Research on the mechanism of the anti-myocardial infarction effect of the Qiliqiangxin capsule on heart failure rats via nontargeted metabolomics and lipidomics

Instrument

Laryngoscope (YL04-IIG, Jiangsu Yongle Medical Technology Co., Ltd.), Animal Respiratory Machine (HX-300 S, Chengdu Taimeng Software Co., Ltd.), Biological Signal Acquisition and Analysis System (BL-420 N, Chengdu Taimeng Software Co., Ltd.), VisualSonics Vevo 2100 Imaging System (VisualSonics, Canada), Fully Automatic Digital Slice Scanning and Applied Imaging (iScan Coreo, Roche, USA), SHIMADZU Ultra High Performance Liquid Chromatography System (Shimadzu, Japan), SCIEX 5600 + Quadrupole Time of Flight Mass Spectrometer (SCIEX, USA) were used.

Reagents and animals

QLQX capsule were purchased from Shijiazhuang Yilin Pharmaceutical Co. (Hebei, China). NTpro-BNP and hs-cTn-I ELISA test kits were purchased from Shanghai Yuanju Biotechnology Co., Ltd. (Shanghai, China). A hematoxylin and eosin (H&E) staining kit was purchased from Biosharp, Inc. (Beijing, China). The Masson trichrome staining kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Methanol (chromatographic grade) and acetonitrile (chromatographic grade) were purchased from Thermo Fisher (USA), ammonia aqueous (mass spectrometry grade) was purchased from Sigma (USA). Watsons distilled water.

Forty adult male SD rats, SPF grade, with a body weight of 200 ± 20 g, were purchased from the Experimental Animal Center of Jiangxi University of Chinese Medicine, with the animal production license number SCXK (Gan) 2023-0001. The animals were allowed to freely feed standard feed and tap water, and feeding environment at 20–25 °C room temperature, 45–65% humidity, and 12 h of dark/light cycling. One week before the start of the experiment, all the rats were adaptively fed for one week and fasted for 12 h before surgery.

Animals and experimental design

SD male rats were anesthetized and placed in a supine position fixed on the operating table. Tracheal intubation was performed under laryngoscopy and assisted by a ventilator for ventilation. The left anterior chest surgical area was routinely prepared for skin disinfection. A horizontal incision was made in the fourth intercostal space on the left side, the muscles were separated layer by layer, hemostatic forceps were used to open the intercostal space, the pleura was broken to expose the heart, then opened the pericardium, and the anterior descending branch of the left coronary artery was ligated with a 6–0 suture line below the left atrial appendage 2–3 mm. After ligation, the myocardium on the anterior wall of the left ventricle turned pale to the naked eye, and a typical myocardial infarction pattern was observed on the electrocardiogram. After closure, the chest wall muscles and skin were sutured layer by layer. Preoperative and postoperative electrocardiogram changes were recorded. After surgery, a ventilator was used to assist in ventilation, the mice were placed on a constant-temperature blanket at 37 °C, and their condition was closely monitored. After the mouse was fully awake and turned over, it was returned to the cage.

We demonstrated the successful establishment of the heart failure model through the following indicators. The inclusion criteria were as follows: electrocardiogram changes; and postoperative electrocardiogram showing ST segment elevation in corresponding leads, accompanied by pathological Q waves for sometimes. Cardiac function parameters such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (lvfs) should also be measured. Histological changes, such as cardiomyocyte hypertrophy and interstitial fibrosis, and the determination of plasma NT proBNP (N-terminal pro brain natriuretic peptide) levels. Behavioral changes, such as reduced activity and weight loss. The exclusion criteriawere as follows: if no pale discoloration of the tissue in the anterior descending branch blood supply area and no electrocardiogram changes, such as ST segment elevation in the corresponding lead, were observed during the ligation process in the rats, the model was considered to have failed. Moreover, it is necessary to observe the death or near death status, mental state, behavioral activity, weight and other changes of the animals after surgery and eliminate rats that experienced extreme emaciation or death.Through the above standards, the successful construction and stability of the rat myocardial infarction model can be ensured, providing a reliable experimental basis for related research.

Valsartan is an AngII receptor antagonist that is commonly used in clinical practice and is known to have cardioprotective effects against LV remodeling after myocardial infarction. Therefore, we chose valsartan as the positive control drug. The day after modeling, the medication was administered by gavage, and the rats were randomly divided into a SHAM group, an MI group, a QLQX group, and a VAL(valsartan) group, with 8 rats in each group. The SHAM group underwent the same surgical procedure without ligation, whereas the MI, QLQX, and VAL groups underwent ligation of the left anterior descending coronary artery according to the modeling criteria. The SHAM group and MI group were given physiological saline at a dose of 1 ml/kg/d, while the QLQX group was given Qili Qiangxin capsule at a dose of 1.3 g/kg/d, and 1 g of QLQX was equivalent to 6.3 g of the total amount of 11 herbs contained in the QLQX. The positive control drug valsartan capsules were administered to the VAL group at a dose of 80 mg/kg/d starting from the day after modeling and were administered by gavage for a total of 4 weeks.

After the experiment, the rats were euthanized via cervical dislocation. An external force was used to dislocate the animal’s cervical spine, causing the spinal cord and brain to disconnect, resulting in painless death of the experimental animal. Owing to its advantages of quickly losing consciousness, reducing pain, easy operation, and not damaging animal organs, it is considered a good method of euthanasia for experimental animals.

Evaluation of cardiac function

Echocardiography was performed on the rats in each group via the VisualSonics Vevo 2100 imaging system (VisualSonics, Canada). At the 4th week after surgery, under anesthesia, cardiac color Doppler ultrasound was performed via M-mode ultrasound to measure the left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular end diastolic diameter (LVIDd), interventricular septal diastole (IVSd), and systolic thickness at the level of the mitral chordae tendineae. Rats with an LVEF < 60% were considered to have heart failure. Among the 40 rats, 4 died during surgery, and 4 did not meet the criteria for chronic heart failure after surgery. The remaining 24 rats were divided into 4 groups (n = 8/group), namely, the SHAM group, MI group, QLQX group, and VAL group.

Biochemical analysis and histopathological examination

The concentrations of NT-proBNP and hs-cTn-I in the serum were detected via an enzyme-linked immunosorbent assay kit according to the manufacturer’s protocol (Shanghai Yuanju Biotechnology Co., Ltd., China). The collected heart tissue was fixed in 4% paraformaldehyde for 24 h, embedded in a paraffin block, sliced and stained with H&E and Masson’s trichrome. H&E staining was used to observe the pathological morphological changes in the myocardial cells, and Masson staining was used to observe the interstitial fibrosis of the myocardium. Stained sections were scanned and imaged via fully automated digital slicing technology (Roche, USA, iScan Coreo), and relevant parts of the tissues at different magnifications were captured via VENTANA Image Viewer software. The fibrosis score of the infarct boundary area was analyzed via Image Pro Plus software.

Plasma sample pretreatment

For metabolomics, after the last dose, 100 µL of orbital plasma supernatant from each rat was taken, and 300 µL of methanol: acetonitrile (1:1) solution was added to precipitate the protein. Vortex shaking was performed for 2 min, and high-speed centrifugation was performed at 16,000 r/min for 10 min. The supernatant was collected and analyzed. For lipidomics, after the last dose, 100 µL of orbital plasma supernatant from each rat was taken, and 400 µL of isopropanol solution was added to precipitate the protein. Vortex shaking was performed for 2 min, followed by overnight freezing at -20 °C. The supernatant was centrifuged at a speed of 16,000 r/min for 10 min at ultrahigh speed and analyzed.

UPLC-QTOF-MS conditions

For metabolomics, an Acquity UPLC BEH amide column (100 mm × 2.1 mm, 1.7 μm, Waters) were used, and the mobile phases were composed as 10 mM ammonium acetate and 0.1% ammonia aqueous water solution (A) - acetonitrile (B), gradient elution procedure: 0–1 min, 95% B, 1–1 min, 95–65% B, 14–16 min, 65–40% B, 16–18 min, 40% B, 18–18.1 min, 40–95% B, 18.1–23 min, and 95% B. The flow rate was 0.3 mL/min, the column temperature was set at 40 °C, and the injection volume was 3 µL. For lipidomics, an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm, Waters) with a mobile phase of 5 mM ammonium acetate methanol: acetonitrile: aqueous solution (1:1:1) (A) -5 mM ammonium acetate isopropanol solution (B), gradient washing procedure: 0–0.5 min, maintaining 20% B, 0.5–1, 5 min, 20–40% B.

The MS data were collected by using a SCIEX Triple TOF 5600 + mass spectrometer. ESI electric spray ion source mode, positive and negative ion scanning, positive ion voltage 5500 V, negative ion voltage − 4500 V. Gas 1/Gas 2 50 psi; Cur gas 35 psi. The first-level TOF MS scanning range is 100–1500 Da, with a DP of 60 V. The second level uses IDA non-data-dependent mode for data collection, with a secondary collision energy of 30 V in metabolomics experiments and 45 ± 20 V in lipidomics experiments. The MS/MS scanning range was 50–1500 Da. In the overall scanning mode, the data collection runs in dynamic background subtraction (DBS) mode.

MS data processing

Metabolomics data processing involves importing raw data into MetDNA (Metabolite identification and Dysregulated Network Analysis) online data processing software. After peak recognition, extraction, and normalization, compound result identification is performed on the data. After screening the results for mass errors < 5 ppm, the data were imported into SIMCAP software (Umetrics, Umea, Sweden) for principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) of the data matrix. PCA is an unsupervised dimensionality reduction method used to observe global clustering trends and dispersion within populations. OPLS-DA, as a supervised modeling method, can remove data variables independent of the independent variable X and categorical variable Y and distinguish different metabolites between different groups. According to the OPLS-DA model, potential biomarkers were screened under the threshold conditions of a VIP value > 1 and a t-test < 0.05. The screened biomarkers were imported into MetaboAnalyst online data processing software for pathway enrichment analysis. Lipidomic data processing involves importing raw data into MS-Dial software for peak recognition, extraction, and normalization, identifying and analyzing lipid composition results, and importing data into SIMCAP software for processing and analysis. Statistical analysis was conducted via SPSS 2.0 software (IBM SPSS Inc., Chicago, USA). All the data are expressed as the means and standard deviations (± s), with P < 0.05 indicating significant differences and P < 0.01 indicating extremely significant differences.

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