Abstract Background: Renshen-Fuzi herb pair (RS-FZ) is often used in the clinical treatment of heart failure (HF) and has a remarkable therapeutic effect. However, the mechanism of RS-FZ for treating HF remains unclear. In our study, we explored the mechanism of RS-FZ for treating HF. Methods: Evaluation of RS-FZ efficacy by cardiovascular pharmacology. Moreover, Global metabolomics profiling of the serum was detected by UPLC-QTOF/MS. Multivariate statistics analyzed the specific serum metabolites and corresponding metabolic pathways. Combining serum metabolomics with network pharmacology, animal experiments screened and validated the critical targets of RS-FZ intervention in HF. Results: RS-FZ significantly ameliorated myocardial fibrosis, enhanced cardiac function, and reduced the serum HF marker (brain natriuretic peptide) level in rats with HF. Through topological analysis of the “Metabolite-Target-Component” interaction network, we found that 79 compounds of RS-FZ directly regulated the downstream specific serum metabolites by acting on four critical target proteins (CYP2D6, EPHX2, MAOB, and ENPP2). The immunohistochemistry results showed that RS-FZ observably improved the expression of CYP2D6 and ENPP2 proteins while decreasing the expression of EPHX2 and MAOB proteins dramatically. Conclusion: The integrated cardiovascular pharmacological assessment with serum metabolomics revealed that RS-FZ plays a crucial role in the treatment of HF by intervening in CYP2D6, EPHX2, MAOB, and ENPP2 target proteins. It provides a theoretical basis for RS-FZ for treating HF. Keywords: Panax ginseng C.A.Mey., Aconitum carmichaeli Debeaux, heart failure, combination mechanisms, cardiovascular pharmacological, serum metabolomics Introduction Heart failure (HF) is the final stage in the progression of most cardiovascular diseases, and it is a common, disabling, and fatal disease ([48]Dyck et al., 2019). Currently, the primary drugs used in the clinical treatment of HF are mainly diuretics, cardiotonic, vasodilators, and angiotensin-converting enzyme inhibitors. However, improper use of these therapeutic drugs often causes side effects such as arrhythmia and ion disturbance, thus affecting the curative effect. ([49]Zhang, 2015). Recent studies have shown that traditional Chinese medicine (TCM) treatment of HF has achieved a remarkable impact in alleviating symptoms, avoiding adverse drug reactions, and improving patients’ quality of life ([50]Wang et al., 2017). Renshen-Fuzi herb pair (RS-FZ) is made up of Panax ginseng C.A.Mey. (RS, Renshen in Chinese) and Aconitum carmichaeli Debeaux (FZ, Fuzi in Chinese). RS-FZ is a classic herb pair often used for HF treatment and contained in many medical preparations ([51]Huang et al., 2020). Furthermore, a large amount of pharmacological experimental studies have shown that both RS and FZ have an independent anti-HF effect ([52]Liu et al., 2021; [53]Wang et al., 2021), and the therapeutic effect was enhanced after the compatibility of RS and FZ by increasing myocardial contractility, inhibiting the expression of inflammatory factors and improving hemodynamics, etc ([54]Liu et al., 2020). However, the underlying mechanism of this herb pair is still unclear. Through the combination of serum metabolomics, network pharmacology, molecular biology, and other systematic pharmacological research methods ([55]Wei et al., 2018; [56]Guo et al., 2022), the potential target of RS-FZ treatment of HF was excavated, and the molecular mechanism of RS-FZ treatment of HF was revealed. At the same time, the critical targets were verified by immunohistochemistry. This study provided a theoretical basis for the clinical application of RS-FZ ([57]Figure 1). FIGURE 1. [58]FIGURE 1 [59]Open in a new tab The detailed flowchart of the study design. Materials and methods Chemicals and instruments RS and FZ (Danfupian) were purchased from Anhui People’s Chinese Herbal Pieces Co., Ltd (Anhui, China). The source and quality of the two medicinal materials were identified according to the 2020 edition of Chinese Pharmacopoeia. Rat brain natriuretic peptide (BNP) enzyme-linked immunosorbent assay (ELISA) Kit (No. E-EL-R0126c, Elabscience). Masson staining Kit (No. 20180804). Cytochrome P450 2D6 (CYP2D6), soluble epoxide hydrolase 2 (EPHX2), Monoamine oxidase-B (MAOB), and Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) antibodies were purchased from Wuhan Seville Biotechnology Co., Ltd. and Wuhan Sanying Biotechnology Co., Ltd. Vivid E9 Color Doppler ultrasound diagnostic apparatus (GE Norway). Water extract of RS-FZ preparation The ratio of RS-FZ (RS: FZ) in TCM prescriptions for the clinical treatment of HF was 1:2 ([60]Luo et al., 2008). In addition, according to the 2020 edition of Chinese Pharmacopoeia, the common human doses of RS and FZ were determined to be 5 g/d and 10 g/d, respectively. RS-FZ was prepared by extracting the mixture of these two herbs twice with water for 1 h. The extract was then decanted, filtered, and dried under reduced pressure. The final yield of dry extract was 18.2%. Animal experimentation Healthy SD male rats (SPF grade, 220 ± 10 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (SCXK 2016-0011). The Experimental Animal Ethics Committee approved animal feeding and experimental procedures of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine (No. YFYDW2017005). After 1 week of adaptation, 30 rats were randomly divided into the sham operation (Sham) group (n = 8) and the myocardial infarction group (n = 22). According to the previous study ([61]Zhu et al., 2015), rats prepared the myocardial infarction model by ligating the anterior descending (LAD) branch of the left coronary artery. Rats in the sham group were not ligated, and the operation was the same. The surviving rats, after modeling, were fed independently for 4 weeks. The left ventricular function of rats in the myocardial infarction group was evaluated by ultrasound, and the left ventricular ejection fraction (LVEF) < 50% was the successful preparation of the HF model (A. [62]Chen et al., 2015). Sixteen rats with successful modeling (Six failed in modeling) were randomly divided into the heart failure model (HF) group and RS-FZ group, with eight rats in each group. Through the dose conversion between human and rat body surface area ([63]Reagan-Shaw et al., 2008), rats in RS-FZ group were given dried extract (0.25 g/kg/d) by gavage for 4 weeks. At the same time, Sham and HF groups received the same volume of distilled water for 4 weeks. Effect of RZ-FZ on cardiac function and biochemical index in rats with heart failure Transthoracic echocardiography was used to detect and record the cardiac function of rats before, and 4 weeks after, RS-FZ was administered. From the long-axis parasternal view, the cardiac cycle was recorded by M-mode ultrasound. The rats were weighed and anesthetized with a 20% ethyl carbamate solution. The ventricular septal thickness (IVS), left ventricular end-diastolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured by echocardiography, and the LVEF and left ventricular short-axis shortening (LVFS) were calculated. Blood was taken from the abdominal aorta and serum was centrifuged to determine the content of brain natriuretic peptide (BNP) in serum by an enzyme-linked immunosorbent assay (ELISA). Then the rats were sacrificed, and the heart was quickly removed. After removing the capsule, atrium, and right ventricular tissue, the myocardial tissue in the ischemic risk area was placed in a −80°C refrigerator and 4% paraformaldehyde for standby. Left ventricular mass index (LVMI) (‰) = left ventricular mass (mg)/body weight (g) was calculated. Distribution of collagen in myocardial tissue Masson staining was used. In the same part of the left ventricle, cut about 2 mm thickness of myocardial tissue, 4% paraformaldehyde-fixed, paraffin-embedded tissue sections, staining procedures strictly following the kit instructions. Moreover, take pictures under the mirror. Immunohistochemistry Tissue sections were placed in EDTA antigen repair buffer (PH9.0) for antigen repair and washed three times with PBS (PH7.4). Then they were incubated in 3% hydrogen peroxide for 25 min and washed three times with PBS. Add 3% BSA to cover the tissue evenly and block for 30 min. Add PBS to slices in a certain proportion of the first antibody (CYP2D6, EPHX2, MAOB, and ENPP2) overnight. Then the slides were washed in PBS 3 times and covered with double-antibody. DAB chromogenic agent color, washing, hematoxylin re-staining nucleus, dehydration sealing, microscopic examination. The signal of immunohistochemical staining was analyzed with Image-Pro Plus 6.0 software. Preparation of serum samples for metabolomics analysis The collected rat blood was centrifuged at 4°C and 3,500 r/min for 10 min, and the serum was collected. Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) analyzed the serum samples after solvent extraction, protein precipitation, and high-speed centrifugation. Then, 10 μl serum samples were taken from the three groups and mixed as quality control (QC) samples to validate the stability of the UPLC-QTOF/MS system. Chromatography and mass spectrometry The UPLC-QTOF/MS system comprised an Acquity Ultra-Performance Liquid Chromatography (UPLC) system and a Xevo G2-XS QTOF-MS mass spectrometer (Waters, United States). The chromatographic separation conditions are as follows. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution: 0–6 min, 5%–45% B; 6–8 min, 45%–75% B; 8–12 min, 75%–85% B; 12–12.5 min, 85%–100% B; 12.5–16 min, 5% B. Column temperature 40°C, flow rate 0.3 ml/min, injection volume 3 μl. To enable high sensitivity, selectivity, speed, and precision, full information tandem mass spectrometry (MSE) technology was used in this experiment. An Electron Spray Ionization source was used for data collection in MSE continuum mode, a uniform high-frequency data acquisition mode. The accurate mass determination was performed using Leucine Enkephalin as the locking mass solution. The following electrospray source parameters were used: the electrospray capillary voltages were 2.5 kV (negative ionization mode) and 3.0 kV (positive ionization mode), the desolventizing gas temperature was 250°C, the cone gas flow was 50 L/h, the desolventizing flow was 800 L/h, the collision energy was 6 V, the mass range ranged from m/z 50-1,200. Data extraction and multivariate analysis The UPLC-QTOF/MS experimental data were collected by MassLynx software (v4.1) of Waters company and imported into Progenesis QI software (v2.4) for chromatographic peak alignment, peak extraction, and normalization, and the retention time (RT) was recorded. Normalized data matrix containing sample name, RT-m/z pair, and abundance were obtained by Progenesis QI software. Then, the normalized dataset was pre-processed using Excel software based on the “80% rule” to reduce the input of missing values and to remove features with relative standard deviations (RSDs) > 30% from all the samples ([64]Dunn et al., 2011). After editing, the normalized dataset was transformed (Log-transformation) and scaled (Pareto scaling), and analyzed by principal component analysis (PCA) and orthogonal partial least-squared discriminant analysis (OPLS-DA) using SIMCA-P 14.1 software. Variable important in the projection (VIP) and S-plot were used to predict the contribution of each data to the model. The variables with VIP >1 and |p(corr)| ≥0.5 in the OPLS-DA analysis were further evaluated with an independent sample t-test. Each feature’s formula and accurate mass were submitted to ChemSpider ([65]http://www.chemspider.com/). Furthermore, we conducted an identification analysis based on the Human Metabolome Database ([66]https://hmdb.ca/) ([67]Wishart et al., 2018) and Kyoto Encyclopedia of Genes and Genomes ([68]https://www.genome.jp/kegg/) ([69]Kanehisa and Goto, 2000) databases identification analysis. The specific metabolite with the most references was considered the