Clinic Analysis And Mechanism Of Tonifying Qi,warming Yang,Activating Blood And Promoting Diuresis In The Prevention Of Chronic Heart Failure

ObjectiveFirstly,clinic meta-analysis was used to objectively evaluate the clinical efficacy of tonifying Qi,warming Yang,activating blood and promoting diuresis in the prevention and treatment of chronic heart failure.Then,scRNA-seq data mining of chronic heart failure mice,the RNA-seq of primary cardiomyocytes with the intervention of XinYang Tablet,and network pharmacology of Xinyang Tablet were combined to explore the key targets and potential mechanisms of Xinyang Tablet in the prevention of cardiac hypertrophy in chronic heart failure,and verified by animal experiments.Methods1.A Clinical Meta-analysis of researches on the prevention of CHF with traditional Chinese medicine which utilized the treatment of tonifying Qi,warming Yang,activating blood and promoting diuresis.The Chinese and English databases were searched by subject key words and free words to obtain relevant literatures,and the literatures were screened according to the established inclusion and ranking criteria to obtain the qualified requirements.Then the literature data extraction and quality evaluation were carried out,and the corresponding outcome indicators were evaluated by statistical analysis.2.Aortic arch coarctation was used to construct CHF model with pressure overload.60 C57/6J mice were randomly divided into sham operation group(Sham),model group(TAC),Xinyang tablet with low-dose group(LXY),Xinyang tablet with middle-dose group(MXY),and Xinyang tablet with high-dose group(HXY).And perindopril positive control group(PDPL),10 mice per group.After 8 weeks of drug intervention,small animal ultrasound was used to detect cardiac function,the heart weight,body weight and heart weight ratio of mice were measured,HE staining was used to evaluate cardiac hypertrophy,Masson staining was used to detect the degree of myocardial fibrosis,and qPCR was used to detect ANP and BNP,Myh7,Colla,Col3a mRNA expression levels,Western Blot detection of ANP,BNP,Myh7,Collagen Ⅰ,Collagen Ⅲ protein expression levels in mouse myocardium.The above tests were combined to evaluate the effect of Xinyang tablet on cardiac function and ventricular remodeling in mice with CHF.3.Mouse scRNA-seq data(GSE120064)downloaded from the GEO database which had 16 mouse samples.The data were preprocessed by the Seurat package in R language before downstream differential analysis;then primary cardiomyocytes were isolated from the mouse hearts of the model group(TAC)and the middle-dose Xinyang tablet group(XYT).And then we extracted the RNA from the two groups of primary cardiomyocytes for RNAseq,and conduct differential expression analysis after data quality control.UPLC-MS method was used to analyze the main compounds in Xinyang Tablet.And then the "Compound-target-disease" network in pharmacology was used to predict the target of Xinyang Tablet for the prevention and treatment of CHF.The above-mentioned differential expression genes(DEGs)obtained from scRNA-seq data mining,DEGs obtained from the primary cardiomyocytes RNA-seq,and the predicted targets obtained from network pharmacology analysis of Xinyang tablets were intersected to obtain Xinyang tablets for the prevention CHF.These DEGs and prediected targets of CHF were adopted to enrichment analysis,and then we intersected their biological processes in GO term to get the mechanism of Xinyang tablet for prevention CHF.4.These experiments of WGA staining,Calcium yellow green staining,Tunel staining,Immunofluorescence assay,Western Blot were used to evaluate the effect of Xinyang tablets on cardiomyocyte hypertrophy and apoptosis in CHF.Phalloidin staining and Western Blot were used to further evaluate the specific mechanism of Xinyang tablet regulating the hypertrophic signaling pathway of PI3K/Akt/GSK-3β in HL-1.And we detected the level of ROS,the levels of SOD and MDA in myocardial tissue,the changes in the mitochondria by transmission electron microscopy,and evaluated the expression of oxidative stress and ferroptosis by Western blot detection of GPX4,NOX1,COX2,xCT,and Nrf2 in the myocardium.death situation.Results1.Meta-analysis on the prevention CHF with tonifying Qi,warmingYang,activating blood and promoting diuresis.A total of 26 qualified literatures were included in this metaanalysis after screening and inclusion criteria,25 Chinese literatures and 1 English literature.The analysis results showed that compared with the conventional western medicine treatment,the traditional Chinese medicine combined with the conventional western medicine treatment could significantly improve the efficacy of the patients’ cardiac function and TCM syndromes,reduce the expression of BNP and NT-proBNP in the patients,improve the level of LVEF,reduce the size of LVEDD,and improve 6-MWT and Heart Failure Minnesota Quality of Life Score.2.Xin Yang Tablet could improve cardiac function and ventricular remodeling in mice with CHF.(1)Xinyang tablets significantly increased the levels of LVEF and LVFS in mice with CHF(P<0.01),The levels of LVEDD and LVESD were significantly reduced in MXY and HXY group(P<0.01),the LVEDD was also reduced(P<0.05)and the LVESD was reduced(P<0.01)in LXY group,but the effect of middle-dose Xinyang tablets(MXY)was more obvious than that of low-dose and high-dose Xinyang tablets.(2)Different doses of Xinyang Tablets could reduce the heart/body weight ratio(P<0.01),cardiac transverse cross-sectional area(P<0.01);And the improvement effect of middle dose Xinyang Tablet was better.But just middle-dose Xinyang tablets could reduce the circumference of heart(P<0.01).(3)Xinyang Tablets could significantly improve the myocardial fibrosis in mice with chronic heart failure(P<0.01).(4)Xinyang tablets could significantly reduce the mRNA expression levels of ANP and BNP which related to the severity of heart failure(P<0.01),It also could reduce the expression of Myh7 mRNA related to myocardial hypertrophy(P<0.01),and reduce the expression levels of Colla and Col3a mRNA related to myocardial fibrosis(P<0.01).(5)Xinyang tablets could significantly reduce the protein expression of ANP and BNP related to the severity of heart failure(P<0.01),It reduced the protein expression of Myh7 which related to myocardial hypertrophy(P<0.01)and the protein expression of cTnt which related myocardial damage in CHF(P<0.05);The protein expression of Collagen Ⅰand Collagen Ⅲ which related to myocardial fibrosis were significantly reduced in MXY group and HXY group(P<0.01),the protein expression of Collagen Ⅰ was also reduced in LXY group(P<0.05),and the protein expression of Collagen Ⅲ was also reduced in LXY group(P<0.01).3.Some key targets and potential mechanisms were detected based on scRNA-seq data mining,primary cardiomyocytes RNA-seq and network pharmacology analysis of Xinyang tablet for prevention CHF.After scRNA-seq data mining,1055 DEGs were observed in 0-week and 8week chronic heart failure mice.A total of 2568 DEGs were observed in primary cardiomyocytes RNA-seq between the Xinyang tablet group and the TAC group of mice.UPLC-MS was used to analyze 81 compounds containing PubChem ID in Xinyang tablet,and these compounds were subjected to network pharmacology to analyze,1248 predictive targets of Xinyang Tablets for the prevention of CHF were obtained.After intersecting the above DEGs and predicted targets,18 key targets of Xinyang Tablets for CHF were observed,including“HDAC2”,“ACSL1”,“DARS”,“EZR”,“F2R”,“FECH”,“HMGCL”,“HSD11B1”,“LARS2”,“MMP2”,“NPPB”,“RAMP2”,“RBP1”,“S100A8”,“S100A9”,“SERPINE1”,“SOD2”,and“XDH”.Enrichment analysis showed the common GO items,some biological processes involved in the top-ranked GO items.They were fatty acid metabolism,lipid oxidation,oxidative stress,carbohydrate metabolism,mitochondrial Biological processes involved in tissues,oxidative phosphorylation,calcium cycling,apoptosis,etc.HDAC2,which actively regulates myocardial pathological hypertrophy,was selected as the key target for verification in this experiment.Based on the primary cardiomyocytes RNA-seq KEGG signaling pathway and previous literatures,it is speculated that Xinyang Tablet may regulated the expression of HDAC2 and then mediate PI3K/Akt/GSK-3βpathway to reduce cardiomyocyte hypertrophy and apoptosis,and it may also regulate the expression of HDAC2 to improve the level of oxidative stress and ferroptosis during chronic heart failure by activating Nrf2 which palys a important role in antioxidative system.4.Xinyang tablet could regulate the expression of HDAC2 to mediate PI3K/Akt/GSK-3 β pathway to prevent cardiomyocyte hypertrophy and opoptosis,and mediate Nrf2 antioxidative pathway to reduce oxidative stress and ferroptosis.(1)Xinyang tablets can significantly reduce myocardial cell hypertrophy in CHF.WGA staining and Tunel staining showed that the myocardial cell hypertrophy in the TAC group was significantly increased(P<0.01)and the apoptosis rate was increased(P<0.01)compared with the Sham group.While the cardiomyocyte hypertrophy was reduced(P<0.01)and the apoptosis rate was reduced after the intervention of Xinyang Tablet(P<0.01).(2)Xinyang tablets can reduce the protein expression of p-HDAC2 and p-GSK-3 β in cardiomyocytes.Immunofluorescence assay showed that the protein expressions of p-HDAC2 and p-GSK-3 β in the cardiomyocytes of the TAC group were increased compared with the Sham group(P<0.01).While the protein expression of p-HDAC2(P<0.05)and p-GSK-3β(P<0.01)were decreased compared with TAC group.(3)Xinyang tablets may regulate HDAC2 to inhibit the activity of PI3K/Akt/GSK-3 β pathway to reduce cardiac hypertrophy.Western Blot detection showed that compared with Sham group,the expression level of Inpp5f protein in the TAC group was decreased(P<0.01),and the ratios of P-HDAC2/HDAC2,p-Akt/Akt and p-GSK-3 β/GSK-3 β were significantly increased(P<0.01);After Xinyang Tablet intervention,the protein expression of Inpp5f protein increased(P<0.01),and the ratios of pHDAC2/HDAC2,p-Akt/Akt and p-GSK-3β/GSK-3 β were significantly decreased(P<0.01).(4)Xinyang tablet could inhibit the activity of Akt and improve the hypertrophy of HL-1 cells induced by AngII.Compared with the Control group,the size of CSA and nucleus of the AngII group were significantly increased(P<0.01,P<0.01);Compared with the AngII group,the size of CSA and nucleus in the XYT+AngII group were smaller(P<0.01,P<0.01);and Compared with XYT+AngⅡ group,after adding SC79 to activate Akt,the size of CSA and nucleus were increased(P<0.01,P<0.01).(5)Xinyang tablet could improve HL-1 cell hypertrophy induced by AngII through reducing the expression of the activity of Akt/GSK-3 βsignaling pathway.Compared with the control group,the protein expression levels of p-Akt and p-GSK-3β in AngⅡ group were significantly increased(P<0.01);Compared with the AngⅡ group,the protein expression levels of p-Akt and p-GSK-3β in AngⅡ+XYT group were significantly decreased(P<0.01);And compared with AngⅡ+XYT group,after adding SC79 to activate Akt,the expression levels of p-Akt,p-GSK-3 β was significantly increased(P<0.01).(6)Xinyang tablets could reduce the activation of HDAC2 to activate Nrf2 antioxidative pathway to improve oxidative stress and ferroptosis in myocardium.Transmission electron microscopy showed that the mitochondria became smaller and atrophied,the density of mitochondrial membrane increased,and some mitochondrial membranes were ruptured compared with Sham group.Compared with Sham group,the content of SOD which has antioxidant effect was significantly decreased(P<0.01),the content of MDA which represented the level of lipid peroxidation was significantly increased(P<0.01),and the level of ROS was significantly increased(P<0.01)in TAC group.Western blot detection suggested that the ratio of p-HDAC2/HDAC2 was increased(P<0.01),the protein expression of Nrf2,GPX4 and xCT were decreased(P<0.01),and the expressions of COX2 and NOX1 were increased(P<0.05,P<0.01)which were related to ferroptosis and oxidative stress in TAC group.Compared with the TAC group,the SOD content was significantly increased(P<0.01),the MDA content was significantly decreased(P<0.01),and the ROS level was significantly decreased(P<0.01)in MXY group.And with the intervention of XinYang tablets,the size of mitochondria was larger than TAC group,and the density of mitochondrial membrane was improved.The protein expressions of Nrf2,GPX4 and xCT were increased(P<0.01),and the protein expressions of p-HDAC2/HDAC2(P<0.01)、COX2(P<0.05)and NOX1(P<0.01)were decreased compared with the TAC group.Conclusion(1)Tonifying Qi,warming Yang,activating blood and promoting diuresis combined with conventional western medicine to prevent these patients with chronic heart failure had better clinical efficacy in cardiac function,traditional Chinese medicine syndromes,BNP,NT-proBNP,LVEF,LVEDD,6-MWT,and MLHFQ than those of conventional western medicine.It suggested that the method of tonifying Qi,warming Yang,activating blood and promoting diuresis played a positive role in improving the clinical efficacy of CHF.(2)XinYang tablets played a vital role in improving ventricular remodeling of CHF through multiple targets and multiple pathways.XinYang tablets could improve the hypertrophy and apoptosis of cardiomyocytes by regulating the transcriptional activity of HDAC2 in cardiomyocytes and then mediated the activity of the PI3K/Akt/GSK-3β pathway.And it may mediate the expression of HDAC2 to improve the level of oxidative stress and ferroptosis in CHF by activating Nrf2.