Mechanism of jianxin granules in the treatment of heart failure based on proteomics and metabolomics

Abstract

Background: Heart failure (HF) is associated with high mortality and rehospitalization rates, highlighting the need for novel therapeutic approaches. Jianxin (JX) granules, a Traditional Chinese Medicine formulation, have been patented for the treatment of HF. However, the specific therapeutic effects and underlying mechanisms of JX granules have not been fully elucidated. This study aimed at investigating the effects and mechanism of JX granules in the treatment of HF based on proteomics and metabolomic profiling.

Methods: HF model was established in rats by ligation of left coronary artery. The successfully modeled rats were randomly divided into three groups: the model group, the JX granules group, and Sacubitril/Valsartan (S/V) group. Four weeks after treatment, left ventricular (LV) function was evaluated via echocardiography. LV fibrosis and apoptosis were examined through histological analyses, while mitochondrial morphology was assessed using transmission electron microscopy. Quantitative assessment of oxidative stress was also conducted. Proteomics was used to identify the differentially expressed proteins and potential pathways. Metabolomics was utilized to elucidate the variations in metabolism. Then western blotting and in vitro analyses were performed.

Results: A rat model of HF was established, evidenced by a decrease in left ventricular ejection fraction (LVEF), stroke volume (SV), and left ventricular fractional shortening (LVFS), alongside diminished adenosine triphosphate (ATP) content, elevated oxidative stress, augmented apoptosis, and disrupted pyruvate metabolism. Treatment with JX granules ameliorated these effects, improving systolic function, reducing ventricular chamber size, and increasing LVEF, SV, and LVFS, as assessed by echocardiography. Additionally, JX granules attenuated cardiac fibrosis and improved mitochondrial structure, as evidenced by less vacuolation and clearer mitochondrial cristae, when compared to the model group. The treatment also regulated apoptosis-related protein expression, partially reversing the increase in cleaved Caspase-9, cleaved Caspase-3, and Bax and the suppression of Bcl-2 observed in the heart failure rats. All of these effects were similar to S/V. Proteomic and metabolomic analyses identified key differential genes, such as triosephosphate isomerase 1 (TPI1), lactate dehydrogenase B (LDHB), pyruvate kinase M (PKM), protein kinase B (Akt), Pyruvate Dehydrogenase Beta (PDHB) and lactate dehydrogenase A (LDHA), as well as vital pathways including carbon metabolism, the PI3K-Akt signaling pathway, pyruvate metabolism, and HIF-1α signaling pathway. Moreover, JX granules mitigated oxidative stress, inhibited apoptosis, and activated Akt in H9c2 cells exposed to angiotensin II, which could be reversed by the PI3K inhibitor LY294002.

Conclusion: JX granules improve HF in parallel to the efficacy of S/V, at least in part, through enhancing pyruvate metabolism, inhibiting oxidative stress and activating PI3K/Akt pathway.

Keywords: Heart failure; JX granules; Metabolomics; PI3K/Akt signaling pathway; Proteomics; Pyruvate metabolism.

Fig. 1

Effects of JX granules on left ventricular histopathology in rats with HF. A Representative M-mode echocardiographic images. B Representative images of HE staining. C Representative images of Masson trichrome staining

Fig. 2

Effects of JX granules on mitochondrial structure and oxidative stress in HF rats. A Representative images of mitochondrial structure, depicted by transmission electron microscopy. B Reactive oxygen species (ROS) levels in LV myocardium detected by DCFH-DA staining. C Relative DCFH-DA fluorescence intensity for ROS. D–F The levels of T-AOC, SOD, and ATP in myocardium determined by specific assay kits. Data were expressed as mean ± SD; ∗p < 0.05 versus control group; ^#p < 0.05 versus model group

Fig. 3

Effects of JX granules on apoptosis in HF rats. A TUNEL (TdT-mediated dUTP Nick-End Labeling) assay for apoptosis detection. Brown nuclei indicated cell apoptosis. B The apoptotic index in LV myocardium. C–F The protein expressions of caspase-9, cleaved-caspase-9, caspase-3, cleaved-caspase-3, Bcl-2, and Bax were analyzed by western blotting. The protein densitometry was normalized with caspase-3, caspase-9, or GAPDH. And they were quantified of three experiments. ∗p < 0.05 versus control group, ^# p < 0.05 versus model group

Fig. 4

Effects of JX granules on differentially expressed proteins in HF rats. The volcano plots (A) and Heatmaps (B) of differentially expressed proteins.Their expression is calculated by log2 method and displayed in the volcano plots and heatmaps in different colors, where red represents significantly up-regulated proteins, blue represents significantly down-regulated proteins, and gray part represents no quantitative information of proteins. DEPs differentially expressed proteins, M model group, J JX granules, Z control group

Fig. 5

Effects of JX granules on signal pathway. The GO functional and KEGG enrichment of differentially expressed proteins. GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes, BP biological processes, MF molecular function, CC cell composition

Fig. 6

Effects of JX granules on differential metabolites in HF rats. Metabolomics analysis identified possible for JX granules. A PCA-3D, B PCA and C PLS-DA analysis in the positive and negative modes. The different colors represent the different groups (red for the control, blue for model, purple for JX granules. D KEGG analyze the metabolic the metabolic pathway of the potential biomarkers in the negative and positive modes. E Heat map analysis of 110 and 49 differential metabolites in the positive and negative modes

Fig. 7

Effects of JX granules on hub genes and critical regulatory pathway by integrated analysis of proteomics and metabolomics. A PPI network of HF targets. The colour circles represent the key genes with the different degree values. B Top 20 significantly enriched pathways enrichment by KEGG analysis. C PPI network of main genes with pathways. The green rectangle nodes represent the pathways and the yellow circle present the relative genes associated with JX granules pharmacologic action. A The Venn analysis showed the crosstalk of DEGs in two omics. The GO enrichment analysis, BP (B), MF (C), CC (D). E PPI network of HF targets, The colour circles represent the key genes with the different degree values. F Top 20 significantly enriched pathways enrichment by KEGG analysis. G PPI network of main genes with pathways, The green rectangle nodes represent the pathways and the yellow circle present the relative genes associated with JX granules pharmacological action

Fig. 8

Effects of JX granules on the protein expression of p-PI3K/PI3K, p-Akt/Akt, HIF-1α, PDHB, LDHA, and PKM2. (A-G) The protein expressions of p-PI3K, p-Akt, HIF-1α, PDHB,LDHA and PKM2 were analyzed by western blotting. The protein densitometry was normalized with PI3K, Akt, or GAPDH. And they were quantified of three experiments. ^*p < 0.05 vs the control group, ^# p < 0.05 vs the model group

Fig. 9

Effects of Jianxin granules on ROS, apoptosis, Akt activity and ATP in response to Ang II incubation. A Fluorescence microscopic images of intracellular ROS showed by 2,7′,7-dichlorofluorescein diacetate (DCFH-DA) staining (green, original magnification, × 200). B Representative images of TUNEL assay for apoptosis. Brown nuclei indicated cell apoptosis (original magnification, × 200). C Corresponding DCF fluorescence value of ROS. D TUNEL-positive nuclei were counted in 500 nuclei from random fields per slide and expressed as the percentage of apoptotic cells (apoptotic nuclei/total nuclei × 100%). E The expression of Akt and p-Akt were analyzed by Western blotting. Quantification of the ratios of band intensity of p-Akt relative to Akt. F The levels of ATP in H9c2 cells. Data are expressed as mean ± SD obtained from three independent experiments. *p < 0.05 compared with blank control. ^#p < 0.05 compared with Ang II challenge cells. ^&p < 0.05 compared with Ang II + LY294002 treated cells

Fig. 10

Schematic overview of the mechanism of JX granules in managing heart failure