Abstract Cardiac hypertrophy is a cardiac structural remodeling and dysfunction resulting from chronic hypertension and is an independent risk factor for cardiovascular morbidity and mortality. Oxymatrine (OMT), an alkaloid extracted from the traditional Chinese medicine Sophora flavescens, can ameliorate cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unclear. In this study, we investigated the underlying mechanism of OMT on cardiac hypertrophy in spontaneously hypertensive rats (SHRs). Echocardiography was used to assess cardiac function of SHRs. Wheat germ agglutinin (WGA) and Masson staining were employed to evaluate the effects of OMT on cardiac fibrosis and cardiomyocyte hypertrophy in SHRs. Transcriptomics and metabolomics were performed to explore the underlying mechanisms of OMT’s improvement of cardiac hypertrophy. The results were further verified by RT-qPCR, immunohistochemistry, and ELISA. The results show that OMT significantly alleviates myocardial hypertrophy and improves myocardial remodeling in SHRs, as evidenced by reduced left ventricular wall thickness, myocardial enlargement and collagen deposition. Transcriptomic analysis revealed that OMT reversed the expression of 10 genes associated with linoleic acid/arachidonic acid metabolism, cytochrome P450, steroid hormone biosynthesis, and myocardial adrenergic signaling. Metabolomic analysis indicated that OMT reversed the levels of 16 metabolites related to steroid hormone biosynthesis, aldosterone synthesis and secretion, and tryptophan metabolism. Integrated analysis revealed that the gene Cyp2e1 and the metabolite desoxycortone (DOC) were enriched in overlapping transcriptomic and metabolomic KEGG pathways, and both were reversed by OMT treatment. Our study underscores the potential molecular and metabolic mechanisms of OMT against pressure overload-induced myocardial hypertrophy and provides theoretical support for its exploration in other indications. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-15930-9. Keywords: Oxymatrine, Myocardial hypertrophy, Metabolome, Transcriptome, Rat Subject terms: RNA sequencing, Metabolomics, Cardiac hypertrophy Background Myocardial hypertrophy is a compensatory response of the cardiovascular system to long-term pressure overload or pathological stimuli, characterized by an increase in cardiomyocyte size, thickening of the myocardial wall, and reduction of the heart chamber^[34]1. Prolonged myocardial hypertrophy can eventually lead to heart failure, arrhythmias, and other serious cardiovascular events^[35]2. The mechanisms underlying myocardial hypertrophy are highly complex, involving multiple factors such as cellular signal transduction, gene expression regulation, and metabolic dysfunction^[36]3. Therefore, understanding the molecular and metabolic mechanisms of myocardial hypertrophy is crucial for formulating effective therapeutic strategies. Oxymatrine (OMT), an alkaloid extracted from the traditional Chinese medicine Sophora flavescens, has garnered increasing attention due to its broad pharmacological activities, particularly its cardiovascular protective effects. OMT has been demonstrated to exert beneficial effects in various cardiovascular disease models through antioxidant, anti-inflammatory, and cell signaling modulation activities^[37]4. The study found that OMT may play a role in diabetic cardiomyopathy by regulating metabolic imbalance^[38]5. OMT protect against isoproterenol-induced cardiac hypertrophy and heart failure in rats by modulating metabolic pathways^[39]6. These findings suggest the important role of OMT in metabolic regulation. Previous research suggested that OMT prevents ventricular remodeling in spontaneously hypertensive rats (SHRs)^[40]7. However, the precise molecular mechanisms and metabolic regulatory effects of oxymatrine in myocardial hypertrophy remain unexplored. Metabolomics offers new insights into metabolic disturbances, enabling comprehensive capture of the changes in metabolites under pathological conditions^[41]8, thus helping to elucidate the metabolic dynamics in myocardial hypertrophy. Furthermore, metabolic abnormalities interact closely with gene expression, and transcriptomics can reveal global gene expression changes associated with myocardial hypertrophy. The integration of these “omics” tools provides unprecedented opportunities to unravel the molecular basis of myocardial hypertrophy. This study aims to evaluate the effects of oxymatrine on myocardial hypertrophy in SHRs by assessing changes in myocardial-associated metabolic profiles and its regulatory role in gene expression through the integration of metabolomics and transcriptomics. Our findings will provide new insights into the potential mechanisms by which oxymatrine alleviates myocardial hypertrophy, offering a theoretical foundation for the development of targeted therapies for cardiovascular diseases. Materials and methods Animals The male Wistar-Kyoto (WKY) rats (12-week-old, 260–280 g) and SHRs (12-week-old, 260–280 g) were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China). All rats were housed under room temperature and standard humidity (50 ± 5%) with a 12 h day/night circle with laboratory chow and water ad libitum. All procedures performed in this study were in accordance with national animal research regulations, and all animal experimental protocols complied with the ARRIVE guidelines and were approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (No: 2021347). The rats were randomly divided into three groups of 6 each group: WKY group, SHR group, and SHR + OMT group. Starting at 12 weeks of age, the rats in the SHR + OMT group were gavaged with OMT (No. [42]B21470, Shanghai yuanye Bio-Technology Co., Ltd, Shanghai, China) at a dose of 50 mg/kg/day for 12 weeks, while the other rats were gavaged with normal saline. The selected dose was based on a previous study^[43]7, which reported effective results with 30 and 60 mg/kg/day; thus, 50 mg/kg/day was chosen as a moderate and potentially effective dose for long-term administration. The systolic (SBP) and diastolic blood pressure (DBP) were measured at the endpoint using a non-invasive tail-cuff method (BP-2000, Visitech Systems, Apex, NC, USA). The body weight was measured at 24 weeks of age, prior to sacrifice. Echocardiography The VINNO 6 ultrasonic diagnostic instrument (VINNO, Suzhou, China) equipped with a X6-16L probe (frequency 18-MHz) was used for the echocardiography analysis. Briefly, rats were anaesthetized with inhalation of 2% isoflurane (RWD Life Science, Shenzhen, China) and restrained supine on a warm platform (37 °C). 2D M-mode cardiac ventricular images were obtained from the parasternal long-axis view. Then the heart rate, left ventricle (LV) internal dimension in systole (LVIDs) and diastole (LVIDd), ejection fraction (EF) and fractional shortening (FS), LV anterior wall thicknesses at end-systole (LVAWs) and end-diastole (LVAWd), LV posterior wall thicknesses at end-systole (LVPWs) and end-diastole (LVPWd) were measured. All measurements were averaged over at least three cardiac cycles per animal. All rats were then sacrificed by intraperitoneally injecting a lethal dose of pentobarbital sodium (100 mg/kg). The left ventricles were rapidly dissected and stored at -80 °C for further analysis. RNA sequencing Total RNA was extracted from left ventricle of WKY rats and SHRs using Trizol reagent (No. 15596018, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The RNA concentration, quality and integrity were determined using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The sequencing library construction, RNA-seq, read mapping and transcript quantification were performed by BGI (Shenzhen, Guangdong, China) with the BGISEQ-500 sequencing platform (BGI). After enrichment of mRNA with oligo (dT) magnetic beads, the mRNA was fragmented and reverse-transcribed into double-stranded cDNA using the RevertAid RT Kit (No. K1691, Thermo Fisher Scientific). The cDNA was amplified by PCR after adding poly A tails and RNA adapters, and products were purified and dissolved with Agencourt® AMPure® XP beads (Beckman Coulter, Inc., Brea, CA, USA) and EB solution (10 mM Tris–Cl, pH 8.5). Then the products were qualified using the Agilent 2100 bioanalyzer, then heat-denatured into single-stranded DNA, followed by circularization to obtain single-stranded circle DNA library. The final library was amplified with Φ29 DNA polymerase to make DNA nanoballs (DNBs), and then the DNBs were loaded into a patterned nanoarray to generate 50 single-end base reads on the BGISEQ-500 sequencing platform. Transcriptomic data analysis The Dr. Tom analysis system (BGI) was used to conduct the transcriptomic data analysis. The raw data were filtered with SOAPnuke (v1.5.2) to obtain the clean reads. The clean reads were first mapped to the reference genome using HISAT2 (v2.0.4), and then aligned to the reference coding gene set using Bowtie2 (v2.2.5). The Fragments per Kilobase per Million (FPKM) method was used to calculate the expression level of gene. The differentially expressed genes (DEGs) were filtered using DESeq2 (v1.4.5) with adjusted P value (Q-value) ≤ 0.05 and log2 (fold change [FC]) ≥ 1). The Kyoto Encyclopedia of Genes and Genomes pathway (KEGG, [44]https://www.kegg.jp/)^[45]9,[46]10 enrichment analysis of DEGs was performed to gain insight into the biological function by Phyper ([47]https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on the Hypergeometric test. Significantly enriched KEGG pathways were identified by the thresholds Q-value < 0.05. The heat map, volcano plot and venn plot were generated using the BGI online tool (Dr.Tom). Metabolomic analysis Untargeted metabolome analysis of left ventricle was performed by BGI. Briefly, 25 mg of left ventricular was mixed with a pre-cooled extraction solvent (methanol: Acetonitrile: Water, 2: 2: 1, v/v/v), homogenized, and precipitated at − 20 °C for 2 h. The sample was then centrifuged at 25,000 g and 4 °C, and the supernatant was collected, freeze-dried, reconstituted in 50% methanol, and centrifuged again to obtain the final supernatant for analysis. After metabolite extraction, the separation and detection of metabolites were performed using a Waters 2777C UPLC system (Waters, USA) coupled with a Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific) to analyze the metabolic profiles in both ESI positive and negative ion modes. Using Compound Discoverer 3.3 (Thermo Fisher Scientific) software, combined with the BGI Metabolome Database (BMDB), mzCloud database, and ChemSpider online database, for analysis and metabolite identification. Use metaX for data preprocessing to remove compounds with a coefficient of variation (CV) > 30% in the relative peak area of the quality control (QC) samples. The classification and functional analysis of identified metabolites through The Human Metabolome Database (HMDB) and KEGG database. The differential abundant metabolites (DAMs) were filtered with the thresholds P value ≤ 0.05 and log2 (FC) ≥ 1.2. The KEGG pathway enrichment analysis of DAMs was performed to gain insight into the biological function. Significantly enriched KEGG pathways were identified by the thresholds P value < 0.05. The heat map, volcano plot, Venn plot and pathway score map were generated using the BGI online tool (Dr.Tom). The Network Analysis in MetaboAnalyst 6.0 ([48]https://www.metaboanalyst.ca/MetaboAnalyst/) was used to predict the related genes of overlapping metabolites in SHR and SHR + OMT. Integrated analysis of metabolomic and transcriptomic The canonical correlation analysis (CCA) was performed using the mixOmics package ([49]http://mixomics.org/) to calculate the correlation coefficients (r) between the top 20 metabolites and the transcriptomics indicators. The correlation chord diagram was plotted for the metabolites and associated transcriptomics indicators with the correlation coefficients |r|> 0.8 and P < 0.05. The Venn diagram for KEGG pathway ([50]https://www.kegg.jp/)^[51]9,[52]10 enrichment was generated using the online tool Xiantao Academic ([53]https://www.xiantaozi.com/) to identify overlapping key pathways between DAMs and DEGs. RNA extraction and real-time quantitative PCR (RT-qPCR) Total RNA was extracted from left ventricles of WKY rats and SHRs using Trizol reagent according to the manufacturer’s protocol. After gDNA removal, the RNA (1 μg) was reversely transcribed into cDNA using 5×All-in-One RT MasterMix with gDNA Removal (No. G592, Applied Biological Materials Inc., Richmond, BC, Canada) according to the manufacturer’s protocol. Target genes were then amplified by RT-qPCR in a CFXConnect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using BlasTaq™ 2 × qPCR MasterMix (No. G891; Applied Biological Materials Inc.), in accordance to the manufacturer’s instructions. The resultant data was carried out by the 2^−ΔΔCt method and normalized to β-actin mRNA expression level. All primers were designed using Primer-BLAST ([54]https://www.ncbi.nlm.nih.gov/). Details of all primers are listed in Table [55]1. Table 1. Sequence of rat primers used in RT-qPCR experiment. Gene Accession No Primer sequence Product length ANP [56]NM_012612.2 F: 5′-ACACAGCTTGGTCGCATTGCCA-3′ 215 bp R: 5′-CGTCTGTCCGTGGTGCTGAAGTTT-3′ BNP [57]NM_031545.1 F: 5′-ACAATCCACGATGCAGAAGCT-3′ 87 bp R: 5′-GGGCCTTGGTCCTTTGAGA-3′ CYP2e1 [58]NM_031543.2 F: 5′-TGGCTACAAGGCTGTCAAGGAG-3′ 102 bp R: 5′-ATAATCCCCTTGTTCTTGTACTCC-3′ GAPDH [59]NM_017008.4 F: 5′-CTGGAGAAACCTGCCAAGTATG-3′ 138 bp R: 5′-GGTGGAAGAATGGGAGTTGCT-3′ [60]Open in a new tab F, forward primer; R, reverse primer Histopathology The left ventricular myocardial tissues were fixed with 4% neutral buffered formalin and cut into 3-μm-thick sections by Shandon™ Finesse™ 325 Microtomes (Thermo Fisher Scientific). Wheat Germ Agglutinin (WGA) staining was performed to examine the cross-sectional areas of cardiomyocytes. Briefly, after being deparaffinized and rehydrated, the heart sections were incubated with FITC-conjugated WGA (No. L4895, Sigma-Aldrich, Inc., St. Louis, MO, USA) at 5 μg/ml in PBS (No. 21-040-CV, Corning, New York, USA) for 1 h at 37 °C in the dark. Masson staining was carried out using Masson’s Trichrome Stain solution (G1006, Wuhan servicebio technology CO., LTD, Wuhan, Hubei, China) to evaluate the cardiac fibrosis. The staining was photographed under Nikon ECLIPSE Ti microscope equipped with a DS-Fi1c-U3 camera (Nikon, Tokyo, Japan) and evaluated by Image-pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). For immunostaining of Cyp2e1, after being deparaffinized and rehydrated, the heart sections were antigen retrievable in 0.01 M citrate buffer (pH 6.0) at 95 °C for 10 min. The sections were blocked with 10% goat serum at room temperature for 1 h, then incubated with primary anti-Cyp2e1 (1:100, [61]GB115524, Servicebio) at 4 °C overnight. After washing three times in PBS, the sections were incubated with GTVision™ III Detection System Mouse & Rabbit Kit (HRP/DAB) (GeneTech, Shanghai, China) to yield a brown crystalline antigen–antibody complex product. All images were captured using a microscope (DM4B, Leica, Germany) equipped with a camera (LAS X, Leica) and analyzed with Image-pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Enzyme linked immunosorbent assay (ELISA) The levels of deoxycorticosterone in serum and left ventricle were measured using a rat deoxycorticosterone ELISA kit (JRX393496, RUIXIN BIOTECH, Quanzhou, Fujian, China) according to the manufacturer’s instructions. Absorbance was read at 450 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific). Statistical analysis The Shapiro–Wilk normality tests of all data were conducted with the SPSS statistical software. The data were presented as means ± SEM. Statistical evaluation was conducted using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered significant. Results Oxymatrine alleviates myocardial hypertrophy in SHR Blood pressure measurement, echocardiography, Masson staining, and WGA staining were performed to evaluate the effect of OMT on blood pressure as well as cardiac structural and functional remodeling in SHRs. Compared to WKY rats, SHRs exhibited significantly elevated SBP, DBP, and mean arterial pressure (MAP) (Fig. [62]1a), which were markedly reduced by OMT treatment. As shown in Fig. [63]1b, SHRs developed concentric cardiac hypertrophy, evidenced by increased LV mass, LV mass/body weight ratio (Fig. [64]1e), LV wall thickness (LVAW and LVPW in both diastole and systole) (Fig. [65]1c), along with decreased LV internal dimensions (LVIDd and LVIDs) (Fig. [66]1d). Heart rate was also elevated in SHRs (Fig. [67]1f), while EF% and FS% remained unchanged (Fig. [68]1g), suggesting preserved systolic function. OMT treatment partially improved cardiac remodeling, as evidenced by reduced LV wall thickness, without significantly altering heart rate. Masson staining (Fig. [69]1h) revealed substantial collagen fiber proliferation in the myocardial interstitium and perivascular space in SHRs, with the most pronounced accumulation around perivascular space. While OMT treatment reduced collagen fiber deposition in SHRs. Additionally, WGA staining (Fig. [70]1i) demonstrated that OMT decreased the enlarged cross-sectional area of cardiomyocytes in SHRs, alleviating myocardial cell hypertrophy. These findings suggest that OMT improves cardiac structural remodeling. Fig. 1. [71]Fig. 1 [72]Open in a new tab Effect of OMT on myocardial hypertrophy of SHRs. (a) Effect of OMT on the blood pressure of SHRs. (b) Representative images and measurement parameters (c–g) of M-mode echocardiography from each group. The red and blue double-headed arrows indicate LVAWs and LVAWd, respectively, while the white and black double-headed arrows represent LVPWs and LVPWd, respectively. Scale bar = 0.5 cm. (h) Masson staining of myocardial tissue (left); Scale bar = 100 μm. Quantification of myocardial fibrosis area (right). (i) WGA staining of myocardial tissue (left); Scale bar = 100 μm. Quantification of cardiomyocyte cross-sectional area in sections of hearts (right). ****P < 0.001; ***P < 0.005; **P < 0.01; *P < 0.05. EF: ejection fraction; FS: fractional shortening; DBP: Diastolic blood pressure; LV mass: Left ventricular mass; LVAWs: Left ventricular anterior wall thicknesses at end-systole; LVAWd: Left ventricular anterior wall thicknesses at end-diastole; LVPWs: Left ventricular posterior wall thicknesses at end-systole; LVPWd: Left ventricular posterior wall thicknesses at end-diastole; LVIDs: Left ventricle internal dimension in systole; LVIDd: Left ventricle internal dimension in diastole; MAP: Mean arterial pressure; OMT: Oxymatrine; SBP: Systolic blood pressure; SHR: Spontaneously hypertensive rat; WGA: Wheat Germ Agglutinin. Transcriptomic characteristics of myocardial tissue in oxymatrine-treated SHRs RNA-seq was conducted to explore the transcriptomic characteristics of the left ventricular myocardial tissue in WKY rats and SHRs (with or without OMT). Compared to WKY rats, a total of 465 DEGs were identified in the left ventricle of SHRs, consisting of 225 upregulated and 240 downregulated genes. Additionally, 35 DEGs were found between OMT-treated SHRs and untreated SHRs, including 18 upregulated and 17 downregulated genes (Fig. [73]2a,b,c). The expression clustering heatmap displays the expression profiles of DEGs across different samples (Fig. [74]2b). Further analysis indicated that OMT treatment reversed 10 DEGs in SHRs, including 6 upregulated and 4 downregulated genes (Fig. [75]2a). These genes reversed by OMT were displayed in Fig. [76]2c, Table [77]2, and Supplementary Figure [78]S1 online. Fig. 2. [79]Fig. 2 [80]Open in a new tab Transcriptomic landscape profile in the left ventricle. (a) Venn diagram of overlapping DEGs. (b) Cluster heatmap of the DEGs in the rat left ventricle of WKY, SHR and SHR + OMT groups. Red represents up-regulated genes, blue represents down-regulated genes. (c) Volcano plot of DEGs between SHRs and WKY rats (left), and between OMT-treated SHRs and untreated SHRs (right), highlighting significant up- (red) and downregulated (green) genes. Genes marked by arrows are DEGs between SHR and WKY rats whose expression is reversed in OMT-treated SHR. (d) KEGG pathway enrichment analysis of DEGs in SHRs vs. WKY rats (left), SHR + OMT vs. SHRs (middle), and Overlapping DEGs (right). DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; OMT: Oxymatrine; SHRs: Spontaneously hypertensive rats; WKY: Wistar-Kyoto rats. Table 2. The reversed differential expression genes. Gene Symbol SHR vs. WKY Regulate SHR + OM vs. SHR Regulate log2FC Q-value log2FC Q-value LOC100911615 22.240768 3.90E−07 Up − 22.972592 4.14E−09 Down Tbx15 3.061959 3.47E−07 Up − 2.23946 0.001288 Down Kcne1 2.551242 4.46E−08 Up − 1.26379 0.022513 Down LOC102556672 2.214051 0.046338 Up − 3.25911 0.009577 Down Cpa6 1.988415 2.33E−06 Up − 1.42129 0.009641 Down Dnah7 1.424969 3.84E−04 Up − 1.30334 0.016695 Down Cyp2e1 − 1.66147 2.40E−08 Down 1.35812 1.28E−06 Up Sostdc1 − 1.57962 0.004339 Down 1.762168 0.001783 Up Oasl − 1.40071 2.35E−07 Down 1.109062 0.001297 Up Ddn − 1.37959 7.11E-09 Down 1.076597 5.94E-06 Up [81]Open in a new tab KEGG pathway analysis (Fig. [82]2d, left) indicated that DEGs of the left ventricle between SHR and WKY group mainly enriched in biological processes related to immune response and inflammation (including antigen processing and presentation, phagosome, endocytosis, cell adhesion molecules, reactive oxygen species), metabolism and cellular function (including arachidonic acid metabolism, metabolism of xenobiotics by cytochrome P450, cellular senescence and folate biosynthesis). The prominent pathways between the SHR + OMT and SHR groups (Fig. [83]2d, middle) included circadian rhythm, adrenergic signaling in cardiomyocytes, proximal tubule bicarbonate reclamation, cAMP signaling pathway, aldosterone-regulated sodium reabsorption, linoleic acid metabolism, carbohydrate digestion and absorption, mineral absorption, endocrine and other factor-regulated calcium reabsorption, and drug metabolism via cytochrome P450. KEGG pathway enrichment analysis of overlapping DEGs (as shown in Fig. [84]2d, right and Table [85]2) identified key pathways, including linoleic acid metabolism, cytochrome P450 drug metabolism and xenobiotic metabolism, steroid hormone biosynthesis, and arachidonic acid metabolism. These results suggest that OMT may exert its biological function in alleviating myocardial hypertrophy by influencing the enrichment pathways of the overlapping genes. Metabolomic characteristics of myocardial tissue in oxymatrine-treated SHRs A total of 870 metabolites were identified through metabolomic analysis, which were classified into 28 categories based on the HMDB database (Fig. [86]3a). The largest group was lipids, making up 30.48% of the total, followed by amino acids, peptides, and their analogues (12.48%). Furthermore, the identified metabolites were categorized into 12 functional groups based on KEGG pathways, with amino acid metabolism and lipid metabolism being the most prominent, accounting for 23.03% and 20.39%, respectively (Fig. [87]3b). Fig. 3. [88]Fig. 3 [89]Open in a new tab Metabolomic landscape profile in the left ventricle. (a) HMDB categorization of identified metabolites in the dataset. (b) KEGG pathway categorization of identified metabolites in the dataset. (c) Venn diagram of overlapping DAMs. (d) Volcano plot of DAMs between SHRs and WKY rats (left), and between OMT-treated SHRs and untreated SHRs (right), highlighting significant up- (red) and downregulated (green) metabolites. (e) Cluster heatmap of the DAMs in the rat left ventricle of WKY, SHR and SHR + OMT groups. Red represents up-regulated metabolites, blue represents down-regulated metabolites. (f, h) Top KEGG pathway enrichment analysis of DAMs in SHRs vs. WKY rats (f) and SHR + OMT vs. SHRs (h). (g, i) Pathway score chart of DAMs enriched pathways in SHRs vs. WKY rats (g) and SHR + OMT vs. SHRs (i), highlighting significant up- (red) and down- (green) regulated pathways. (j) Venn diagram of overlapping KEGG pathways. DAMs: Differentially abundant metabolites; HMDB: Human Metabolome Database; KEGG: Kyoto Encyclopedia of Genes and Genomes; OMT: Oxymatrine; SHRs: Spontaneously hypertensive rats; WKY: Wistar-Kyoto rats. Based on a log2 (FC) ≥ 1.2 and P value ≤ 0.05, we identified 50 DAMs (39 upregulated and 11 downregulated) between the SHR and WKY groups, as well as 44 DAMs (9 upregulated and 35 downregulated) between the SHR + OMT and SHR groups (Fig. [90]3c,d,e). The expression clustering heatmap displays the expression profiles of DAMs across different samples (Fig. [91]3e). The Venn diagram showed that OMT treatment reversed the upregulation of 16 metabolites in the SHR group (Fig. [92]3c, Table [93]3 and Supplementary Fig. [94]S2a online). KEGG enrichment analysis showed that these 16 metabolites were primarily enriched in steroidogenesis and tryptophan metabolism (Supplementary Fig. [95]S2b online). Pathway enrichment analysis indicated that the DAMs between SHR and WKY were enriched in 20 pathways (Supplementary Tab. [96]S1 online). The top 10 pathways were presented in Fig. [97]3f, which include steroid hormone biosynthesis, aldosterone synthesis and secretion, ABC transporters, parathyroid hormone synthesis/secretion and action, regulation of lipolysis in adipocytes, sphingolipid metabolism, and oxidative phosphorylation. The pathway scoring analysis indicated that among these 10 pathways, 7 were upregulated and 3 were downregulated (Fig. [98]3g). In contrast, the DAMs between SHR + OMT and SHR were enriched in 6 pathways (Fig. [99]3h and Supplementary Tab. [100]S2 online), with 2 upregulated and 4 downregulated (Fig. [101]3i). The Venn diagram showed that OMT treatment reversed the 3 KEGG pathways (aldosterone synthesis and secretion, steroid hormone biosynthesis and tryptophan metabolism) in the SHR group (Fig. [102]3j). Table 3. The reversed differential abundant metabolites. Name SHR vs. WKY Regulate SHR + OM vs. SHR Regulate log2FC P value log2FC P value Desoxycortone 4.65732 1.87E−04 Up − 2.07728 0.010358 Down NP-011548 1.78125 0.025835 Up − 1.84752 0.025955 Down Indoleacetic acid 1.66280 9.91E-04 Up − 0.68767 0.047282 Down Pivaloylcarnitine 1.40275 0.003446 Up − 1.01760 0.016360 Down 14-hydroxy-12-tetradecenoic acid 1.18886 0.013925 Up − 1.32010 0.008301 Down (R)-3-Hydroxy myristic acid 0.99838 0.019578 Up − 1.35239 0.010091 Down NP-003692 0.99790 0.044030 Up − 1.39320 0.012629 Down (1S,2S)-3-oxo-2-pentyl-cyclopentanehexanoic acid 0.93464 0.037393 Up − 1.18098 0.014096 Down 2alpha-(3-Hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D3 0.92181 0.006314 Up − 0.71314 0.026404 Down 16-Hydroxyhexadecanoic acid 0.86631 0.038897 Up − 1.03519 0.025712 Down 7-Ethyl-3,5-dimethyl-2E,4E,6E,8E-decatetraene 0.84283 0.036625 Up − 0.98439 0.018504 Down Heptadecanoyl carnitine 0.79108 0.028904 Up − 1.13377 0.008778 Down PC(18:2(9Z,12Z)/19:0) 0.76807 0.016721 Up − 0.42592 0.031530 Down NP-021797 0.65196 0.032161 Up − 0.78957 0.019832 Down Ricinoleic acid 0.46809 0.01593 Up − 0.515600 0.012908 Down 1-O-(2R-methoxy-4Z-tetradecenyl)-sn-glycerol 0.46809 0.01593 Up − 0.515600 0.012908 Down Decanoylcarnitine − 1.15309 0.009779 Down − 1.78046 0.015920 Down [103]Open in a new tab Integrated transcriptomics-metabolomics analysis The CCA chord diagram was used to explore the correlation between transcriptomics and metabolomics. In the CCA chord diagram, the genes and metabolites are arranged around the circle, with connections demonstrating their interaction patterns. As shown in Fig. [104]4a, 20 DEGs and 19 DAMs exhibited strong positive (red chords) or negative (blue chords) correlations between SHR and WKY (|r|> 0.8 and P < 0.05). Similarly, Fig. [105]4b displayed strong positive and negative correlations between 12 DEGs and 19 DAMs in the SHR + OMT vs. SHR comparison. Here, red and blue chords again represented positive and negative correlations, respectively, highlighting the interconnected genes and metabolites in this comparison (|r|> 0.8 and P < 0.05, Fig. [106]4b). The Venn diagram was used to perform a functional enrichment analysis of DEGs and DAMs in SHR vs. WKY and SHR + OMT Versus SHR comparisons (Fig. [107]4c). The Venn diagram showed that there were 16 overlapping pathways in the enrichment of DEGs and DAMs between SHR and WKY groups. In contrast, there were 2 overlapping pathways in the enrichment of DEGs and DAMs between SHR + OMT and SHR. These two pathways were also the commonly enriched pathways across all four comparisons (Fig. [108]4c). Further analysis of the DEGs and DAMs involved in these two overlapping pathways (Tab. [109]4 and Tab. [110]5) revealed that several genes (Atp1a3, Cacna1d, Cyp1a1, Cyp11a1, Cyp2e1, Kcnk3) and metabolites (Corticosterone, Desoxycortone) were enriched. However, among them, Cyp2e1 (DEG) and desoxycortone (also known as deoxycorticosterone, DOC) (DAM) were the only ones significantly reversed by OMT treatment. Fig. 4. [111]Fig. 4 [112]Open in a new tab Functional association analysis of transcriptomics and metabolomics. (a) CCA chord diagram of DEGs and DAMs between SHR and WKY groups. (b) CCA chord diagram of DEGs and DAMs between SHR + OMT and SHR groups. (c) Venn diagram of overlapping pathways. CCA: Canonical correlation analysis; DAMs: Differentially abundant metabolites; DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; OMT: Oxymatrine; SHRs: Spontaneously hypertensive rats; WKY: Wistar-Kyoto rats. Table 4. The DEGs related to the 2 overlapping pathways. KEGG pathway SHR vs. WKY Gene symbol SHR + OM vs. SHR Gene symbol Rich ratio Q-value Rich ratio Q-value Steroid hormone biosynthesis 0.037500 0.885739 Cyp1a1, Cyp2e1, Cyp11a1 0.012500 0.258185 Cyp2e1 Aldosterone synthesis and secretion 0.031579 0.907839 Kcnk3, Cyp11a1, Cacna1d 0.010526 0.258185 Atp1a3 [113]Open in a new tab Table 5. The DAMs related to the 2 overlapping pathways. KEGG Pathway SHR vs. WKY Metabolites SHR + OM vs. SHR Metabolites Count P value Count P value Steroid hormone biosynthesis 2 2.27E-04 Corticosterone, Desoxycortone 1 0.012494 Desoxycortone Aldosterone synthesis and secretion 2 0.004993 Corticosterone, Desoxycortone 1 0.057679 Desoxycortone [114]Open in a new tab To further ascertain the reliability of the above results, RT-qPCR, immunostaining, and ELISA were performed. As shown in Fig. [115]5a, the mRNA level of Cyp2e1 in myocardial tissue was lower in the SHRs compared to the WKY group. However, OMT treatment markedly increased Cyp2e1 expression relative to untreated SHRs. As shown in Fig. [116]5b, the hypertrophy-associated genes atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were upregulated in SHRs and showed a downward trend following OMT treatment, which is consistent with the observed attenuation of myocardial hypertrophy. The immunostaining results (Fig. [117]5c, d) were consistent with the changes in the mRNA level, showing that Cyp2e1 expression was lower in the SHR group compared to the WKY group, and that OMT treatment increased Cyp2e1 expression in SHR myocardial tissue. ELISA analysis indicated that DOC levels in both serum (Fig. [118]5e) and myocardial tissue (Fig. [119]5f) were elevated in SHRs compared to WKY rats. However, OMT treatment prevented the elevation of DOC levels in both the serum and myocardial tissue of SHRs (Fig. [120]5e, f), suggesting that OMT may alleviate myocardial hypertrophy in SHRs by inhibiting DOC levels in both circulation and local myocardial tissues. Fig. 5. [121]Fig. 5 [122]Open in a new tab Effect of OMT on the mRNA and protein expression levels of Cyp2e1. (a) RT-qPCR analysis of Cyp2e1 mRNA expression in the left ventricle. (b) RT-qPCR analysis of expression of myocardial-hypertrophy-associated genes (ANP and BNP) in the left ventricle. (c) Immunohistochemistry staining of Cyp2e1 protein in the left ventricle. Scale bar = 50 μm. (d) The H-score statistics of Cyp2e1 immunostaining in panel B. (e) ELISA analysis for DOC level in serum of difference groups. (f) ELISA analysis for DOC level in left ventricle of difference groups. ****P < 0.001; ***P < 0.005; **P < 0.01; *P < 0.05. ANP: Atrial natriuretic peptide; BNP: B-type natriuretic peptide; DOC: Deoxycorticosterone; OMT: Oxymatrine; SHRs: Spontaneously hypertensive rats; WKY: Wistar-Kyoto rats. Discussion OMT is a quinoline alkaloid primarily used for treating hepatitis and related diseases^[123]4,[124]11. In recent years, due to its anti-inflammatory, antioxidant, and immune-regulating properties, the role of OMT in tumors, diabetes, and cardiovascular and cerebrovascular diseases has been widely explored and researched^[125]4,[126]6,[127]7,[128]11,[129]12. In this study, we investigated the effects of OMT on myocardial hypertrophy in SHRs. Our findings demonstrated that OMT significantly reduces blood pressure and alleviates myocardial hypertrophy in SHRs, consistent with previous studies^[130]7. The analysis using Masson and WGA staining demonstrated that OMT effectively reduces collagen fiber deposition in myocardial tissue and significantly decreases the cross-sectional area of cardiomyocytes. Collagen fiber deposition is recognized as a structural basis for ventricular remodeling and myocardial fibrosis^[131]13, while cardiomyocyte hypertrophy is closely related to cellular stress and metabolic dysregulation^[132]3,[133]14. Therefore, we hypothesize that OMT may alleviate myocardial hypertrophy by reducing ventricular remodeling and enhancing the metabolic state of cardiomyocytes. Transcriptomics, with its high-throughput and high-precision capabilities, is widely used to explore disease targets, evaluate drug mechanisms, and assess toxicity^[134]15,[135]16. In this study, transcriptomic analysis was used to investigate the gene expression mechanisms underlying OMT’s effects on myocardial hypertrophy in SHRs. In our study, OMT treatment significantly enriched several key metabolic and cytotoxic metabolic pathways, including linoleic/arachidonic acid metabolism, cytochrome P450 metabolism. Linoleic acid is the primary polyunsaturated fatty acid in the diet, and its main metabolite is arachidonic acid. Although their impact on cardiovascular health remains controversial, a prospective study^[136]17 involving 13 countries suggests that higher levels of linoleic acid and arachidonic acid are associated with a lower risk of cardiovascular events. Cytochrome P450 (CYP) enzymes, the membrane-bound, heme-containing terminal oxidases, are involved in drug metabolism, endogenous metabolite synthesis and degradation, hormone or fatty acid metabolism, and toxin detoxification. CYP-derived metabolites of arachidonic acid in endothelial cells and vascular smooth muscle cells play a key role in vascular relaxation^[137]18. The heart contains active CYP enzymes, and their arachidonic acid-derived metabolites play a role in maintaining heart health^[138]19. The enrichment of these pathways in our study suggests that OMT may improve myocardial hypertrophy in SHRs by influencing the metabolic processes. Sympathetic nervous system activation is a key pathophysiological mechanism in hypertension^[139]20, which drives disease progression by stimulating the renin–angiotensin–aldosterone system and promoting adrenaline secretion. Our study found that genes reversed by OMT were enriched in the adrenergic signaling in cardiomyocytes and steroid hormone biosynthesis signaling pathways, suggesting that OMT may exert its effects under sympathetic activation. These findings provide novel mechanistic insights into the potential of OMT in treating hypertension and myocardial hypertrophy. Recent studies have increasingly highlighted metabolic abnormalities as key risk factors for cardiovascular diseases^[140]3. These imbalances not only cause direct damage to the cardiovascular system but also exacerbate cardiovascular injury through chronic inflammation and oxidative stress^[141]21. Given that transcriptomic analysis indicates that OMT treatment is enriched in various metabolic pathways, we further employed metabolomics to investigate its impact on metabolites. In this study, we found significant metabolic differences between SHRs and WKY rats. Approximately 90% of the ATP in the heart is derived from mitochondrial oxidative phosphorylation. Study has reported a significant decrease in the expression of genes related to mitochondrial oxidative phosphorylation in the myocardial tissue of mice with chronic kidney disease^[142]22. Consistent with previous study, we observed a reduction in oxidative phosphorylation metabolism in the hearts of SHRs during hypertrophy. Seventeen ABC transporters have been identified to play key roles in the human heart, involved in drug transport, cholesterol/lipid metabolism, cofactor/iron transport, and heme biosynthesis^[143]23. Studies have also shown that ABC transporters can prevent hypertrophy and heart failure induced by pressure overload by promoting angiogenesis and exerting antioxidant effects^[144]24. An increase in metabolites associated with ABC transporter pathways in SHRs is observed in our results, suggesting a potential protective compensatory response. Sphingolipids are a class of complex bioactive lipids and serve as major components of cell membranes, playing critical roles in the development and progression of cardiovascular diseases. It has been shown that abnormal sphingolipid metabolism is closely associated with left ventricular remodeling following acute myocardial infarction (SMI)^[145]25. Our findings align with this, showing disrupted sphingolipid metabolism in SHRs heart tissue, which likely contributes to cardiac remodeling in SHRs. Weber and Brilla^[146]26 reported that elevated circulating aldosterone levels are associated with fibrosis and tissue heterogeneity in the left ventricle during hypertrophy. Moreover, aldosterone can directly inhibit cardiac mitochondrial function by activating reactive oxygen species^[147]27. It has been found that corticosterone increases atrial natriuretic peptide expression and cell surface area in cardiomyocytes, exacerbating isoproterenol-induced cardiac hypertrophy^[148]28. In this study, we observed that both corticosterone and DOC, along with their associated metabolic pathways (including aldosterone synthesis and secretion, steroid hormone synthesis), were significantly upregulated in SHRs hearts, suggesting that these hormones play critical roles in cardiac hypertrophy of SHRs. Further analysis revealed that OMT significantly reversed 16 DAMs, suggesting that OMT may improve cardiac hypertrophy by regulating metabolism, offering new insights for clinical applications. Pathway enrichment analysis showed that these 16 metabolites were enriched in the aldosterone synthesis and secretion, steroid hormone biosynthesis, and tryptophan metabolism pathways. It has been reported that the tryptophan metabolic enzyme indoleamine 2,3-dioxygenase can prevent pathological cardiac hypertrophy by reducing GATA binding protein 4^[149]29. This finding is consistent with our results, in which indoleacetic acid, a metabolite associated with tryptophan metabolism, was upregulated in the hearts of SHRs, while OMT reduced its levels. Interestingly, we found that DOC was the most significantly regulated metabolite by OMT and the metabolite with the most significant alteration among the 16 DAMs in SHRs. ELISA analysis also confirmed that OMT inhibits the elevation of DOC levels in the serum and myocardial tissue of SHRs. DOC is an important intermediate in the steroid hormone biosynthesis, primarily synthesized in the zona fasciculata of the adrenal cortex^[150]30. DOC serves as a precursor to aldosterone, which can be further metabolized into aldosterone. Aldosterone is a key hormone involved in regulating blood pressure and electrolyte balance, promoting increased blood volume and elevating blood pressure. Although in vitro studies have shown that OMT may inhibit aldosterone-induced myocardial fibroblast proliferation and differentiation by attenuating the Smad signaling pathway^[151]31 or the Nrf2/Keap1 pathway^[152]32 and may reduce aldosterone-induced cardiotoxicity by inhibiting calpain/AIF signaling^[153]33, the regulation of aldosterone or deoxycorticosterone levels by OMT and its underlying mechanisms have not been reported. Notably, spironolactone, a well-established mineralocorticoid receptor (MR) antagonist, has been reported to lower blood pressure and improve cardiac hypertrophy in SHR rats^[154]34. Considering that OMT may also alleviate cardiac hypertrophy by modulating aldosterone levels, future studies comparing the effects of OMT and spironolactone—either separately or in combination—may help to better define the therapeutic potential of OMT in hypertensive cardiac remodeling. To further explore the potential mechanisms underlying the effect of OMT on deoxycorticosterone levels, we performed a combined transcriptomics and metabolomics analysis. The results show that both in the SHR vs. WKY group and the SHR + OMT vs. SHR group, there is a strong correlation between the DAMs and DEGs, suggesting that these genes and metabolites may interact to regulate the pathological processes of the cardiac hypertrophy of SHRs. Further integrated analysis revealed that the DEGs and DAMs reversed by OMT treatment were significantly enriched in the Steroid hormone biosynthesis and Aldosterone synthesis and secretion pathways. These findings indicate that OMT may regulate these two pathways, promoting coordination between transcriptional and metabolic processes, thereby modulating the pathological processes of myocardial hypertrophy in SHRs. Among the genes associated with these pathways, Cyp2e1 was the only one concurrently reversed by OMT treatment. Cyp2e1, a member of the cytochrome P450 family, is involved in various biotransformation processes, including drug metabolism, lipid metabolism, and oxidative stress responses^[155]35,[156]36. Using RT-qPCR and immunohistochemistry, we confirmed that both the mRNA and protein levels of Cyp2e1 were downregulated in SHRs, whereas OMT treatment significantly restored its expression. Regardless of age (4 or 22 months), the expression level of Cyp2e1 in the heart of SHRs is significantly lower than that in the liver^[157]37. However, cardiac Cyp2e1 expression increases with age and has been associated with age-related cardiac dysfunction^[158]37,[159]38. Cyp2e1 overexpression has been reported to promote cardiomyocyte apoptosis^[160]39. Notably, heart-specific Cyp2e1-knockdown transgenic mouse exhibit significantly greater left ventricular wall thickness compared to nontransgenic mice (Table [161]1 in^[162]40). Furthermore, Cyp2e1 expression was also found to be downregulated in isoproterenol-induced myocardial hypertrophy^[163]41. These findings support the notion that the decreased expression of Cyp2e1 in SHRs hearts may represent a compensatory mechanism to reduce apoptosis and preserve cardiac function during the early stages of hypertension—albeit at the cost of increased ventricular wall thickness. In contrast, OMT-induced upregulation of Cyp2e1 may facilitate apoptosis of hypertrophied cardiomyocytes, thereby contributing to improved cardiac remodeling. Moreover, Cyp2e1 expression is hormonally regulated: Insulin and thyroid hormone suppress its expression^[164]42, whereas growth hormone upregulates it^[165]43. In our study, the increase in Cyp2e1 was accompanied by a reduction in DOC levels, suggesting a possible regulatory interaction, which warrants further investigation. Future studies may utilize cardiomyocyte-specific Cyp2e1 intervention models combined with pharmacological modulation of DOC or its receptor, the mineralocorticoid receptor (MR), to further elucidate the regulatory relationship between OMT, DOC, and Cyp2e1, clarify the time-dependent role of Cyp2e1 in the development and progression of myocardial hypertrophy, and correlate these molecular mechanisms with hemodynamic changes through longitudinal blood pressure monitoring. In addition, we acknowledge the systemic effects of OMT, particularly its significant blood pressure-lowering action in SHRs, which raises the possibility that the observed improvements in cardiac hypertrophy may be, at least in part, secondary to hemodynamic changes. While our transcriptomic and metabolomic analyses revealed enrichment in oxidative stress related pathways following OMT treatment, these findings alone do not establish a direct cardiac-specific effect. However, together with previous in vitro studies demonstrating that OMT can directly modulate cardiomyocytes and cardiac fibroblasts^[166]31,[167]32,[168]44, these results support the plausibility of a direct cardioprotective role. Nevertheless, the contribution of blood pressure reduction cannot be excluded. Future studies incorporating antihypertensive comparator drugs and comparing localized versus systemic administration of OMT will be essential to disentangle the relative contributions of hemodynamic versus direct myocardial mechanisms. Among other DEGs and DAMs involved in the Steroid hormone biosynthesis and Aldosterone synthesis and secretion pathways, corticosterone—like DOC—is a hormone synthesized in the adrenal cortex. Previous studies have reported significantly elevated plasma corticosterone levels in SHRs compared to WKY rats^[169]45. In our study, both myocardial corticosterone levels and the mRNA expression of its rate-limiting enzyme Cyp11a1 were higher in the left ventricle of SHRs than in WKY rats. Corticosterone and aldosterone signaling may interact with adrenergic pathways to promote cardiac hypertrophy^[170]28,[171]46. These findings suggest that cardiac-localized Cyp11a1 may serve as a novel target for further investigation into the molecular mechanisms underlying cardiac hypertrophy and potential therapeutic intervention. In addition, Cyp1a1, a member of the cytochrome P450 family, has been reported to be upregulated in the hearts and kidneys of rats with isoproterenol-induced cardiac hypertrophy^[172]41, whereas its expression in the kidneys of SHRs shows no significant difference compared to WKY rats^[173]47. Cacna1d, which encodes the L-type calcium channel subunit Cav1.3, and Kcnk3, which encodes the two-pore potassium channel TWIK-related acid-sensitive potassium channel 1 (TASK-1)—both of which are involved in maintaining cardiac electrophysiological stability and resting membrane potential—were also differentially expressed in SHRs. However, their specific roles in SHR-associated cardiac hypertrophy remain unclear and warrant further investigation. Conclusion Our study, through transcriptomics, metabolomics, and their integrated analysis, reveals that OMT may improve myocardial hypertrophy in SHRs by regulating multiple targets and metabolites and mediating the interaction between transcriptomic and metabolic changes. This finding underscores the potential of OMT in treating pressure overload-induced myocardial hypertrophy and provides theoretical support for its exploration in other indications. Supplementary Information Below is the link to the electronic supplementary material. [174]Supplementary Material 1^ (330.1KB, pdf) Abbreviations ANP Atrial natriuretic peptide BMDB BGI metabolome database BNP B-type natriuretic peptide CCA Canonical correlation analysis CV Coefficient of variation CYP Cytochrome P450 DAMs Differential abundant metabolites DBP Diastolic blood pressure DNBs DNA nanoballs DOC Desoxycortone DEGs Differentially expressed genes EF Ejection fraction ESV End systolic volume FC Fold change FPKM Fragments per Kilobase per Million FS Fractional shortening HMDB The human metabolome database KEGG Kyoto encyclopedia of genes and genomes GO Gene ontology LV Left ventricle LVAWd LV anterior wall thicknesses at end-diastole LVAWs LV anterior wall thicknesses at end-systole LVIDd LV internal dimension in diastole LVIDs LV internal dimension in systole LV mass Left ventricular mass LVPWd LV posterior wall thicknesses at end-diastole LVPWs LV posterior wall thicknesses at end-systole MAP Mean arterial pressure OMT Oxymatrine QC Quality control RT-qPCR Real-time quantitative PCR SBP Systolic blood pressure SHR Spontaneously hypertensive rat WGA Wheat germ agglutinin WKY Wistar-kyoto Author contributions C.H.L and J.H contributed to the study conception and design. Material preparation, data collection and analysis were performed by C.H.L, X.Y.L, J.T, Q.H.F and M.Z.Z. The first draft of the manuscript was written by C.H.L and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by National Natural Science Foundation of China to JH (Grant number 82170445). Data availability The datasets generated and/or analysed during the current study are available in the [NCBI’s Gene Expression Omnibus (GEO)] repository, [[175]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = [176]GSE2827 67] (Enter token klqdqicylrajbiz into the box). Declarations Competing interests The authors declare no competing interests. Ethical approval All procedures performed in this study were in accordance with national animal research regulations, and all animal experimental protocols complied with the ARRIVE guidelines and were approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (No: 2021347). Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References