Introduction

Heart failure (HF) is the terminal stage of various cardiovascular diseases, with over 64 million cases worldwide1, causing severe social and economic burdens. The phenomenon of altered myocardial energy metabolic pathways, which results in structural and functional abnormalities in HF, is termed metabolic remodeling of the failing myocardium and plays a crucial role in the progression of HF2. Based on the 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure3, first-line treatments for HF include angiotensin receptor/neprilysin inhibitors (ARNI), β-blockers, mineralocorticoid receptor antagonists (MRA), and sodium-glucose cotransporter 2 (SGLT-2) inhibitors. However, patients undergoing these treatments still face unfavorable prognoses, with the 5-year survival rate remaining unsatisfactory4. Myocardial metabolic remodeling implies that optimizing myocardial energy metabolism constitutes an efficacious therapeutic method to prevent HF5. There is a limited selection of pharmaceuticals exhibiting protective effects on cellular energy metabolism, with common examples including coenzyme Q10, trimetazidine, and cyclophosphoadenosine. Therefore, attempts to research and develop new medicines for HF and elucidate their underlying mechanisms have become a major global public health priority.

Traditional Chinese Medicine (TCM) plays a significant role as a complementary treatment for HF. More than 71.2% of patients undergoing treatment for HF prefer a combination of Western and Chinese medicine6. Numerous Chinese patent medicines have demonstrated the ability to restore normal energy metabolism, such as Compound Danshen dripping pills, Qiliqiangxin Capsules and Naoxintong Capsules7,8,9, etc. Zhilong Huoxue Tongyu capsule (ZL) (patent No. 200810147774.1) includes five components that provide a rich pharmacodynamic material basis and is composed of Huangqi (Astragalus membranaceus Fisch. ex Bunge.), Guizhi (Cinnamomum cassia (L.) J. Presl), Daxueteng (Sargentodoxa cuneata (Oliv.) Rehder & E.H.Wilson), Earthworm and Leech, is extensively utilized in the treatment of HF, due to its anti-inflammatory, anti-apoptotic, and improving vascular endothelial function10,11,12. There is a lack of clarity regarding the impact of ZL on myocardial metabolism, and most studies on ZL in the treatment of HF have focused on rodent models. Hence, it is imperative to explore the mechanism of ZL from metabolic perspectives and to conduct more relevant clinical trials.

Untargeted metabolomics, which analyzes alterations of endogenous metabolites in a biological system, has been increasingly used to investigate metabolic changes in cardiovascular diseases13. Ultra-high performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) technology is a widely applied technique in untargeted metabolomics, providing enhanced coverage of the metabolome space in biology and medicine14. In this study, 80 subjects were enrolled to receive conventional treatment (CT) in combination with ZL. The clinical effects of integrative medicine in treating HF were evaluated using cardiac ultrasound, pertinent biochemical markers, and appropriate assessment tools. LC–MS/MS was conducted to explore the key compounds, core targets and pathways that mediate the effects of ZL against HF. Untargeted metabolomics has been used for the first time to elucidate the potential mechanism of action of ZL in treating HF, shedding new light on the mechanism of ZL as a Chinese patent medicine. The research flow of this study is shown in Fig. 1.

Fig. 1
figure 1

Research framework of this study.

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Materials and methods

Study design

A prospective, single-center clinical trial was designed to evaluate the effects and safety of ZL in the treatment of HF with Qi deficiency and blood stasis syndrome. The study was conducted in strict compliance with the ethical principles outlined in the Declaration of Helsinki. All investigators had completed standardized training prior to the trial. The protocol and informed consent for the trial were approved by the Ethics Committee of the Affiliated TCM Hospital of Southwest Medical University (SWMU) (Ethical Approval No. KY2022037-FS01). The clinical trial registration number (ChiCTR230070345) was assigned on 10 April 2023 by the Chinese Clinical Trial Registry (www.chictr.org.cn).

From May 2021 to May 2023, a total of 80 participants who were treated at the Affiliated TCM Hospital of SWMU were enrolled. All participants gave informed and signed consent for the anonymous use of their information. The diagnostic criteria of HF were performed in accordance with the 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure3. The Criteria for Diagnosis and Treatment of Heart Disease was first published by the New York Heart Association (NYHA) to determine the classification of heart function15. The criteria for TCM syndromes were Guiding Principles for Clinical Research of New Chinese Medicines16 and National Standard of the People’s Republic of China: Clinical terminology of traditional Chinese medical diagnosis and treatment-Syndromes17.

Key inclusion and exclusion criteria for the subject

Patients with the following criteria were included 1. Met the diagnostic criteria of HF; 2. Met the diagnostic criteria of HF in TCM and the syndrome differentiation criteria of Qi deficiency and blood stasis; 3. NYHA functional classification II-IV; 4. Length of hospitalization ≤ 16 h; 5. Age ranged from 18 to 75 years, gender not limited; 6. Participated voluntarily, understood and signed the statement of consent before enrollment.

Patients were excluded if they had: 1. Complex congenital heart disease, serious valvular heart disease, cardiomyopathy, or pericardial disease; 2. Acute stage of coronary atherosclerotic heart disease, requiring surgery or percutaneous intervention; 3. Serious arrhythmia with variation in hemodynamics; 4. Non-cardiac etiologies of dyspnea; 5. Significant neurological events occurred within 60 days of the present study; 6. Mechanical cycle support planned or being received; 7. Laboratory examination: hematocrit under 25%, hemoglobin (Hb) under 8.0 g/dl, white blood cell count under 3.0 × 109/L, alanine aminotransferase (ALT) or aspartate transaminase (AST) levels exceeded 10 times upper limit of normal value, estimated glomerular filtration rate (eGFR) below 25 mL/min/1.73 m2; 8. Planned or completed primary organ transplantation; 9. Malignant tumor; 10. Mental disorders; 11. Planned pregnancy, gestation, or lactating; 12. Used the investigational drug in the prior 3 months, or participated in other trials in the recent 30 days; 13. Patients with known hypersensitivity to therapeutic medicine.

Study drugs and Intervention

ZL was produced by the manufacturing laboratory of the Affiliated TCM Hospital of SWMU and authorized for production by Sichuan Medical Products Administration (Approval No. Z20,220,463,000). The process route for the preparation of ZL involved crushing Cassia Twig and Hirudo, and Astragalus, Pheretima, Sargentodoxae Caulis were extracted with water, concentrated to thick paste, dried to dry extract, followed by mixed with Cassia Twig and Hirudo powder, and divided into packages18. The dosage of ZL was 1.2 g orally three times a day (30 min before meals) for 14 days. CT refers to the 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure3 and China National Heart Failure Guideline 202319. Medication treatment was chosen based on the severity of the subject’s condition and relevant indications, with strict adherence to guidelines and recommendations in determining specific drugs, dosage, and treatment duration. Detailed information is provided in Supplementary Table 1.

The study period consisted of visit (V) 1 to V4, with V1 (hospitalization to 0-day) for screening, V2 (0-day) for baseline, V3 (0 to 14 ± 1-day) for treatment, V4 (30 ± 7-day) for follow-up.

Efficacy indicators and safety indicators

Efficacy indicators included left ventricular ejection fraction (LVEF), brain natriuretic peptide (BNP) level, 6-min walking distance (6MWD), Minnesota Living with Heart Failure Questionnaire (MLHFQ), and TCM syndrome scores, which were observed after administration. The above indicators were all completed at the Affiliated TCM Hospital of SWMU. Echocardiography (LVEF) was performed in Department of Ultrasound. BNP was performed in Clinical Laboratory. 6MWD, MLHFQ, and TCM syndromes tests were conducted in Department of Cardiology. TCM syndromes consist of primary and secondary symptoms, tongue and pulse conditions. The highest TCM syndromes score is 83 points. The specifics of the TCM syndromes score are shown in Supplementary Table 2.

Safety indicators: physical examination (temperature, respiration, heart rate, blood pressure, and body mass index), blood examination (Hb), liver function (ALT, AST), and renal function (creatinine (CR), brain natriuretic peptide (BUN)). Subjects need to fill out daily diary cards containing symptoms of discomfort. Routine laboratory tests were performed in Clinical Laboratory of the Affiliated TCM Hospital of SWMU.

Samples collection

After fasting for 8 h, 5 mL of venous blood was collected and left at room temperature for 1 h. Subsequently, the blood was centrifuged at 3000 rpm for 10 min at 25 °C, and then, 1 mL of liquid supernatant was transferred to a cryopreservation tube. Samples were stored in a refrigerator at − 80 °C prior to measurements and experiments.

Samples preparation

First, 100 μL of samples were transferred to EP tubes, resuspended with 400 μL of prechilled 80% methanol, vortexed thoroughly, incubated on ice for 5 min, and then centrifuged at 15,000 g for 20 min at 4 °C. Second, some of supernatants were diluted to final concentration containing 53% methanol using LC–MS grade water20,21. The samples were then placed into fresh EP tubes and centrifuged at 15,000 g, 4 °C for 20 min. Finally, the supernatants were injected into the LC–MS/MS system for analysis22,23. Quality control (QC) samples were prepared by mixing equal aliquot of experimental samples. Blank samples were replaced with 53% methanol and underwent the same pre-treatment process as the experimental samples.

Metabolomic analysis

The experimental method was referenced from “Material basis and integrative pharmacology of danshen decoction in the treatment of cardiovascular diseases”24. UHPLC-MS/MS analysis was conducted using the Vanquish UHPLC system (Thermo Fisher, Germany) coupled to the Q Exactive™ HF or Q Exactive™ HF-X mass spectrometer (Thermo Fisher, Germany). Chromatographic analysis was performed using the Hypesil Gold column (Thermo Fisher, USA) (100 mm × 2.1 mm, 1.9 μm) using a 12-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive and negative polarity modes were 0.1% formic acid in water (solvent A) and methanol (solvent B). The elution gradient was as follows: 2% B, 1.5 min; 2–85% B, 3 min; 85–100% B, 10 min; 100–2% B, 10.1 min; 2% B, 12 min. The Q Exactive™ HF mass spectrometer was operated in positive/negative polarity mode with a spray voltage as 3.5 kV, a capillary temperature of 320 °C, a sheath gas flow rate as 35 psi and aux gas flow rate as 10 L/min, S-lens RF level of 60, aux gas heater temperature of 350 °C.

To monitor the stability of the UPLC-MS/MS system, all samples were randomized. At the beginning of the sample sequence, three blank samples were injected to confirm baseline stability. The first three QC samples were used for chromatography-mass spectrometry system equilibration, and the following three QC samples were injected to segmented scanning, and then, the remaining QC samples were injected once every group injection to evaluate system stability.

Statistical analysis and data processing

The raw data files obtained from UHPLC-MS/MS were processed using Compound Discoverer 3.3 (CD 3.3, ThermoFisher) for peak discrimination, filtering, alignment, integration, and metabolite identification. The key parameters included: peak area corrected with the first QC sample, actual mass tolerance 5 ppm, signal intensity tolerance 30%, minimum intensity, etc. Subsequently, peak intensities were normalized to the total spectral intensity. The normalized data were utilized for molecular formula identification based on additive ions, molecular ion peaks and fragment ions. Peaks were matched with the mzCloud (https://www.mzcloud.org/), mzVault and MassList databases to obtain the accurate qualitative and relative quantitative results. Statistical analyses were conducted using R statistical software (R version R-3.4.3), Python (Python 2.7.6 version) and CentOS (CentOS release 6.6). When data were not normally distributed, standardization was performed according to the formula: sample raw quantitation value/(the sum of sample metabolite quantitation value/The sum of QC1 sample metabolite quantitation value) to obtain relative peak areas; compounds with coefficient of variation (CVs) of relative peak areas in QC samples exceeding 30% were excluded from analysis. Finally, this process yielded identification and relative quantification results for the metabolites.

Principal components analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) were conducted using metaX25, a flexible and comprehensive software for processing metabolomics data. Univariate analysis (t-test) was applied to calculate the statistical significance (P-value) and the fold change (FC) value of the metabolite between the two groups. Metabolites with variable importance in the projection (VIP) > 1.0, FC > 1.5 or FC < 0.833 and P-value < 0.05 were identified as differential metabolites. Matchstick diagrams were used to filter metabolites of interest based on log2 (FC) and -log10 (p-value) of metabolites by ggplot2 in R language. The differential metabolites were analyzed using cluster analysis. For clustering heat maps, data were normalized using z-scores of the intensity areas of differential metabolites and were plotted by pheatmap package in R language. Finally, explain the biological significance of metabolites through functional analysis of metabolic pathways.

GraphPad Prism software (version: 10.2.3) was used to assess the statistical significance of differences between pre- and post-medication conditions; P < 0.05 was considered significant26.

Result

Study completion

A total of 80 subjects were enrolled, and 76 finished cases with a completion rate of 95%. 4 subjects died during medication period. As depicted in Table 1, the mean age of the total population was 68.84 years, and 55.26% were male, and 39.47% were smoking, and 36.84% were drinking. The average course of HF was 3.76 years. The comorbidities of HF include coronary heart disease (51.32%), hypertension (40.79%), diabetes mellitus (27.63%), hyperlipemia (23.68%), and arrhythmia (36.84%). Combined use of angiotensin converting enzyme inhibitor (ACEI) class (47.37%), β-blocker (61.84%), MRA (69.74%), SGLT-2 inhibitors (31.58%), statins (47.37%), digitalis (18.42%), optimize energy metabolism (27.63%). Combining drugs represents a new approach to the treatment of HF, known as ‘quadruple therapy’, which includes renin–angiotensin–aldosterone system inhibitors, β-blocker, MRA, and SGLT-2 inhibitors. However, SGLT-2 inhibitors are not widely used like the first three drugs due to its late introduction as recommended drugs for heart failure. Only about a quarter of subjects received treatment with medications designed to optimize myocardial energy metabolism.

Table 1 Baseline Characteristics of examinees.
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Efficacy analysis

As shown in Table 2, the average LVEF of subjects after treatment was 57.75%, and there was a statistically significant difference in LVEF (p < 0.001) after administration, suggesting that ZL played a role in enhancing myocardial contractility. Its positive inotropic effect may be attributed to Astragaloside IV. Modern pharmacological studies have demonstrated that Astragaloside IV can enhance myocardial systolic and diastolic function without increasing myocardial oxygen consumption. A favorable effect of ZL was observed on the plasma BNP level (Table 2). After 14 d of treatment, subjects showed a significant decrease in BNP levels from baseline (BNP = 382.80) (p < 0.001). The mean percent reductions in BNP level after CT combined with ZL treatment was 57.45%. At baseline and 30-day, the 6MWD tests were performed. As indicated in Table 2, the average 6MWD of subjects prior to treatment was 274.70 m, while after treatment it increased to an average of 447.50 m (p < 0.001). ZL can improve exercise endurance in patients with HF. Quality of life was assessed through the MLHFQ, which was completed at 0-day and 30-day. In comparison to pre-treatment, the MLHFQ score of subjects after treatment decreased significantly to 29.46 (p < 0.001). After combined CT and ZL treatment, the total scores of TCM syndromes in the subjects decreased significantly (P < 0.001), as shown in Table 2. Both primary and secondary symptoms of TCM showed alleviation, particularly in terms of palpitation, wheezing, lower limb swelling, cyanosis, ventosity, and oliguria.

Table 2 Change in endpoints from baseline to after 30-day of follow-up.
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Safety analysis

ZL did not demonstrate any discernible effect on blood routine (Hb), liver function (ALT, AST), or kidney function (Cr, BUN). Additionally, none of the subjects experienced any adverse reactions. The results display that ZL exhibits good safety.

Metabolomics method validation

Pearson’s correlation analysis was performed on QC samples, and the correlation coefficients of nine QC samples exceeded 0.979, indicating good data quality (Supplementary Fig. 1). PCA is a statistical method that converts a set of observations of correlated variables into a set of linearly uncorrelated variables through orthogonal transformation27. It can report the overall metabolic distinctions among each group of samples and the degree of variability within each group. The results of PCA illustrated that QC samples clustered together (Fig. 2A,B), indicating high stability and reproducibility of the LC–MS/MS system. Furthermore, the overlapped total ion chromatograms (TICs) of QC samples demonstrated satisfactory reproducibility of the sample analysis (Supplementary Fig. 1). In conclusion, the stability and reproducibility of current metabolomics methods were satisfactory for metabolomics analysis.

Fig. 2
figure 2

Multivariate statistical analysis. (A) Positive ion mode PCA scores plots. (B) Negative ion mode PCA scores plots. (C) Positive ion mode PLS-DA scores plots. (D) Negative ion mode PLS-DA scores plots. (E) Positive ion mode validation plots after 200 replacement tests. (F) Negative ion mode validation plots after 200 replacement tests.

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Metabolomics analysis

First, the unsupervised pattern recognition method PCA was used to analyze the serum metabolic profile before and after treatment. As shown in Fig. 2A, there is a discernible separation trend in the serum metabolic profile in positive ion mode, while no significant differences were observed in negative ion mode (Fig. 2B). To further elucidate the disparities in serum metabolic profile, we employed PLS-DA for data analysis. PLS-DA is used to solve the problem of insensitivity to less correlated variables, thereby playing a pivotal role in the identification of different metabolites. Figure 2C,D have illustrated palpable differences before and after treatment in both positive and negative ion modes. These results suggest that the metabolic profiles in the plasma of patients with HF were ameliorated following CT combined with ZL treatment. Moreover, to validate the PLS-DA model and avoid overfitting, 200 × permutation tests were obtained, and the intercepts were R2 = 0.56, Q2 = − 0.43 (positive ion mode) and R2 = 0.41, Q2 = − 0.40 (negative ion mode), respectively, illustrating the model’s robustness in terms of usability and predictability (Fig. 2E,F).

Screen and identification of differential metabolites

Using untargeted metabolomics in conjunction with online databases such as KEGG (https://www.genome.jp/kegg/pathway.html), HMDB (https://hmdb.ca), and LIPIDMaps (http://www.lipidmaps.org/), a total of 919 and 470 metabolites were identified in the positive and negative ion modes, respectively, from 152 samples. All metabolites were carefully screened based on VIP > 1.0, FC > 1.5 or FC < 0.833 and P-value < 0.05 to identify potential differential metabolites. 33 and 24 metabolites with important contributions after CT combined with ZL treatment were screened in positive and negative ion mode. In positive ion mode, 13 metabolites were upregulated, and 20 metabolites were downregulated, and in negative ion mode, 12 metabolites were upregulated, and 12 metabolites were downregulated. The detailed information of the top 20 up- and down-regulated metabolites among the differential metabolites is summarized in Table 3. Detailed information on the 57 differential metabolites is presented in Supplementary Table 3. Matchstick diagrams can clearly illustrate the upward and downward adjustments of metabolites. The FC values for differential metabolites were subjected to a logarithmic transformation with a base of 2, and the top 20 metabolites with up- and down-regulation were selected in this order for display (Fig. 3A,B). To better understand the metabolic differences and characterize the metabolites changes before and after treatment potential biomarker data were analyzed through clustering analysis (Fig. 3C,D). To assess the consistency of metabolite and metabolite trends, differential metabolite correlation analyses were performed. The correlation between individual metabolites was analyzed by calculating the Pearson’s correlation for all metabolites pairs (Fig. 3E,F).

Table 3 Potential biomarkers in HF based on UHPLC-QE-MS analysis in serum.
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Fig. 3
figure 3

Analysis of differential serum metabolites in subjects before and after treatment. (A) Positive ion mode Matchstick diagrams. (B) Negative ion mode Matchstick diagrams. (C) Positive ion mode clustering heatmap. (D) Negative ion mode clustering heatmap. (E) Positive ion mode differential Metabolites Correlation Chart. (F) Negative ion mode differential Metabolites Correlation Chart.

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Metabolic pathway analysis

The 33 and 24 differential metabolites were uploaded to the Metaboanalyst 6.0 (https://www.metaboanalyst.ca/) website for investigation of metabolic pathways. The results observed that CT combined with ZL mainly regulates riboflavin metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism, arginine biosynthesis, butanoate metabolism, lipoic acid metabolism, and fatty acid biosynthesis. A summary of the pathways analysis is shown in Fig. 4.

Fig. 4
figure 4

Pathway analysis.

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Discussion

This study observed 76 patients with HF who received combined treatment with CT and ZL. After treatment, patients exhibited an increase in levels of LVEF and 6MWD, as well as a reduction in BNP levels, MLHFQ score, and TCM syndromes score. This suggested that the combination of CT and ZL has a positive effect on left ventricular systolic function, exercise endurance, quality of life, and traditional Chinese medicine syndrome of patients with HF.

UHPLC-MS/MS analysis revealed that the top three differences in positive ion mode were PC 20:2_20:2, 2-[(2-chlorobenzyl)sulfanyl]-4,6-dimethylnicotinonitrile, and LPC O-19:1, while in negative ion mode, the top three differences were Cyclic acid, N-Acetyl-1-aspartylglutamic acid, and Naringenin. PC 20:2_20:2 is a phosphatidylcholine, which can have many different combinations of fatty acids of varying lengths and saturation attached at the C-1 and C-2 positions. PC 20:2_20:2, consists of one chain of arachidic acid at the C-1 position and one chain of eicosadienoic acid at the C-2 position. Lysophosphatidylcholine (LPC) is one of the major products of phospholipid catabolism28. Phospholipids are key components of the lipid bilayer of cells, as well as being involved in metabolism and signaling. Glycerophospholipids have a role in regulating cardiac contractility29. Phosphatidyl choline (PC) is a primary component of glycerophospholipid and plays a crucial role in maintaining the structure and function of cardiomyocytes30. PC can catalyze the production of phosphatidylethanolamine via both the CDP choline pathway and the phosphatidylethanolamine N-methyltransferase (PEMT) pathway31. The PC produced by the PEMT pathway can regulate the production or secretion rate of cholesterol and low-density lipoprotein, thereby ensuring the normal metabolism of high-density lipoprotein32,33,34. In addition, it has been found that decreased LPO levels are associated with an increased risk of acute coronary syndrome events35. ZL can elevate LPO levels. Hence, in this experiment, the utilization of ZL led to a modification in the metabolism of PC 20:2_20:2 and LPC O-19:1, indicating that ZL has an impact on the biosynthesis of phospholipids. 2- [(2-chlorobenzyl) thioalkyl] -4,6-dimethylnicotinamide is not included in the HMDB database, and information on its relationship with HF is not yet available. This study found that the level of 2- [(2-chlorobenzyl) thioalkyl] -4,6-dimethylnicotinamide decreased after ZL treatment. Cyclamic acid, an organic compound known as cyclamates, is a potent sweetener suitable for use in pharmaceutical preparations. The notable rise in cyclamic acid levels in subjects after ZL treatment was attributed to the presence of cyclamate in the medication. N-Acetyl-L-carnosine is a natural dipeptide containing histidine. Histidine is known to dilate blood vessels and lower blood pressure36,37. Histidine levels were found to be low in patients with heart failure, suggesting that histidine may be associated with cardioprotective effects38, while N-Acetyl-L-carnosine levels were elevated after ZL treatment. Naringenin is a flavanone found in a variety of herbs that has anti-oxidant, anti-inflammatory, anti-osteoporosis, and anti-tumor39,40. The utilization of ZL led to a modification in the metabolism of Naringenin, indicating that ZL has anti-oxidant, anti-inflammatory effect.

Three metabolic pathways, riboflavin metabolism, citrate cycle, and alanine, aspartate and glutamate metabolism may play major roles in the treatment of HF with ZL. Riboflavin serves as a common cofactor for oxidase and dehydrogenase in the body, participating in numerous intricate energy metabolic processes41. Riboflavin is phosphorylated by riboflavin kinase to produce flavin mononucleotide (FMN), FMN produces flavin adenine dinucleotide (FAD) through the action of flavin adenine dinucleotide synthase, which can catalyze redox and dehydrogenase reactions42. There is a close association between FAD and acyl coenzyme A (CoA) dehydrogenase, which is involved in mitochondrial fatty acid β-oxidation43. Fatty acid β-oxidation supplies 60–90% of the heart’s energy44. In HF, a decrease in fatty acid oxidation capacity leads to reduced adenosine triphosphate (ATP) production, resulting in cardiomyocyte damage and myocardial systolic and diastolic dysfunction45. The treatment of HF with CT and ZL is associated with the enhancement of mitochondrial fatty acid β-oxidation in cardiomyocytes through riboflavin metabolism. Mitochondria are the energy factories of eukaryotic cells and the main organelles supplying ATP to the organism. In HF, mitochondria become the main site where metabolic remodeling occurs. And the Citrate cycle (TCA cycle), which is one of the main pathways of mitochondrial aerobic respiration and energy metabolism, is also damaged. TCA cycle serves as the common oxidative pathway for the three major nutrients and represents the primary means by which the body obtains energy. Fatty acids are catalyzed by acyl CoA synthetase to form acyl CoA, which combines with carnitine to form acyl carnitine46. The resulting compound enters the mitochondria for β-oxidation, yielding acetyl CoA and releasing energy through the citrate cycle. Glucose is oxidized aerobically to produce pyruvate, which produces acetyl CoA, and ATP via citrate cycle47. CT and ZL have the ability to improve mitochondrial TCA cycling, which may improve mitochondrial energy metabolism. When abnormal glucose and lipid metabolism results in inadequate energy supply, the body will mobilize amino acid metabolism to enhance energy provision48. Branched chain amino acids undergo catabolism to produce branched chain α-keto acids, which undergo irreversible oxidative decarboxylation to ultimately generate acetyl CoA and succinyl CoA49,50. These compounds enter the citrate cycle to produce energy. Alanine, aspartate and glutamate can be interconverted with α-keto acids under the catalysis of aminotransferase. α-keto acids can enter the tricarboxylic acid cycle, releasing energy to support physiological activities in the body51. Arginine inhibits the expression of pro-inflammatory genes by regulating the protein kinase signaling pathway, thus exerting an inhibitory effect on the body’s inflammatory response52. Proline inhibits apoptosis through antioxidant effects and resists damage to the heart caused by inflammatory responses53. Arginine is derived from glutamic acid and aspartic acid. Although this study identified the key compounds, core targets, and pathways of ZL in the treatment of HF, several aspects require further improvement. 1. Since this study is an exploratory trial and the number of cases in the hospital is limited, resulting in a relatively small sample size, the reliability of the research findings is constrained. 2. Animal experiments have demonstrated that ZL improves cardiac function in rats with HF, but the benefits of ZL for patients with HF remains unclear. Therefore, this study did not establish a control group. 3. While untargeted metabolomics was used to explore the differential metabolites and core pathways of ZL therapy for HF, this method cannot conduct in-depth analysis and research on specific metabolites and pathways. 4. This study conducted a clinical trial involving patients with HF. Within the population, there are significant metabolic variations among different individuals, which may lead to the possibility that the differential metabolites identified are influenced by other factors. Therefore, future studies of ZL in HF include the following: 1. Conducting prospective, multicenter, large-scale, randomized, controlled clinical trials to validate these observations. 2. Targeted metabolomics will be used to analyze differential metabolites and core pathways identified by untargeted metabolomics, combined with transcriptomics or proteomics to systematically and comprehensively elucidate the pharmacodynamic material basis and mechanism of action of ZL. 3. Carrying out in-vivo and in-vitro experiments to verify the key compounds, core targets, and pathways of ZL in treating HF.

Conclusion

In conclusion, CT combined with ZL treatment has improved heart function, exercise endurance, quality of life, and TCM syndromes in HF patients. UHPLC-MS/MS analysis revealed that the combination of CT and ZL regulates riboflavin metabolism, citrate cycle, alanine, aspartate and glutamate metabolism, arginine biosynthesis, butanoate metabolism, lipoic acid metabolism, and fatty acid biosynthesis in the treatment of HF. The efficacy, safety and mechanisms of CT combined with ZL have been verified in this study, which is conducive to the further research and application of ZL in the treatment of HF.