| Objective:(1)To provide evidence-based medical evidence for the efficacy and safety of reinforcing qi,nourishing Yin and eliminating phlegm represented by Deng’s Yangxin decoction in the treatment of heart failure.(2)To reveal whether Dengshi Yangxin decoction can enhance the clearance of apoptotic and necrotic cells,reduce myocardial fibrosis,protect myocardial cells and combat heart failure by regulating the autophagy function of macrophages.(3)To clarify the signal transduction mechanism of Dengshi Yangxin decoction in regulating the activity of macrophage apoptosis,so as to expand the theoretical connotation of the method of replenishing qi,nourishing Yin and eliminating phlegm established by Professor Deng Tietao,a master of traditional Chinese medicine,and provide theoretical basis for the treatment of heart failure with traditional Chinese medicine.Methods:In this study,we conducted a series of experiments.(1)Efficacy and safety of Dengshi Yangxin decoction in the treatment of chronic heart failure:A prospective randomized controlled clinical study was conducted.A total of 128 patients who were diagnosed with heart failure and met the inclusion criteria in the First Affiliated Hospital of Guangzhou University of Chinese Medicine from June 2022 to February 2023 were enrolled.The patients were randomly divided into experimental group and control group by random number table method.One patient dropped out in the control group.The control group was treated with standard western medicine treatment,and the experimental group was treated with Dengshi Yangxin decoction(once a day)on the basis of standard western medicine treatment.The follow-up period was 12 weeks,and the follow-up time node was the run-in period,4 weeks,8 weeks and 12 weeks of treatment.After 12 weeks of treatment,the left ventricular ejection fraction(LVEF)(biplanar Simpson method),left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),NT-proBNP level and New York Heart Association(NYHA)cardiac function classification were compared between the two groups before and after treatment to evaluate the cardiac structure and function.The improvement of TCM syndrome score and Minnesota Living Quality Questionnaire(MLHFQ)were compared to evaluate the clinical symptoms and quality of life.The rehospitalization rate and mortality rate due to heart failure during the treatment were recorded.In addition,the levels of serum aspartate aminotransferase(AST),alanine aminotransferase(ALT)and creatinine(CREA)were compared to evaluate the safety of treatment.So as to verify the efficacy and safety of Dengshi Yangxin decoction in the treatment of chronic heart failure.(2)The effect of Dengshi Yangxin decoction on cardiac dysfunction in adriamycin-induced heart failure rats:Sprague-Dawley(SD)rats with adriamycin-induced heart failure were selected and randomly divided into blank group,model group,low dose group,middle dose group and high dose group.Echocardiography was used to measure the LVEDD,LVESD,LVEF,LVFS and other indicators after intervention,and PCR method was used to measure the level of BNP to evaluate the cardiac function of rats.HE staining and Masson staining were used to compare the histopathological changes of myocardial cells,so as to verify the effect of Dengshi Yangxin decoction on the improvement of cardiac dysfunction in adriamycin-induced heart failure rats.(3)Network pharmacology study of Dengshi Yangxin decoction in improving cardiac dysfunction:The active ingredients of Dengshi Yangxin decoction were screened by network pharmacology,and the target prediction was performed.By mapping with "heart failure" related pathogenic genes,the protein-protein interaction(PPI)network of mapping targets was constructed.KEGG pathway enrichment analysis was used to screen out the potential signaling pathways of Dengshi Yangxin decoction in the treatment of heart failure.(4)The mechanism of Dengshi Yangxin decoction in the treatment of heart failure based on PI3K/Akt and AMPK autophagy signaling pathways:The primary rat macrophages were extracted and treated with adriamycin to establish the model.The optimal intervention time and concentration of Dengshi Yangxin decoction were selected by CCK8 method,and the cells were divided into blank group,model group and Dengshi Yangxin decoction group.The changes of autophagosome in macrophages were 62 and PI3K,Akt,AMPK,p-PI3K,p-Akt,and p-AMPK in macrophages.The mRNA expressions of LC3B,Beclin-1,p62,AMPK,AKT,and PI3K in macrophages were detected by qRT-PCR.At the same time,the expression of LC3B,Beclin-1 and p62 in myocardial tissue was detected by Western Blot,so as to explore the potential mechanism of Dengshi Yangxin decoction in the treatment of heart failure based on PI3K/Akt and AMPK autophagy signaling pathways.Results:(1)Efficacy and safety of Dengshi Yangxin decoction in the treatment of chronic heart failure:62 subjects in the trial and 63 subjects in the control group were enrolled.There were no significant differences in baseline data(age,gender,combined medication)between the experimental group and the control group(P>0.05).①Cardiac function(experimental group VS.control group):(A)LVEF:Before treatment,which was comparable.(6.74±4.875%VS.0.33±5.355%,P<0.01).(B)NT-proBNP:Compared with the control group,the NT-proBNP test results of the experimental group decreased more significantly after treatment,and the difference was statistically significant(3844.62±2989.84pg/ml vs.2345.94±2947.06pg/ml,P<0.01).(C)NYHA classification:Before treatment,there was no significant difference in NYHA classification between the two groups(P>0.05),and the two groups were comparable.After treatment,the NYHA classification of the two groups was improved(P<0.01).②Cardiac structure(experimental group VS.control group):(A)LVEDD:decreased in both groups,and there was no significant difference between the two groups(57.9±8.672mm vs.60.63±6.785mm,P>0.05).The LVEDD in the treatment group decreased more significantly than before,and the difference was statistically significant(1.29±4.198mm vs.0.35±3.686mm,P<0.05).(B)LVESD:were comparable.There was no significant difference in LVESD between the two groups after treatment(48.74±9.295mm vs.51.63±7.936mm,P>0.05).③Clinical symptoms and quality of life(experimental group VS.control group):(A)TCM syndrome score:the NYHA classification of the two groups was improved,and the improvement of the treatment group was more obvious,and the difference was statistically significant(13.86±3.50VS.15.26±4.12,P<0.01).(B)Minnesota Quality of Life Questionnaire:Before treatment,there was no significant difference in Minnesota Quality of Life questionnaire between the two groups(76.45±5.14VS.76.44±5.22,P>0.05)and the improvement was more significant in the treatment group(34.37±6.392VS.29.22±6.632,P<0.01).①Incidence of adverse events:8 patients were hospitalized due to heart failure in the treatment group,and 12 patients were hospitalized due to heart failure in the control group.④Safety evaluation(experimental group VS.control group):(A)Liver function(ALT,AST):Before treatment,there was no significant difference in ALT and AST between the experimental group and the control group(24.55±14.66mmol/L VS.24.54± 13.04mmol/L,23.98± 12.13mmol/L VS.23.65±9.025mmol/L,P>0.05).The results were comparable between the two groups.(B)Renal function(CREA):Before treatment,there was no significant difference in CREA between the experimental group and the control group(86.71±27.32mmol/LVS.89.866±22.28mmol/L,P>0.05),which was comparable between the two groups.After treatment,CREA decreased in both groups(73.58±20.20mmol/LVS.82.19±15.04mmol/L,P<0.05),but the difference was not clinically significant.(2)Dengshi Yangxin decoction improved cardiac dysfunction in adriamycin-induced heart failure rats:SD rats with chronic heart failure were induced by intraperitoneal injection of adriamycin-induced heart failure.①Echocardiography:After 8 weeks of intervention,6 rats were randomly selected from each group according to the random number table method for echocardiography.The results showed:The LVEF level(44.63± 1.91%VS.81.87±2.25%,P<0.01),and the LVEF level(54.67±5.97%VS.44.63± 1.91%,P<0.05).②HE staining:HE staining showed that there was no obvious morphological change in the myocardial tissue of the blank group reduced compared with the model group.③Masson staining showed that there was no fibroblast proliferation in the blank group,but collagen deposition and a large number of fibroblasts were observed in the myocardial interstitium of the model group.Compared with the model group,the proliferation of myocardial fibroblasts and the degree of myocardial interstitial fibrosis in the low-,medium-.and high-dose groups were significantly reduced.④Serum BNP levels:the BNP level in the model group was significantly increased(0.70±0.07ng/ml vs.0.23±0.02ng/ml,P<0.01),and the low-dose group(0.56±0.03ng/ml vs.0.70±0.07ng/ml,P<0.01).P<0.01),medium dose group(0.50±0.03ng/ml vs.0.70±0.07ng/ml,P<0.01),and high dose group(0.428±0.506ng/ml vs.0.70±0.07ng/ml,P<0.01),and showed a concentration-dependent manner.The higher the concentration of BNP,the better the effect of reducing the BNP level.(3)Network pharmacology study of Dengshi Yangxin decoction improving cardiac dysfunction:a total of 377 chemical components of YX were obtained by searching TCMID and TCMSP databases.These included RS group(n=134),MD group(n=8),FBX group(n=116)and SQ group(n=119).According to the ADME threshold OB≧30%and DL≧0.18,a total of 45 active ingredients were obtained.Targets corresponding to the 80 active compounds were predicted based on chemical similarity using PharmMapper server.A total of 289 active ingredient-related targets were obtained,including 53 targets corresponding to RS,143 targets corresponding to MD,43 targets corresponding to FBX and 87 targets corresponding to SQ.In addition,by integrating the results obtained from GC and OMIM databases with the keyword "heart failure",a total of 1798 disease-causing genes of heart failure were obtained.Then,the two sets of data and the active targets of YX that may be involved in HF were crossed,and 84 mapped targets were obtained.Protein-protein interaction analysis of these 84 mapped targets was performed using STRING database,PPI pathways were screened.These included TNF signaling pathway,MAPK signaling pathway,PI3K/Akt signaling pathway.(4)Study on the mechanism of Dengshi Yangxin decoction in the treatment of heart failure based on PI3K/Akt and AMPK autophagy signaling pathways:serum containing Dengshi Yangxin decoction was prepared,SD rats were grouped as in(2),and primary rat macrophages were extracted.CCK8 assay showed that the optimal pretreatment time of DST was 8 hours,and the concentration of adriamycin 1μmol/L and 320μg/mL DST were more suitable for subsequent experiments.①The changes of autophagosomes in macrophages observed by transmission electron microscopy showed that the autophagosomes showed obvious double membrane structure,and the cleft between the inner and outer membranes was wide.The number of autophagosomes in the model group was lower than that in the control group,and the number of autophagosomes in the Dengshi Yangxin decoction group was higher than that in the model group.②Western Blot was used to detect the expressions of LC3B,Beclin-1,p62 and PI3K,Akt,AMPK,p-PI3K,p-Akt,62 was increased.Dengshi Yangxin decoction can up-regulate the expression of LC3B and Beclin-1,and inhibit the expression of p62 protein.Compared with the control group,the levels of p-PI3K/PI3K and p-Akt/Akt were increased,and the level of p-AMPK/AMPK was decreased in the model group.Dengshi Yangxin decoction could down-regulate the expression of p-PI3K/PI3K and p-Akt/Akt,and up-regulate the expression of p-AMPK/AMPK protein.③qRT-PCR was used to detect the mRNA expressions of LC3B,Beclin-1,p62 and Akt,AMPK,PI3K in macrophages:compared with the blank group,the expression levels of LC3B and Bcclin-1 mRNA in the model group were decreased,and the expression level of 62.Compared with the control group,the levels of PI3K.Akt and AMPK were increased in the model group.Dengshi Yangxin decoction can inhibit the expression of PI3K,Akt and AMPK genes.④The expression of Caspase-3 in myocardial tissue was detected by immunofluorescence(IF):compared with the blank group,the level of Caspase-3 in the model group was increased.Dengshi Yangxin decoction at middle and high doses can down-regulate the expression of Caspase-3,and the effect of Dengshi Yangxin decoction at high dose is more significant.⑤The expressions of LC3B,Beclin-1 and p62 in myocardial tissue detected by Western Blot:compared with the blank group,the levels of LC3B and Beclin-1 in the model group decreased,and the level of p62 increased.Dengshi Yangxin decoction can of Dengshi Yangxin decoction has the most significant effect.Conclusion:Dengshi Yangxin Decoction high dose group has the most significant effect.In this study,we first verified from a clinical perspective,and then verified the myocardial protective effect of Dengshi Yangxin decoction in a rat model of heart failure from the perspective of animal experiment and cell experiment,and further verified that Dengshi Yangxin decoction improved the autophagy function of macrophages in myocardial tissue of heart failure through Akt/mTOR and AMPK signaling pathways.It can enhance the ability of macrophages to clear apoptotic and necrotic cells in myocardial tissue of heart failure,reduce macrophage apoptosis and necrosis,and reduce myocardial fibrosis. |
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