Verapamil hydrochloride was purchased from Shanghai Hefeng Pharmaceutical Co., Ltd. (Shanghai, China). Barium chloride was obtained from Chongqing Maoye Chemical Reagent Co., Ltd. Optimal LC grade acetonitrile was obtained from Merck (Merck, Darmstadt, Germany). Isopropanol, formic acid and nucleotides for metabolite analyses were purchased from Sigma-Aldrich (Spruce St., St Louis, MO, United States).

PRP was obtained from Shaanxi Xingshengde Co., Ltd. (Shannxi, China) and identified by Prof. Ji-Qing Bai. The specimens were deposited at Shaanxi University of Chinese Medicine (Shaanxi, China). PRP was prepared via the following steps: precise weighing of 0.8 g PRP in a 50 ml conical bottle, dilution with 25 ml methanol, weighing, soaking for 30 min, ultrasonic processing for 60 min, cooling, and weighing again. Next, the solution was brought up to the desired weight with methanol and filtered. A 15 ml aliquot was then absorbed by continuous filtration, evaporated to approximately 1 ml in an evaporation dish in a water bath, diluted with methanol in a 5 ml capacity bottle, shaken, evaporated in an evaporating pan in a water bath, and then dissolved in deionized water for animal experiments.

Zebrafish embryos 72 h post fertilization (hpf) were purchased from Shanghai Fish Biotechnology Co., Ltd. and maintained in Holt buffer solution (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 0.15 mM KH₂PO₄, 0.05 mM Na₂HPO₄, 0.7 mM NaHCO₃, 5% methylene blue; pH 7.5) on a 14 h light/10 h dark cycle at 28 ± 1°C and pH 7.5 ± 0.5.

Zebrafish embryos were randomized into six groups and placed on 48-well cell culture plates (ten embryos per group, five embryos per well): control, model (barium chloride sterile saline solution 2.1 µg/ml, 2 ml), positive control (model + 0.45 µg/ml verapamil hydrochloride), and three treatment groups (PRP-H: model + PRP extract 162.60 µg/ml, PRP-M: model + PRP extract 121.95 µg/ml, PRP-H: model + PRP extract 97.56 µg/ml). Technical details of the establishment protocol for the barium chloride-induced arrhythmia model establishment protocol and the PRP treatment dosage design are shown in Supplementary File S1. After 24 h of incubation with sterile saline solution of barium chloride, verapamil hydrochloride, or PRP extract, the morphology of the zebrafish embryo hearts was observed. All animal handling and experimental conditions were approved by the Laboratory Animal Care and Use Committee of Shaanxi University of Chinese Medicine.

The concentration of PRP extract was 9.6 mg/ml. The PRP extract solution was analysed on a Waters Acquity H-Class UltraPerformance LC (Waters, MA, USA)/tandem 5600⁺ triple-quadruple time-of-flight mass spectrometer (QTOF-MS) system (AB SCIEX, MA, United States) using a Waters BEH C₁₈ column (50 mm × 2.1 mm, 1.7 µm) with a gradient mobile phase of 0.1% formic acid aqueous solution (A) and 0.1% formic acid-acetonitrile solution (B) at a flow rate of 0.3 ml/min and a 30°C column temperature. The gradient program was as follows: 0–1 min, 2% B; 1–42 min, 2–100% B; 42–44 min, 100% B; 44–48 min, 100–2% B; 48–50 min, 2% B. The full scan and MS/MS experiments were performed under positive and negative modes by using an electrospray ionization (ESI) ion source. Information-dependent data (IDA) acquisition mode was used to switch automatically between MS and MS/MS acquisition with the following parameters: ion spray voltage floating: +5500 or −4500 V, declustering potential (DP): 40 V, ion source gas 1 and ion source gas 2 both set as nitrogen: 50 psi, curtain gas: 35 psi, source temperature: 500°C. The scanning range of parent ions (TOF-MS) was 100–2000 m/z. The eight strongest peaks exceeding 100 cps were collected by MS², and the scanning range was 100–2000 m/z. Data were acquired using Analyst TF software (version 1.7.1, AB SCIEX). Component identification was performed by using PeakView™ software (version 2.2, AB SCIEX) and MasterView™ software (version 1.1, AB SCIEX) coupled with the TCM library database (version 1.0, AB SCIEX).

According to the UPLC-TOF-MS/MS identification results, the screening for active components of PRP and the PRP target prediction for arrhythmia treatment were performed by network pharmacology analysis on ETCM (http://www.tcmip.cn/ETCM/index.php/Home/Index/), BATMAN-TCM (http://bionet.ncpsb.org.cn/batman-tcm/index.php) , Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://tcmspw.com/tcmsp.php), Therapeutic Targets Database (TTD, http://db.idrblab.net/ttd/) and Genecards (https://www.genecards.org/) (Liu et al., 2016).

Common targets were identified between the screened active component-related targets and disease targets (‘arrhythmia’) by VENNY 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/), and a relationship among these targets was generated by the STRING 11.0 database (https://string-db.org/). Then, the component-disease-target association network was constructed by Cytoscape software (version 3.7.2). Finally, the enrichment of Gene Ontology (GO) functions and pathways of selected key targets were examined by using the clusterProfiler package of R software (version 3.6.1) for RPR treatment function prediction (Yu et al., 2012).

After incubation with Holt buffer solution from 60 to 72 hpf, zebrafish embryos were randomly selected (n = 10 each group) for the observation of cardiac morphology. The zebrafish embryos were fixed with 3% methylcellulose (AlfaAesar, Shanghai, China), and cardiac morphology observations (pericardial oedema, abnormal circulation, thrombosis, haemorrhage, etc.) and sinus venosus-bulbus arteriosus (SV-BA) distance measurement for heart tube looping quantification were performed on an ABX51 electron microscope (Olympus, Tokyo, Japan), and images were acquired under a DD71 digital camera (Olympus, Tokyo, Japan). The pericardial congestion area and the SV-BA straight line distance were calculated with ImageJ software (version 1.8.0, NIH, USA). Each experimental treatment was repeated three times (Lin et al., 2007; Gut et al., 2017; Echeazarra et al., 2020).

Acridine orange (AO) is a nucleic acid-selective metachromatic dye: AO-stained apoptotic cells show yellow-green fragments, while normal cells show uniform green or yellow-green fluorescence (Antonio et al., 2017). To evaluate the cardiomyocyte apoptosis of zebrafish embryos after barium sodium exposure and rescue treatment, five live zebrafish embryos at 96 hpf from each group were randomly selected and washed with phosphate-buffered saline (PBS, pH 7.4) to remove exposure residues and then incubated in 2.5 mg/L AO solution at 28°C in the dark for 30 min. Next, the embryos were rinsed thoroughly in PBS twice and fixed in 3% methyl cellulose solution. Images of the dorsal side of fixed zebrafish were acquired under a BX51 push-around-type electronic fluorescence microscope (Tokyo, Japan) coupled with a DD71 digital camera under a green light source. The average fluorescence density of the particle number was calculated by Image J software (Jin et al., 2019).

To analyse the targeted metabolomic selection results, potential metabolite markers were quantitated by comparison to the areas of the peaks for the external standards (method validation is shown in Supplementary File S2, Supplementary Table S1).

After treatment at 96 hpf, zebrafish embryos were randomly selected (n = 30 each group) for qRT-PCR analysis. Thirty milligrams of zebrafish embryos was homogenized in TRIzol reagent (Servicebio Technology Co., Ltd., Wuhan, China) to extract total RNA. The extracted RNA was used as a template, and cDNA was synthesized by reverse transcription with appropriate primers according to the instructions of the Servicebio® RT First Strand cDNA Synthesis Kit (G3330, Servicebio, Wuhan, China). qRT-PCR was performed in an ABI 7900HT Fast Real-Time PCR system (Bio-Rad Bole Gradient, California, America). The primer sequences were as follows (ref. NM_001128584.1, 242 bp, 60°C, Servicebio): TTC​TGA​CCC​AAA​GTT​CCA​TCC​T (forward) and CTC​AAA​CTG​GCA​GGT​GAC​GAT (reverse). Levels of the Adenosine A1 Receptor (ADORA1) mRNAs were normalized to the GAPDH mRNA level and determined using the 2^−ΔΔCt method, and all reactions were performed in triplicate.

Statistical Program for the Social Sciences (SPSS) 26.0 was used for statistical analysis. All the data are expressed as the mean ± SD. One-way analysis of variance (ANOVA) was used to compare the data among the groups. GraphPad Prism 8 software was used to generate all graphics, and p < 0.05 was considered statistically significant.

Under the optimized chromatographic and mass spectrometry conditions, UPLC/Q-TOF-MS in positive and negative detection modes was used to analyse the PRP extracts (Figures 1A,B). The results showed that 31 compounds were identified in positive ion mode and 5 in negative ion mode (Supplementary File S3, Supplementary Table S2). According to the molecular weight, retention time, and MS/MS data, four compounds were identified: pachymic acid, glycyrrhetinic acid, oleanolic acid and adenosine (Figures 1C–F) (Supplementary File S3, Supplementary Table S3) (Qian et al., 2018).

By combining UPLC/Q-TOF-MS identification and database search results, 11 compounds were identified and selected (Table 1). For these compounds, 606 putative targets were predicted from the ETCM, TCMSP and Batman-TCM databases (Supplementary File S4, Supplementary Table S4). A total of 316 targets related to arrhythmia were obtained against the ETCM, TTD, and GeneCards databases (Supplementary File S4, Supplementary Table S5). In Figure 2A, we first extracted 58 intersection targets from the overlaps between the disease gene set and PRP target sets (Supplementary File S4, Supplementary Table S6). In the following study, the relationships of these 58 targets were connected as a PPI network for importance evaluation (Supplementary File S4, Supplementary Table S7). As shown in Figure 2B, the PPI k-means classification results suggested that 25 genes, iADORA1, ADORA2B, ADRA1A, ADRA1B, and ADRA1D, play a more important role in arrhythmia pathology and PRP treatment.

To further explore PRP multicomponent and multitarget therapy functions, all 58 targets of the PPI network were mapped to “component-disease” connections, and then the “component-disease-target” network of PRP therapy was constructed by Cytoscape software (Figure 2C). This network included 70 nodes, which illustrated potential interplay among 1 disease type, 11 chemical components, and 58 targets. Since the node size is proportional to the importance of nodes involved in the pathway, 7 components involved in the therapy pathway, including adenosine, palmitic acid, caprylic acid, lauric acid, and adenine. In addition, 58 targets, such as ADORA1, ADORA2B, and CACNA1B, were considered potential therapeutic targets (Supplementary File S4, Supplementary Table S8).

GO analysis revealed that the targets were significantly enriched in 581 pathways for PRP therapy, specifically “complex ion channels and transmembrane transporters”, “ion channel activity and metal ion transmembrane transport protein activity”, and “myocardial contraction, the regulation of myocardial contractility and the regulation of blood circulation” (Figures 2D–F, Supplementary File S4, Supplementary Table S9). Similarly, KEGG pathway enrichment analysis showed that the targets were significantly enriched in 193 pathways for PRP therapy, specifically the “cGMP-PKG signalling pathway” and “calcium ion signalling pathway” (Figure 2G, Supplementary File S4, Supplementary Table S10). As the GO and KEGG analyses suggested that most adenosine receptors (ADORAs) under PRP treatment were involved in arrhythmia-related pathways, these 58 targets were used to perform metabolomic joint pathway analysis.

In the morphological phenotype observation, PRP extracts showed a significant and dose-dependent rescue function against barium chloride-induced morphological phenotypes (Figure 3A). Calculating the cardiac congestion area in zebrafish showed that the cardiac congestion area was 605.42% larger in the zebrafish model group than in the control group. However, the cardiac congestion areas in the positive control group, PRP-H group, PRP-M group and PRP-L group showed decreases of 81.44, 78.05, 74.22 and 47.54%, respectively, compared to that in the model group. These findings further proved that PRP can lessen the degree of cardiac congestion induced by barium chloride in zebrafish (Figure 3B, Supplementary File S5, Supplementary Tables S11, S11-1, S11-2). Compared with the control group, the model group zebrafish showed a 58.10% increase in the SV-BA distance. Compared to the model group, the SV-BA distance in the verapamil exposure group led to a significant 31.30% decrease, while the PRP-H, PRP-M, and PRP-L treatment groups showed 27.83, 25.96, and 16.82% decreases, respectively (Figure 3D, Supplementary File S5, Supplementary Tables S12, S12-1, S12-2).

After AO staining, compared with the control group, a large number of apoptotic cells (green debris particles) were found in zebrafish embryo hearts, and the average fluorescence density was 51.18% higher than that in the control group. At 96 hpf, the numbers of apoptotic cells in zebrafish embryo hearts were significantly and dose-dependently decreased in the PRP treatment group. Compared to the model group, the average fluorescence density of the PRP-H, PRP-M and PRP-L groups showed declines of 25.13, 22.20 and 14.50%, respectively. These results indicated that PRP could rescue barium chloride-induced apoptosis in the hearts of zebrafish embryos in a concentration-dependent manner (Figures 3C,E, Supplementary File S5, Supplementary Tables S13, S13-1, S13-2).

PLS analysis of the untargeted metabolome showed a distinct change in metabolite profiles between the model embryos and control embryos, represented by the substantial metabolomic perturbation of the cardiac system observed in these embryos (three experiments, Figures 4A,B). Then, significant features were selected based on a selected criterion with an adjusted p-value cut-off of <0.05. Statistical analysis of the metabolomics data from the barium chloride-treated group and the control group indicated significant differences in ions between these two groups, as shown in the loading plot (Figures 4C,D). The VIP values were also used to calculate the feature importance by the cut-off value of weight VIP > 1 (Figures 4E,F). Following the selection flow, a total of 17 potential differential metabolites were identified in positive and negative ion modes (Table 2), including amino acid components such as L-tyrosine, L-valine, L-tryptophan, indoleacrylic acid, and L-histidine and choline components such as glycerophosphocholine, L-acetylcarnitine and adenosine triphosphate.

Furthermore, to clarify whether PRP administration can regulate barium chloride-induced metabolite alterations, comparisons among the control group, model group, positive control group and PRP-H, PRP-M and PRP-L groups were analysed by using the MetaboAnalyst database. As shown in Figure 4G, the PCA score plot showed obvious sample clusters among the control, model, positive control and PRP-treated groups, where the PRP-H group and PRP-M group were close to the control group but separated from the model group. The results indicated that PRP treatment could improve barium chloride-induced metabolomic profile imbalance in zebrafish embryos.

To further investigate the roles of the enriched metabolite pathways and dissect which of the screened targets contributed to these pathways, the candidate targets from the network pharmacology selection were mapped to 17 differential endometabolites by joint-pathway analysis on the MetaboAnalyst database (Figure 4H). Gene-metabolic pathway correlation analyses showed that the “cGMP-PKG signalling pathway” (p < 0.001) was highly enriched among the 72 enriched metabolic pathways. In this pathway, ADORA1 is a highly ranked protein that acts as an adenosine receptor to reduce cardiomyocyte calcium overload in the occurrence of arrhythmia diseases, and adenosine and cGMP are highly ranked metabolites (Szentmiklosi et al., 2015; Deb et al., 2019).

Next, by quantifying the levels of adenosine and cGMP in embryo samples using LC-MS in multiple reaction monitoring (MRM) mode, barium chloride induced a significant decreasing trend in adenosine by 63.30% and cGMP by 78.86% compared with the control ones. PRP therapy significantly reversed these changes: the adenosine and cGMP levels in the PRP-H group were increased by 75.84 and 231.35%, in the PRP-M group were increased by 66.71 and 159.57%, and in the PRP-L group were increased by 41.71 and 78.86%, respectively (Figures 5A,B, Supplementary File S6, Supplementary Tables S14–S15-2). ADORA1 expression was determined using qRT-PCR assays to examine how PRP treatment influenced barium chloride-induced arrhythmia in zebrafish embryos (Figure 5C, Supplementary File S6, Supplementary Tables S16, S16-1, S16-2). Interestingly, ADORA1 expression was significantly decreased by 81.65% (p < 0.01) in the model group. PRP therapy restored ADORA1 expression at three dosages, with increases of 320.53, 261.96, 184.56 and 154.58%, respectively. The results showed that PRP could increase the expression level of ADORA1 in the cGMP-PKG signalling pathway. These findings indicated that barium chloride disturbed the cGMP-PKG signalling pathway, which resulted in arrhythmia. This was attributed to ADORA1 downregulation associated with adenosine and cGMP decline and PRP treatment rescued these trends towards an imbalance.