometry before and after treatment in healthy subjects and IHF patients.Results:A total of 30 patients with IHF and 30 healthy subjects were enrolled,and all patients with IHF completed the treatment for 8 weeks.1.Main curative effect index1.1 LVEF:After treatment,the LVEF of IHF patients was higher than that before treatment,but there was no statistical difference(P>0.05).1.2 LVEDD:LVEDD value of IHF patients after treatment was not significantly different from that before treatment(P>0.05).1.3 BNP:The level of BNP in IHF patients after treatment was significantly lower than that before treatment(P<0.05).2.Secondary efficacy indicators2.1 Score of Qi Deficiency Symptom Scale:After treatment,the total score of Qi Deficiency Symptom Scale of IHF patients decreased significantly compared with that before treatment,with statistical difference(P<0.05).2.2 MLHFQ:After treatment,MLHFQ scores of IHF patients decreased significantly compared with those before treatment(P<0.05).3.Immune indicators3.1 CD4+T lymphocyte subsets:Compared with healthy subjects,Th2 decreased,Th17 and Th17/Treg increased in IHF patients,with statistical difference(P<0.05),while Thl did not change significantly,Th1/Th2 and Treg increased,with no statistical difference(P>0.05);After 8 weeks treatment,Th1,Th17,Th1/Th2,Th17/Treg in IHF patients decreased significantly,while Th2 increased significantly(P<0.05),while Treg decreased,but there was no statistical difference.3.2 Cytokines:The levels of IL-4 and IL-10 in serum of IHF patients decreased,while the levels of IL-2,IL-6,IL-17,TNF-α and IFN-γ increased,the difference was statistically significant(P<0.05);After 8 weeks of treatment,the serum levels of IL-10 increased,IL-2,IL-6,IL-17,TNF-α,IFN-γ decreased(P<0.05).Although IL-4 increased,there was no statistical difference(P>0.05)4.Correlation analysis between the score of Qi deficiency symptom scale and BNP,immune index:Pearson correlation analysis showed that the score of Qi deficiency symptom in IHF patients was significantly positively correlated with the levels of BNP,Th17,IL-2,IL-17,TNF-α and IL-6;It was negatively correlated with IL-4 and IL-10,and the difference was statistically significant(P<0.05).5.Safety:No adverse events occurred in IHF patients during treatment.Conclusions:1.ShenQi Compound Recipe can effectively reduce the BNP level of IHF patients,relieve clinical symptoms and improve the quality of life.2.CD4+T lymphocyte subsets in IHF patients were disordered,which were mainly manifested by the increase of Th17 and Th17/Treg,and the increase of pro-inflammatory factors IL-2,IL-6,IL-17,TNF-α and IFN-γ.The levels of anti-inflammatory factors IL-4 and IL-10 decreased.3.Shenqi Compound Recipe can effectively regulate the immune balance of CD4+T lymphocyte subsets in peripheral blood of IHF patients,mainly showing the decrease of Th1,Th17,Thl/Th2,Th17/Treg expression and the decrease of pro-inflammatory factors IL-2,IL-6,IL-17,TNF-α and IFN-γ.The expression of Th2 and the level of IL-10 were increased.4.The severity of Qi deficiency symptoms in IHF patients was related to the distribution of CD4+T lymphocyte subsets.Study 2:Discuss the mechanism of Shenqi Compound Recipe in treating Ischemic Heart Failure based on network pharmacology and molecular docking technologyObjective:Based on network pharmacology and molecular docking technology,to explore the active components and targets of Shenqi Compound Recipe in the treatment of IHF,and lay a foundation for the follow-up mechanism research.Methods:TCMSP and SwissTargetPrediction database were used to screen the active components of Shenqi Compound Recipe and predict their targets.Based on TTD,DisGeNet,Genecards,OMIM and Drugbank databases,heart failure-related disease targets were searched;Venn diagram was drawn to get the intersection target of Shenqi Compound Recipe in treating IHF;STRING platform was used to build PPI network and get core targets;Clusterfiler package in R language was used to analyze GO and KEGG enrichment of common targets;Finally,the software Autodock Vina was used for molecular docking.Results:Seven active ingredients,146 drug targets and 1237 IHF disease targets were screened out,with a total of 68 targets.Among them,IL6,TNF-α,IL-1β,CCL2,MMP9,VEGFA,AKT1 and CASP3 are the core targets of Shenqi Compound Recipe in treating IHF.Gene enrichment analysis showed that the treatment of IHF with Shenqi Compound Recipe mainly involved Th17 cell differentiation,IL-17,Toll-like receptor,T cell receptor,hypoxia inducible factor,cell-cytokine receptor interaction and other signal pathways;Molecular docking showed that quercetin and kaempferol,the effective active components of Shenqi Compound Recipe,could bind well with the core targets IL-6 and CASP3.Conclusion:The anti-IHF effect of Shenqi Compound Recipe can be exerted by the way of multi-component,multi-target and multi-pathway.The mechanism may be related to affecting the expression of target proteins such as IL-6,TNF-α,IL-1β and CCL2,which mediates signal pathways such as differentiation of Th17 cells,and inhibits inflammatory response and regulates immune response.Study 3 Explore the immunoregulation mechanism of Shenqi Compound Recipe in the treatment of IHF based on IL-6/STAT3 signaling pathwayObjective:To investigate the effects of Shenqi Compound Recipe on the balance of Th17/Treg cells and IL-6/STAT3 signal transduction in IHF rats based on animal experiments and molecular biological techniques.Methods:Sixty SD male rats were randomly divided into sham operation group,model group,low,medium and high dose group of Shenqi Compound Recipe and fosinopril group,with 10 rats in each group.The model of heart failure after acute myocardial infarction was established by coronary artery ligation in rats,and was given intragastric administration for 5 weeks after operation.Cardiac echocardiography was used to detect the changes of cardiac function and ventricular structure in rats.The pathological changes of rat myocardium were observed by HE and Masson staining;Serum NT-proBNP,cTnT,IL-6,IL-10,IL-17A,TNF-α were detected by ELISA;Flow cytometry was used to detect the proportion of Th17 and Treg cells in peripheral blood and spleen of rats.The positive expression of IL-6,IL-10,IL-17A,STAT3 and phosphorylated STAT3 were observed by immunohistochemistry.The expression of IL-6,STAT3,phosphorylated STAT3,Foxp3 and RORyt in rat myocardium was detected by Western blot.Results:1.Cardiac ultrasound showed that compared with sham operation group,the cardiac function indexes LVEF,LVFS,SV and CO in model group decreased significantly,while ventricular remodeling indexes LVDs,LVDd,LVESV and LVEDV increased significantly.Compared with the model group,the cardiac function and ventricular remodeling indexes in the treated group were significantly improved(P<0.01 or P<0.05).2.Myocardial pathology showed that the myocardial tissue of sham-operated rats was arranged densely and orderly without fibrosis deposition;In the model group,the myocardial tissue was disordered and broken,and there was a large amount of collagen deposition.Myocardial injury was improved in the administration group,and no obvious collagen deposition was found.3.Compared with sham operation,the contents of NT-proBNP and cTnT in model group were increased by Elisa;Compared with the model group,the serum NT-proBNP content in the administration group decreased(P<0.01 or P<0.05).4.Flow cytometry showed that compared with sham operation group,Th17 and Th17/Treg in peripheral blood and spleen of model group increased,while Treg decreased.Compared with the model group,Th17 and Th17/Treg in peripheral blood and spleen of rats in the administration group decreased.Treg increased,and the effect of high dose was most significant(P<0.01 or P<0.05).5.Cytokine test showed that compared with sham operation,serum pro-inflammatory factors IL-6,IL-17A and TNF-α increased and anti-inflammatory factor IL-10 decreased in model group.Compared with the model group,the serum pro-inflammatory factor level decreased and the anti-inflammatory factor IL-10 increased(P<0.01 or P<0.05).6.Immunohistochemistry showed that the high dose group could effectively inhibit the expression of IL-6,IL-17,STAT3 and phosphorylated STAT3,and up-regulate the expression of IL-10(P<0.05).7.Western Blot showed that the expression of IL-6,STAT3,phosphorylated STAT3 and RORyt was inhibited and the expression of Foxp3 was up-regulated in the high dose group of compound ginseng(P<0.05).Conclusion:The functional imbalance of Th17/Treg cells exists in CHF rats after AMI.Shenqi Compound Recipe can inhibit the differentiation of Th17 cells by inhibiting IL-6/STAT3 signaling pathway,restore the balance of Th17/Treg cells,alleviate inflammatory injury,improve cardiac function and reduce ventricular remodeling in rats. |
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