Experimental Animal Models and Handling

Six 9-week-old male Wistar rats (WT) and eighteen 9-week-old male spontaneously hypertensive rats (SHR), weighing 200–250 g, were purchased from the Experimental Animal Centre of Guizhou Medical University, and all animal experimental protocols were approved by the Animal Ethics Committee of Guizhou Medical University (No. 1900572). All animals were fed water ad libitum and housed at 20–25 °C. At 26 weeks, SHR blood pressure was at a stable high value. At this point we started daily administration of ALM (150 mg/kg/d) [35] and captopril (CAP) (50 mg/kg/d) [36] gavage treatment. Each group consisted of 6 rats. The treatment ended after 4 weeks. Body weight was measured weekly, and rats were executed at the end of treatment after pentobarbital sodium anesthesia [37] and blood sampling through the femoral artery, and cardiac tissue was removed and weighed to calculate the heart weight to body weight ratio.

Cell Culture

Rat cardiomyocyte (H9C2) cells were purchased from the American Type Culture Collection (ATCC). They were cultured in Gibco high glycemic medium (DMEM) containing 10% fetal bovine serum and placed in an incubator (5% CO2, 37 °C). Before establishing the myocardial hypertrophy model, H9C2 cells were pretreated with PI3K inhibitor (LY294002) at 10 μM for 1 h [38] and co-treated with 1 μM angiotensin II (Ang II) [5] combined with different concentrations of ALM for 48 h. The groups were control group, Ang II group, Ang II + 10 μM ALM group, Ang II + 50 μM ALM group, AngII + 100 μM ALM group, AngII + 150 μM ALM group, AngII + 200 μM ALM group, and AngII + 10 μM LY294002 + 100 μM ALM group.

Echocardiography

Rats were weighed and anesthetized with 1% pentobarbital sodium. Transthoracic 2-dimensional (2D), M-mode, and Doppler echocardiographic studies were performed with Mylab 50 (Esaote, Italy) using a high-resolution transducer (SL3116) with a frequency of 22 MHz. M-mode images were obtained to measure intraventricular septal thickness during diastole (IVSd) and left ventricular internal dimension (LVID) during diastole (LVIDd) and systole (LVIDs). The following parameters of systolic function were also calculated: left ventricle ejection fraction (LVEF), left ventricle fractional shortening (LVFS), left ventricular end systolic volume (LVESV), and left ventricular end diastolic volume (LVEDV). For each measurement, data from at least three consecutive cardiac cycles were averaged.

Blood Pressure Measurement

Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were assessed on a regular basis in the tails of conscious rats using a noninvasive computerized tailcuf system (Kent Scientific Corporation, CT, USA). Each rat was placed in a heated tube (38 °C) for 10–15 min, after which its body temperature was monitored to increase. Each rat was measured three times, and each set of rats was measured concurrently.

Immunoblotting

Protein samples were extracted from rat cardiac tissue, and protein concentrations were determined by BCA method, and the amount of protein spiked in each protein sample was calculated to be consistent. The protein samples were separated by gel electrophoresis according to the conventional method, transferred and closed at room temperature; primary antibodies Bax (Proteintech 1:2000 dilution), Bcl2 (Proteintech 1:1000 dilution), Fibronectin (Proteintech 1:1000 dilution), Col-III (Proteintech 1:1000 dilution), Cleaved-caspase3 (1:1000 dilution), Nrf2 (Proteintech 1:1000 dilution), HO-1 (Proteintech 1:1000 dilution), PI3K (Proteintech 1:5000 dilution), Akt (Cell Signaling Technology 1:1000 dilution), p-PI3K (Cell Signaling Technology 1:2000 dilution), and p-Akt (S473) (Cell Signaling Technology 1:1000 dilution) were added and incubated at 4 ℃ overnight; secondary antibodies were added and incubated at room temperature for 1 h. The protein bands were analyzed by the ImageJ image processing system with β-actin (Proteintech 1:10,000 dilution) as the internal reference, and semi-quantitative analysis was performed.

Histological Analysis

After echocardiographic examination, the rats were deeply anesthetized with pentobarbital sodium solution and executed by blood sampling through the femoral artery, and the heart tissues of each group were dissected and collected, fixed and then dehydrated, and embedded and sectioned (5 μm) to produce myocardial histopathological sections for hematoxylin–eosin (H&E) and Masson staining, and the myocardial histopathological changes were observed by light microscopy.

WGA Staining

The tissue sections underwent dewaxing in xylene and hydrating through a graded series of ethanol to water and were incubated for 1 h with WGA dying in the dark. Phosphate-buffered saline was used to washing the slides 3 times. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. The slides were sealed with antifade mounting medium and then observed using a fluorescence microscope.

ELISA

Myocardial tissues were collected and stored at − 80℃, 200 mg of myocardial tissues was grinded and prepared as homogenate, centrifuged at 3000 r/min for 15 min, and the supernatant was extracted, and the levels of IL-1β, IL-6, and TNF-α in myocardial tissues were measured by enzyme-linked immunosorbent assay (ELISA) kit (Mlbio, Shanghai China) according to the manufacturer’s instructions. A microplate absorbance reader (Thermo, USA) was employed to measure the absorbance at 450 nm. Standard curves were applied to calculate the analyte concentrations.

RNA Extraction and Quantitative Real-Time PCR (qPCR) Analysis

qPCR was used to detect the mRNA expression levels of hypertrophic markers ANP and BNP in Rats. Total RNA was obtained from tissue by the Trizol method, and then cDNA was synthesized using a reverse transcription instrument according to the manufacturer’s instructions. Real-time polymerase chain reaction was performed using SuperReal PreMix (Bio-Rad, USA), and qPCR SYBR Green Master Mix (YEASEN, Shanghai China) was performed. The sequences of the primers used were as follows: ANP forward, 5′-GAGAGTGAGCCGAGACAGCAAAC-3′ and ANP reverse, 5′-GAAGAAGCCCTTGGTGATGGAGAAG-3′; BNP forward, 5′-CCAGTCTCCAGAACAATCCACGATG-3′ and BNP reverse, 5′-GCCTTGGTCCTTTGAGAGCTGTC-3′; and β-actin forward, 5′-TGTCACCAACTGGGACGATA-3′ and β-actin reverse, 5′-GGGGTGTTGAAGGTCTCAAA-3′. β-Actin was used as the internal control. Primers were obtained from Sangon Biotech (Shanghai, China).

Related Index Testing

LDH, SOD, MDA, and CAT detection kits were purchased from Jian Cheng Institute of Biotechnology (Nanjing, China). Weigh 0.02 g of tissue, add saline at the ratio of weight (g): volume (mL) = 1:9, and make 10% homogenate by homogenizer, centrifuge to extract the supernatant. For the detection of MDA, the homogenate was used for the determination; for the detection of SOD, the sample was diluted 50 times with saline on the basis of 10%. The MDA content, SOD activity, LDH activity and CAT activity in rat heart tissues were measured according to the instructions of the LDH, SOD, MDA and CAT detection kits.

CCK8

Cell viability was analyzed by the Cell Counting Kit-8 (CCK8, Beyotime, China) according to the manufacturer’s protocols. Cells were seeded and cultured at a density of 5 × 103/well in 100 μL of medium in 96-well microplates (NEST, USA). Then, the cells were treated with various concentrations of ALM (10, 50, 100, 200, and 400 μmol/L). After treatment for 48 h, 10 μL of CCK-8 reagent was added to each well and then cultured for 2 h. All experiments were performed in triplicate. The absorbance was analyzed at 450 nm using a microplate reader (Thermo, USA) using wells without cells as blanks. The proliferation of cells was expressed by the absorbance.

Measurement of Cell Surface Area

Cells in each group were fixed with 4% paraformaldehyde; thereafter, cells were washed with 0.1%Triton X-100 and stained with Actin-Tracker Red-Rhodamine (1:150 dilution, Beyotime, China). A confocal microscope was used to photograph the cells after nuclear blocking with DAPI staining.

Reactive Oxygen Assay

Cells were inoculated in Confocal dishes (Biosharp, China) and treated with different drugs. 50 μM of reactive oxygen fluorescent dye H2DCFDA (MCE, China) was added to each well, and cells were incubated for 1 h in the incubator and washed with PBS, and the ROS production in each group was observed under a laser confocal microscope after nuclear blocking with DAPI staining.

Statistical Analysis

All data were expressed as mean ± standard deviation (SD) and were analyzed by SPSS Version 27 software. Differences between groups were determined and analyzed using one-way analysis of variance (ANOVA) followed by the LSD, using GraphPad Prism 8.0 (GraphPad Soft-ware, San Diego, CA, USA) to draw statistical charts. Statistical significance was defined as P < 0.05.