Mechanisms of Shengxian Quyu Decoction In Heart Failure Using an Untargeted Metabolomics and Network Pharmacology Approach In Rats

Summary

This study confirmed the therapeutic effect of Shengxian Quyu Decoction on heart failure through animal experiments. Ultra-performance liquid chromatography–mass spectrometry and network pharmacology analyses identified potential therapeutic targets and mechanisms.

Abstract

Shengxian Quyu decoction (SXQY) has been suggested as a potential therapeutic strategy for heart failure (HF), but its therapeutic mechanisms remain unclear. This study investigated the therapeutic effects and underlying mechanisms of SXQY in HF. A rat model of HF was induced by transverse aortic constriction (TAC) and treated with SXQY. Cardiac function was assessed by transthoracic echocardiography, and myocardial structure and fibrosis were evaluated using hematoxylin and eosin and Masson’s trichrome staining. Serum samples were analyzed by untargeted metabolomics using ultra-performance liquid chromatography–tandem mass spectrometry. Blood-entering components were mapped to targets, and intersecting HF-related targets were analyzed using protein–protein interaction (PPI) network analysis, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. SXQY significantly improved cardiac function in TAC rats, as evidenced by decreased left ventricular internal diameter in diastole and left ventricular posterior wall thickness in diastole and increased left ventricular ejection fraction, and alleviated myocardial hypertrophy, inflammation, and fibrosis. A total of 563 blood-entry compounds were identified (367 prototypes and 196 metabolites), with 5 key active compounds identified in the Traditional Chinese Medicine Systems Pharmacology database corresponding to 63 targets. Network analysis revealed 44 overlapping genes, with Formononetin, Timosaponin BII, and Sinensetin as core components, and PTGS2, PPARG, and HSP90AA1 as hub targets. PPI analysis further identified key genes including ESR1, PTGS2, PPARG, HSP90AA1, JUN, and 15 additional genes. KEGG analysis indicated that SXQY mainly acts via Ca²⁺, phosphatidylinositol 3-kinase–protein kinase B, cyclic adenosine monophosphate, and inflammation- and hormone-related pathways. In conclusion, SXQY exerts protective effects against HF by improving cardiac function and attenuating myocardial remodeling through multicomponent, multitarget, and multipathway mechanisms.

Introduction

Heart failure (HF) is a clinical syndrome characterized by impaired ventricular filling and ejection capacity due to cardiac structural or functional abnormalities, resulting in a constellation of clinical signs and symptoms¹. As the terminal stage of various cardiac diseases, HF significantly compromises patients’ quality of life and results in high mortality and frequent hospital readmissions, thereby imposing a substantial disease burden on both families and society^2,3. In clinical management, pharmacotherapy for HF forms the foundation and cornerstone of treatment, primarily comprising key drug classes such as diuretics, renin–angiotensin system inhibitors, beta-blockers, mineralocorticoid receptor antagonists, and sodium–glucose cotransporter 2 inhibitors^3,4,5,6. While these medications have partially improved the prognosis of patients with HF, they each carry certain limitations and adverse effects, such as electrolyte disturbances, bradycardia, and urinary tract infections⁷. Therefore, pharmacological therapies for HF still warrant further investigation.

Traditional Chinese Medicine (TCM) has accumulated extensive clinical experience in the long-term management of HF. Emphasizing holistic regulation and multitarget intervention, it offers a distinctive approach to treatment⁸. In recent years, a number of randomized controlled trials (RCTs) have demonstrated the therapeutic efficacy of Chinese herbal formulas for HF^{9,10,11,12,13,14,15}. Shengxian Quyu decoction (SXQY) has already begun undergoing rigorous RCTs to confirm its therapeutic efficacy, but its specific therapeutic mechanisms still require further and more comprehensive investigation¹⁶. Network pharmacology, as an emerging discipline integrating systems biology, polypharmacology, and computational analysis, offers a powerful tool for elucidating the holistic mechanisms of action of Chinese herbal formulas^17,18,19. Therefore, this study employs an integrated strategy combining untargeted metabolomics and network pharmacology to identify blood-absorbed components of SXQY and elucidate its potential therapeutic mechanisms. This work aims to provide a foundation for the further clinical application of SXQY.

Protocol

All experimental procedures were examined and approved by the Animal Ethics and Welfare Committee of China–Japan Friendship Hospital (approval No. zryhyy21-23-01-09) and were conducted in accordance with the replacement, reduction, and refinement principles to minimize animal use and suffering. Investigators performing data acquisition and analysis (including echocardiography measurements and histological assessment) were blinded to group allocation.

Preparation of SXQY sample

SXQY is composed of the following herbal components: Astragalus membranaceus (Huangqi) (30 g), Codonopsis pilosula (Dangshen; 20 g), Cornus officinalis (Shanyurou; 15 g), Sparganium stoloniferum (Sanleng; 12 g), Curcuma zedoaria (Erzhu; 15 g), Cimicifuga heracleifolia (Shengma; 10 g), Bupleurum chinense (Chaihu; 10 g), Platycodon grandiflorus (Jiegeng; 10 g), Anemarrhena asphodeloides (Zhimu; 15 g), and Leonurus japonicus (Yimu; 15 g), with a total crude herb weight of 152 g per prescription. All herbal materials were purchased from the Department of Pharmacy of China–Japan Friendship Hospital and authenticated by a chief pharmacist according to standard pharmacognostic identification methods. Clinically, SXQY is administered at a dose of one prescription (152 g) per day. Assuming an average adult body weight of 70 kg, the corresponding human dosage is 2.17 g (crude herb)/kg. Based on body surface area normalization between humans and rats, the equivalent rat dosage was calculated to be 13.67 g (crude herb)/kg. For decoction preparation, one prescription of SXQY was soaked in distilled water at a ratio of 10 mL per gram of crude herbs (w/v, based on the initial solvent volume without adjustment after soaking) for 1 h, followed by boiling using an electric heating mantle and maintained at 100°C under continuous boiling for 1 h. The extract was then filtered through gauze (200–300 µm pore size). The residue was then extracted twice more under identical conditions. The pooled filtrates were concentrated under reduced pressure using a rotary evaporator at 60°C to a final volume of 111 mL, yielding a concentration of 1.37 g (crude drug)/mL. The concentrated decoction was filtered again through gauze (200–300 µm pore size) and stored at 4°C and used within 24–48 h. For administration, the solution was used directly, and the gavage volume was calculated based on the concentration of 1.37 g/mL, resulting in an administration volume of approximately 10 mL/kg to achieve the target dose of 13.67 g/kg.

Animal Model

Eighteen 8-week-old male Sprague–Dawley (SD) rats were housed in a standard conditioned environment (23°C ± 2°C, 50 ± 5% relative humidity, and a 12 h light/dark cycle) with free access to food and water. Animals were randomly assigned to three groups (n = 6 per group) using a random number table: a sham-operated group (Sham), a transverse aortic constriction group (TAC), and an SXQY-treated group (SXQY). Rats in the TAC and SXQY-treated groups were anesthetized by inhalation of isoflurane (induction 4%, maintenance 2%) and placed in the supine position on a thermostatically controlled heating pad maintained at 37°C. Adequate anesthesia was confirmed by the absence of reflex responses to paw pinch, along with a stable respiration rate and the absence of spontaneous movement. Ophthalmic ointment was applied to both eyes to prevent corneal drying. Following endotracheal intubation, animals were connected to a small-animal ventilator with a tidal volume of 4–6 mL per 200 g body weight, a respiratory rate of 70 breaths per minute, and an inspiratory-to-expiratory ratio of 1:1.

All surgical procedures were performed under aseptic conditions. Surgical instruments were sterilized by autoclaving, and the operative area was prepared by shaving and disinfecting with povidone–iodine followed by 70% ethanol. After surgical site preparation, a left thoracic skin incision was made, and the pectoralis major and minor muscles were bluntly dissected to expose the ribs. A horizontal incision of approximately 1.0 cm was created at the second to third intercostal space adjacent to the left sternal border. Blood vessels and fascia were bluntly separated, and the thymus was gently retracted to fully expose the aortic arch.

The aortic arch was carefully lifted between the right innominate artery and the left common carotid artery using sterile curved forceps, and a 2-0 silk suture was passed underneath. A blunt-tipped curved 16-gauge needle (outer diameter 1.6 mm) was manually modified by blunting the tip and bending it to an appropriate curvature before use, followed by sterilization with autoclaving. The prepared blunt-tipped curved needle was then positioned parallel to the aortic arch, after which the suture was tied snugly around both the aorta and the needle. The needle was immediately removed to create a standardized constriction, and the thymus was repositioned. After confirming the absence of active bleeding, the chest wall and skin were closed in layers. Postoperatively, mechanical ventilation was continued for approximately 10 min until spontaneous respiration was restored, as indicated by a stable and regular respiratory rate and the ability to maintain spontaneous breathing without ventilatory support. At this point, the endotracheal tube was removed. Sham-operated animals underwent the same surgical procedures except that the aortic arch was not ligated.

After surgery, the incision site was disinfected, and animals were placed in clean recovery cages to reduce the risk of infection. Rats were then returned to the animal facility for recovery and routine housing. Analgesia was administered using buprenorphine (0.05 mg/kg, subcutaneously). Rats were observed every 12 h during the first 72 h for signs of pain, distress, or surgical complications, including reduced mobility, piloerection, abnormal posture, labored breathing, or wound dehiscence.

Three days after surgery, rats in the SXQY-treated group were orally gavaged with 1 mL/100 g body weight of the SXQY decoction for 9 weeks. Meanwhile, rats in the sham-operated and TAC groups received an equal volume (1 mL/100 g body weight) of distilled water by gavage.

Transthoracic Echocardiography

At the end of the treatment period, transthoracic echocardiography was performed in all groups. Rats were anesthetized by inhalation of isoflurane and placed in the supine position on a dedicated imaging platform. The anterior chest was carefully shaved and coated with a generous layer of ultrasound gel. Ultrasound imaging was conducted using a high-resolution small-animal ultrasound system equipped with a high-frequency transducer (18–40 MHz) in B-mode and M-mode. The transducer was positioned gently on the gel-covered area, with a depth setting of 12.29 mm and a gain of 22 dB; the focus was adjusted to optimize image quality. Images and video loops were acquired for subsequent quantitative analysis. Left ventricular end-diastolic internal diameter (LVIDd) and left ventricular posterior wall thickness at end diastole (LVPWd) were measured, and left ventricular ejection fraction (LVEF) was calculated using the Teichholz formula based on M-mode measurements. For each parameter, measurements were averaged over 3–5 consecutive cardiac cycles. All echocardiographic acquisitions and analyses were performed by a certified operator with formal training in echocardiography, in accordance with standardized echocardiographic protocols.

Sample Collection and Processing

After the ultrasound procedure, rats were anesthetized with isoflurane. Animals were placed in the supine position, and the thoracic cavity was opened to expose the heart. Hearts were rapidly excised, rinsed in cold phosphate-buffered saline, and sectioned along the sagittal plane. Cardiac tissues were immediately fixed in 4% paraformaldehyde at 4°C for 24 h, followed by dehydration through a graded ethanol series (70%, 80%, 90%, 95%, and 100%) for 1 h at each step, during which tissues gradually became translucent without noticeable shrinkage. Samples were cleared in xylene, infiltrated with molten paraffin at 60°C for 3 h, and allowed to solidify at room temperature (RT; 20°C–25°C). Paraffin-embedded heart tissue blocks were trimmed and sectioned at 4–6 µm thickness using a microtome. Sections were floated on a water bath maintained at 45°C, mounted onto glass slides, and dried at 37°C–40°C overnight. Slides were stored at RT under low-humidity conditions and used within 1 week for histological staining.

Hematoxylin and Eosin (HE) Staining

Paraffin-embedded sections were processed as follows. Sections were first deparaffinized by immersion in xylene I, II, and III for 5 min each, followed by hydration through a series of anhydrous ethanol I and II and graded ethanol solutions (95%, 90%, 80%, 70%, and 50%) for 5 min each, and finally rinsed in distilled water, with no visible wax residues. The sections were then stained with Harris hematoxylin for 5 min and rinsed under running tap water for 1–2 min. Differentiation was performed in acid alcohol solution for approximately 1–2 s, with the endpoint determined by visual inspection when the background appeared pale while nuclear staining remained clearly defined. This was followed by bluing in water at 37°C–45°C and rinsing with distilled water. Subsequently, the sections were counterstained with alcohol-soluble eosin for 40 s, washed with distilled water, dehydrated through graded ethanol, cleared in xylene, and finally mounted with neutral resin.

Masson Staining

Paraffin-embedded sections were deparaffinized sequentially in xylene I, II, and III for 5 min each, followed by hydration through anhydrous ethanol I and II and graded ethanol solutions (95%, 90%, 80%, 70%, and 50%) for 5 min each, and finally rinsed in distilled water. Sections were immersed in 0.5% acetic acid (pre-cooled to 4°C) for 1 s, followed by staining with the fuchsin solution from a commercial Masson’s trichrome staining kit for 10 min, followed by differentiation in phosphomolybdic acid for 5 min and counterstaining with aniline blue for 3 min. All reagents were components of the same commercial kit and were used according to the manufacturer’s instructions. After staining, sections were rinsed three times in acetic acid pre-cooled to 4°C (approximately 30 s per rinse), dehydrated through graded ethanol, cleared in xylene, and mounted with neutral resin.

Serum Sample Collection for Metabolomics Analysis

Six 8-week-old male SD rats were housed under the same conditions as those in the above in vivo experiment. Animals were randomly assigned to an SXQY-treated group and a vehicle control group (distilled water; n = 3 per group) using a computer-generated randomization method. Rats in the SXQY group were orally administered SXQY at a dose of 1 mL/100 g body weight, while the vehicle control group received an equal volume of distilled water. After seven days of daily gavage, the final dose on the seventh day was doubled. Following the last administration, the animals were fasted for 12 h. Rats were anesthetized with isoflurane until loss of the pedal withdrawal reflex was confirmed as an indicator of adequate anesthesia. Blood samples were collected from the retro-orbital venous plexus using sterile capillary tubes and immediately transferred into 1.5 mL microcentrifuge tubes without anticoagulant. The whole blood was then allowed to stand at RT for 1 h and centrifuged at 1000 × g for 15 min at 4°C to obtain serum. When a clear separation between the upper transparent serum layer and the clot appeared, the supernatant serum was carefully aspirated and transferred into clean microcentrifuge tubes. Serum samples were stored at −80°C until analysis of blood-entering components.

Sample Pretreatment for Metabolomics Analysis

Serum samples were thawed on ice after removal from −80°C storage and briefly vortex-mixed (10 s) to ensure homogeneity. An aliquot of 100 µL serum was transferred into a 1.5 mL microcentrifuge tube, followed by the addition of 500 µL of extraction solvent (20% acetonitrile–methanol containing 2-chloro-L-phenylalanine as an internal standard at a final concentration of 1 µg/mL) to induce protein precipitation. The mixture was vortexed vigorously for 3 min and centrifuged at 12,000 × g for 10 min at 4°C. A 470 µL portion of the resulting supernatant was transferred to a new tube and evaporated to dryness using a nitrogen evaporator under a low, controlled flow of nitrogen. After reconstitution in 200 µL of 70% methanol–water and vortexing for 1 min, samples were centrifuged at 12,000 × g for 10 min at 4°C, and 180 µL of the clarified supernatant was transferred into autosampler vials for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

An aliquot of 200 µL of SXQY sample was then transferred into a pre-labeled 1.5 mL centrifuge tube. The internal standard extraction solution was added to each sample at a volume ratio of 1:1 (v/v). The mixture was vortex-mixed for 15 min and centrifuged at 12,000 × g for 3 min at 4°C. The resulting supernatant was carefully collected and filtered through a 0.22 µm microporous membrane, yielding a transparent filtrate without visible particles. The filtrate was transferred to autosampler vials and stored at 4°C prior to ultra-performance LC-MS/MS (UPLC–MS/MS) analysis.

Preparation of Internal Standard Solutions

The internal standard (2-chloro-L-phenylalanine) stock solution was prepared by dissolving 1 mg of the reference standard in 1 mL of 70% (v/v) methanol–water, yielding a concentration of 1,000 µg/mL. This stock solution was further diluted with the same solvent to obtain a working internal standard solution with a final concentration of 1 ppm, which was freshly prepared, stored at 4°C, and used within 48–72 h.

UPLC–MS/MS Conditions

Chromatographic separation was performed using a UPLC system coupled to a tandem mass spectrometer. Separation was achieved on a C18 column maintained at 40°C, with a flow rate of 0.35 mL/min and an injection volume of 4 µL. The mobile phase consisted of ultrapure water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). Gradient elution was applied as follows: 5% B at 0.0 min; linearly increased to 25% B at 2.0 min, 35% B at 3.0 min, 60% B at 5.0 min, and 99% B at 6.0 min; held at 99% B for 1.5 min; returned to 5% B at 7.6 min; and equilibrated at 5% B until 10.0 min.

Mass spectrometric detection was carried out using a high-resolution mass spectrometer equipped with an electrospray ionization source operating in positive and negative ion modes. The spray voltages were set to 3500 V in positive mode and 3200 V in negative mode. The sheath gas and auxiliary gas were set to 30 and 5 arbitrary units (instrument-defined), respectively. The ion transfer tube temperature and vaporizer temperature were maintained at 320°C and 300°C, respectively. Full MS scans were acquired over an m/z range of 84–1250 at a resolution of 35,000 with an automatic gain control (AGC) target of 1.0 × 10⁶, followed by data-dependent MS/MS acquisition (top 10) at a resolution of 17,500 with an AGC target of 2.0 × 10⁵ and an isolation window of 1.0–1.5 m/z. Stepped normalized collision energies of 30, 40, and 50 were applied. The top 10 most intense precursor ions were selected for fragmentation with a dynamic exclusion duration of 3 s.

Raw Data Processing

Raw mass spectrometry data were converted to mzML format using ProteoWizard. To ensure analytical reliability, pooled quality control (QC) samples were prepared by mixing equal volumes of all serum samples. QC samples were injected at regular intervals (one QC injection every 5–10 analytical samples) throughout the UPLC–MS/MS analysis to monitor instrument stability, retention time consistency, and signal reproducibility. Reproducibility was evaluated based on the relative standard deviation of peak intensities in QC samples.

Peak detection, retention time correction, and peak alignment were performed using the XCMS package. Features with a missing rate greater than 50% within any group were excluded from further analysis. Signal drift correction was subsequently applied to peak areas using a support vector regression (SVR)-based normalization method implemented in the R statistical software using QC-based modeling to correct signal drift across the analytical run.

Metabolite identification was carried out by matching the corrected features against an in-house reference standard library (containing approximately 5,000 compounds with both MS1 and MS2 spectral data; proprietary and not publicly available), as well as publicly available spectral databases including Human Metabolome Database (HMDB), Metabolite and Tandem Mass Spectrometry Database (METLIN), MassBank, and Global Natural Products Social Molecular Networking (GNPS), based on accurate mass matching, retention time alignment, and MS/MS spectral similarity. Mass matching was performed using a tolerance of ±25 ppm for precursor (MS1) and fragment ions (MS2), with a retention time tolerance of ±6 s. For library searching, a tolerance of ±25 ppm was applied to precursor ions (Q1) and ±50 ppm to MS/MS fragments, with a minimum MS/MS matching score of 0.3. Only metabolites with a comprehensive identification score greater than 0.5 were retained for non-targeted identification. The final score was calculated as a weighted sum of fragment (0.1), forward (0.3), and reverse (0.6) matching scores. Data acquired in positive and negative ionization modes were merged at the compound level. For duplicated annotations, the entry with the highest identification confidence and the highest score was selected. The finalized annotations were compiled into a metabolite annotation file.

Criteria for Identification of Absorbed Compounds

Absorbed prototype constituents were defined as compounds detected in the SXQY extract and the SXQY-treated group with matching retention times within a tolerance of ±0.1 min. In addition, these compounds were required to exhibit a peak area in the SXQY-treated group that was at least two-fold higher than that in the vehicle control group or to be absent from the vehicle control samples.

Absorbed metabolites were identified using a different set of criteria. These compounds were not detected in the SXQY extract but were present in the SXQY-treated group. Their mass spectrometric features were required to be consistent with characteristic metabolic transformations, including hydroxylation (Δm/z = +15.9949 Da) and demethylation (Δm/z = −14.0157 Da). Furthermore, the peak area in the SXQY-treated group was required to be at least two-fold higher than that in the vehicle control group based on normalized peak areas after signal drift correction, or the corresponding signal had to be undetectable in the vehicle control samples.

Retrieval of Absorbed Compound-Related Genes

Blood-entering components were interrogated in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; https://old.tcmsp-e.com/tcmsp.ph; accessed in January 2026)²⁰. Compounds documented in TCMSP were screened as candidate bioactive constituents based on established pharmacokinetic thresholds of oral bioavailability ≥ 30% and drug-likeness ≥ 0.18²¹. The putative targets corresponding to the retained compounds were subsequently retrieved, and all target identifiers were standardized to official gene symbols using the UniProt database²².

Retrieval of Disease-Related Targets and Intersection Genes

HF-related genes were collected from the GeneCards database (https://www.genecards.org/; accessed in January 2026) using “heart failure” as the search term, and genes with a relevance score ≥ 10 were selected for further analysis. The HF-related targets were subsequently intersected with compound-associated targets identified from TCMSP using the Weishengxin platform (https://www.bioinformatics.com.cn; accessed in January 2026). The overlapping genes were defined as intersection targets and were used for subsequent protein–protein interaction (PPI) network construction and functional enrichment analyses.

Network Construction and Functional Enrichment Analyses

PPI analysis of the identified targets was performed using the STRING database (http://cn.string-db.org/; accessed in January 2026)²³ with the species set to Homo sapiens and a confidence score threshold of ≥0.7. The interaction data were imported into the Cytoscape visualization tool for network visualization. Topological analysis was performed using the NetworkAnalyzer plugin in the visualization tool, and hub genes were identified using the CytoHubba plugin^24,25. Gene Ontology (GO) enrichment analysis was performed on the intersection targets, including biological process (BP), cellular component (CC), and molecular function (MF) categories. GO terms with a P value < 0.05 were regarded as significant, and the top 10 enriched terms from each category were selected for visualization²⁶. The intersection targets were further subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the clusterProfiler package in the statistical software. Pathways with a P value < 0.05 were considered statistically significant, and the top 20 enriched pathways were selected for visualization. These pathways were subsequently used to construct a target–pathway interaction network²⁷.

Statistical Analysis

Data are presented as mean ± SD. Normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test or Brown-Forsythe test prior to analysis. Statistical comparisons among groups were performed using one-way analysis of variance followed by Tukey’s post hoc test using GraphPad Prism. P < 0.05 was considered statistically significant.

Results

Transthoracic Echocardiography

Echocardiographic analysis revealed pronounced cardiac remodeling and dysfunction in TAC rats (Figure 1). Compared with Sham controls, TAC significantly increased LVIDd and LVPWd, while LVEF was markedly decreased. Notably, SXQY administration partially reversed these pathological changes, as reflected by a significant reduction in LVIDd and LVPWd and a concomitant improvement in LVEF compared with the TAC group.

...

Discussion

SXQY has been extensively used in clinical practice for over two decades, demonstrating efficacy in treating various cardiovascular conditions. Recently, its use has been extended to patients with heart failure with reduced ejection fraction and mildly reduced ejection fraction¹⁶. In the present study, a rat model was established by TAC surgery to mimic pathological changes of HF, and meanwhile some rats were treated with SXQY to examine its efficacy. Transthoracic echocardiography, HE staining, a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-chloro-L-phenylalanine	Aladdin	C105993-1g	Internal standard; ≥98%
Acetonitrile (LC-MS grade)	Aladdin	A120771-1L	Extraction solvent
Acetic acid	Solarbio	G1340	Masson staining; pre-cooled to 4 °C
Autosampler vials	Agilent	5190-9590	For LC-MS/MS sample loading
Buprenorphine	Cerilliant	B-044S	Analgesic
C18 column (1.8 μm, 2.1 mm × 100 mm)	Waters	176001125	ACQUITY Premier HSS T3 column
Capillary tubes, sterile	Fisherbrand	22-362-566	Retro-orbital blood collection
Centrifuge (refrigerated)	KUBOTA	N61196-F000	For serum and sample processing
clusterProfiler package	Bioconductor	—	R package for KEGG enrichment analysis; v4.0.2
Curved forceps, sterile	HL-SQX	HL-JP	Aortic arch manipulation
Cytoscape software	NHGRI	NIH NHGRI U24 HG012107	Version 6.4.1
Dedicated imaging platform	Fujifilm VisualSonics	VisualSonics Vevo 1100 ultrasound imaging system	Animal positioning during echocardiography
Distilled water	N/A	N/A	Decoction preparation and vehicle control
Electric heating mantle	Midea	WK2102	Decoction boiling
Endotracheal tube	CWE Inc., USA	SAR-1000	Endotracheal intubation; The endotracheal tube used in this study was integrated into the ventilator configuration and was not a separate or standalone device
Ethanol (absolute)	Beijing Yili Fine Chemicals Co., Ltd.	64-17-5	Dehydration and sterilization
Formic acid	Aladdin	O930702	Mobile phase additive
Gauze	Solarbio	YA0720	Decoction filtration; 200–300 μm pore size
GeneCards database	GeneCards	—	Disease-related target retrieval; accessed January 2026
GNPS database	University of California San Diego	—	Spectral database; version 1.3.16, data archived in December 2025
GraphPad Prism software	GraphPad Software	—	Version 10.1.2; SCR_002798
Heating pad (thermostatic)	Physitemp	HP-4M	Maintained at 37 °C
Hematoxylin and Eosin (H&E) Staining Kit	Solarbio	G1120	Histological staining
High-frequency transducer	VisualSonics	Vevo F2	18–40 MHz
High-resolution mass spectrometer	Thermo Fisher Scientific	Q Exactive	Q Exactive HF-X
High-resolution small-animal ultrasound system	Fujifilm VisualSonics	VisualSonics Vevo 1100 ultrasound imaging system	Vevo 2100
HMDB database	Wishart Research Group / The Metabolomics Innovation Centre, University of Alberta	—	Spectral database; HMDB current version 5.0; release 5.0; January 2022
In-house reference standard library	METWARE	—	Composition of the in-house reference standard library: The library contains a total of 5,000 compounds and includes both MS1 (precursor) information and MS2 (fragmentation) spectral data. Availability statement: The library data format is .txt. Storage condition: The database is stored on the company's internal server. Access permissions: This self-built standard library is a proprietary in-house spectral library of the company. It is used solely for metabolite identification, result verification, and quality traceability within the company's controlled analytical workflows. The library is not publicly available for download. Clients may obtain annotation results, hit information, and necessary field descriptions generated based on this library.
Isoflurane	Sigma-Aldrich	792632	Inhalation anesthetic
Liquid chromatography system (UPLC)	SCIEX	ExionLC AD	ExionLC AD
MassBank database	MassBank consortium; Mass Spectrometry Society of Japan / MassBank Europe	—	Spectral database; version 2024.06; data archived in December 2025
Masson’s Trichrome Stain Kit	Solarbio	G1340	Histological staining
Membrane filter (0.22 μm)	Beyotime	FF362	Polyethersulfone (PES)
METLIN database	The Scripps Research Institute / Siuzdak Lab	RRID: SCR_010500	Spectral database; this database does not provide a fixed annual version number; data downloaded in 2023
Methanol (LC-MS grade)	Beijing Yili Fine Chemicals Co., Ltd.	67-56-1	Extraction solvent
Microcentrifuge tubes (1.5 mL)	Corning Life Sciences (Wujiang) Co., Ltd.	MCT-150-C	Sample processing
Microtome	Leica	DSC1	Tissue sectioning
Neutral resin	Solarbio	G8590	Slide mounting
Nitrogen evaporator/gas source	Organomation	N-EVAP	Sample drying
Ophthalmic ointment	Aladdin	08-03-8009	Eye protection during anesthesia
Paraffin	Solarbio	YA0010	Tissue embedding
Paraformaldehyde (4%)	Solarbio	P1110	Tissue fixation
Phosphate-buffered saline	DU YOU YOU	DYP1021	Tissue washing
Phosphomolybdic acid	Solarbio	G1340	Masson staining
Povidone–iodine	Solarbio	IP3800	Surgical disinfection
ProteoWizard software	ProteoWizard Foundation	—	Version 3.0.19254
R software	R Foundation	—	Version 4.2.2
Rotary evaporator	QINGDAO JINGCHENG INSTRUMENT CO., LTD	JC-2LZF	Decoction concentration under reduced pressure
Silk suture (2-0)	FULAN	NL30RH26A-76	Aortic constriction
Small-animal ventilator	CWE Inc., USA	SAR-1000	Mechanical ventilation
Sprague–Dawley rats (male, 8 weeks)	SPF Bio Co., Ltd.	—	RGD_70508; License No. SCXK(jing)2024-0001
STRING database	STRING Consortium	—	Protein interaction analysis; accessed January 2026
Support vector regression normalization package/script	MetNormalizer	—	Signal drift correction; v1.3.02
TCMSP database	TCMSP	—	Compound and target retrieval; accessed January 2026
UniProt database	UniProt Consortium	—	Gene symbol standardization
Ultrasound gel	MAGNAFLUX	25-912	Echocardiography
Ultrapure water	Laboratory-prepared	—	Mobile phase solvent
Vortex mixer	Yalin	VM-ST	Sample mixing
Water bath	Julabo	PURA22	Section floating at 45 °C
Weishengxin platform	Weishengxin	—	Target intersection analysis; accessed January 2026
XCMS software	R Foundation	—	Version 3.16.1
Xylene	Aladdin	X775162	Deparaffinization