High-performance liquid chromatography (HPLC) analysis

PNS samples (S27243) and standard solutions of notoginsenoside R1 (B21099) and ginsenoside (Rg1, B21057; Re, B21055; Rb1, B21050; Rd, B21054) were purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). A 25 mg sample of PNS was placed in a 10 mL volumetric flask. Approximately 8 mL of 70% methanol was added, followed by ultrasonic treatment (640 W, 40 kHz) for 30 min. Then diluted to the mark with 70% methanol and mixed thoroughly.

HPLC were performed on the Agilent HPLC 1260 series system (Agilent, CA, USA). Samples were separated using C18 reversed phase Eclipse XDB column (5 μm, 4.6 mm × 250 mm). A flow rate of 1.3 mL/min and sample injection volume of 10 μL were employed. The detection wavelength was set at 203 nm, and the column temperature was maintained at 25 °C. The mobile phase comprised acetonitrile (A) and water (B), with a linear gradient established as follows: 0–20 min for 20% A, 20–55 min for 20% A to 46% A, and 55–60 min for 46% A.

Animal model

Animal experiments were approved by the Animal Ethics Committee of Yangzhou University (202209005). Sprague Dawley rats (male; 6-week-old, weighing 160 ± 20 g; n = 30) purchased from GemPharmatech (Jiangsu, China) were fed in a space at 23 ± 3 °C (12 h/12 h light/dark cycle, 44 ± 2% humidity) with access to water and food ad libitum. All rats were divided into five groups (n = 6): sham, model, model + 50 mg/kg d−1 PNS, model + 100 mg/kg d−1 PNS, and model + 200 mg/kg d−1 PNS groups. The CRS4 rat model was constructed as previously described [5]. Briefly, all rats were anesthetized intraperitoneally with 40 mg/kg ketamine and 5 mg/kg xylazine (Sigma-Aldrich, MO, USA) and fixed at a 37 °C electrical warming pad to keep body temperature. The artery of left kidney was momentarily occluded before the upper and lower poles of this kidney were ligated and removed. The left kidney remained with a third of it in this manner. Buprenorphine (0.03 mg/kg) was subcutaneously injected twice daily for 3 days to maintain postoperative analgesia. One week after, the right kidney was removed following the occlusion of renal pedicle. Sham rats received a similar procedure with exception that only renal envelopes were wiped off. PNS treatment rats were administered intragastrically with 50 mg/kg d−1, 100 mg/kg d−1, or 200 mg/kg d−1 of PNS, while rats in model group received saline solution.

Eight weeks after modeling, blood samples were collected via the tail vein for further experiments. The 24 h urine was collected using metabolic cages, and the urine protein was measured by an automatic chemistry analyzer (Thermo Fisher Scientific, MA, USA).

Echocardiography

All rats were anesthetized by intraperitoneally with 50 mg/kg sodium pentobarbital and put on a 37 °C pad in a supine position. According to manufacturer’s instructions, the 2D M-mode echocardiography was recorded through the Vevo 2100 ultrasound machine (Visual Sonics, Canada) with a 35 MHz ultrasound probe. Left ventricular posterior wall thickness at diastole (LVPWd), left ventricular internal diameter systolic (LVIDs), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were measured thrice and averaged.

Masson staining

All rats were sacrificed by intraperitoneally injecting with 4% sodium pentobarbital (200 mg/kg). After being excised and washed with phosphate-buffered saline (PBS; Beyotime, Shanghai, China), cardiac samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sliced into 4 μm sections. The slices were then stained with Weigert hematoxylin for 10 min and differentiated in 1% molybdenum phosphate acid. After being treated with decolorized blue aniline, slices were differentiated with 0.2% glacial acetic acid. Finally, the sections were dehydrated in absolute ethanol and rinsed in xylene. Light microscope (Olympus) was utilized to observe the slices.

Cell culture and treatment

H9c2 cells were supplied by Stem Cell Bank, Chinese Academy of Science (Shanghai, China) and cultured in high-glucose (33.3 mM) Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, UT, USA) with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C with 5% CO2 and constant humidity.

To construct hypoxia-injured cells, H9c2 cells were incubated in an atmosphere of 94% N2, 5% CO2, and 1% O2 for 4 h. Hypoxia-injured cells were cultured in 100, 200, or 400 μg/mL PNS for a further 4 h after hypoxia injury. Part of the hypoxia-injured cells was treated with 400 μg/mL PNS and/or 50 μΜ VX-765 (an inhibitor of caspase-1) for 4 h, and cellular morphology was surveyed by electron microscope (Olympus, Tokyo, Japan).

Cell transfection

Hypoxia-injured cells (2 × 105 mL/well) were seeded into six-well plates. ANRIL-overexpression (oe-ANRIL) pcDNA3.1 plasmid or negative control (NC, empty plasmid) were transfected into H9c2 cells using Lipofectamine 2000 reagent (Invitrogen, CA, USA). After transfection for 18 h, cells were incubated in a fresh DMEM for 48 h. Subsequently, stably transfected cells were treated with 400 μg/mL PNS for 4 h.

Cell counting kit-8 (CCK-8) assay

H9c2 cells (100 μL/well) were grown in 96-well plates at 37 °C with 5% CO2. Followed by 48 h-corresponding drug treatment, cells were treated with CCK-8 reagent (10 μL; Beyotime, China) for another 2 h. The optical density (OD) value at 450 nm was determined using microplate reader (Wuxi Hiwell Diatek, Jiangsu, China).

Flow cytometry for cell apoptosis

The apoptosis was determined using Annexin V-EGFP/propidium iodide (PI) double staining kit (Beyotime). Briefly, cells were trypsin digested and centrifugated at 1500 rpm for 5 min. After being resuspended with 195 μL Annexin V-EGFP binding solution, cells were treated with 5 μL Annexin V-EGFP and 10 μL PI staining for 20 min in dark. The apoptosis rate was analyzed using the FACSCanto flow cytometer (BD Biosciences, NJ, USA).

Immunofluorescence staining

H9c2 cells (5 × 105 cells/well) were added into 12-well plates. After fixing in 3% paraformaldehyde for 10 min, cells were washed with PBS (Beyotime) and permeabilized by 1% Triton X-100 (Solarbio, Beijing, China) for 5 min. To determine cell pyroptosis, H9c2 cells were stained with the PI solution for 15 min before being stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Beyotime) for 5 min at room temperature in dark. Cells were observed under a fluorescence microscope (Olympus).

For detection of expression on gasdermin D (GSDMD-N) and caspase-1, H9c2 cells were further sealed with 3% BSA solution (Solarbio) for 30 min. Next, cells were incubated with primary antibodies anti-N terminal of GSDMD-N (DF13758; 1:100, Affinity, TX, USA) and anti-caspase-1 (ab286125; 1:100, Abcam, Cambridge, UK) at 4 °C overnight and treated with secondary antibody FITC goat anti-rabbit IgG (H + L) (AS011; 1:500, ABclonal Technology, MA, USA) for a further 30 min at room temperature. Nucleus was stained by DAPI (Beyotime). The images were captured under a confocal microscope (Zeiss Microscopy, Jena, Germany).

Enzyme-linked immunosorbent assay (ELISA)

Corresponding ELISA kits were used to determine the levels of creatine kinase isoenzymes (CK-MB; ml092665; mlbio, Shanghai, China), lactate dehydrogenase (LDH; ml003416; mlbio), brain natriuretic peptide (BNP; ml003039; mlbio), blood urea nitrogen (BUN; ml092695; mlbio), plasma sulfate (IS; ab252894; Abcam), and serum creatinine (Scr; ml092663; mlbio). Cell suspension or cardiac serum was added into 50 μL standards in the testing sample wells, and blank wells were left empty. After treatment with 100 µL horseradish peroxidase-labeled enzyme conjugate working solution, cells or cardiac serum were sealed and incubated at 37 °C for 0.5 h. Subsequently, 50 µL substrates A and 50 µL B were added into each well at 37 °C for 15 min in dark, and 50 µL stop solution was then added to each well for 15 min treatment. OD values (CK-MB at 340 nm, BNP and LDH at 540 nm, Scr at 546 nm, BUN at 520 nm, and IS at 600 nm) were detected with a microplate reader (Wuxi Hiwell Diatek).

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

Total RNA of cardiac tissues or cells were extracted using Trizol reagent (Invitrogen) and reversely transcribed to cDNA with a Prime-Script reagent kit (Tiangen, Beijing, China). SYBR Green kit was used to detect relative mRNA levels on a 7500 Fast RT-PCR System (Biosystem, Singapore). The thermal cycling parameters were: 95 °C for 3 min; 45 cycles of 95 °C for 12 s and 62 °C for 40 s. GAPDH was utilized as an internal reference. Gene expression was determined through the 2−ΔΔCT method. Primer sequences were presented in Table 1.

Table 1 Primer sequences in RT-qPCRWestern blotting

Total proteins were extracted from cardiac tissues and H9c2 cells by radioimmunoprecipitation assay lysis buffer (Beyotime) and measured through a bicinchoninic acid protein assay kit (Beyotime). Protein samples (20 μg) were isolated by 10% SDS-PAGE (Beyotime) and transferred into polyvinylidene fluoride (PVDF; Beyotime) membranes. PVDF membranes were then treated with 5% skimmed milk/TBST for 1 h. Subsequently, the membranes were treated with primary antibodies overnight at 4 °C and secondary antibody for another 2 h at ambient temperature. The primary antibodies were as follows: anti-interleukin (IL)-1β (ab254360; 1:1000, Abcam), anti-nucleotide-binding oligomerization domain (NLRP3; ab263899; 1:1000, Abcam), anti-GSDMD-N (DF13758; 1:1000, Affinity), anti-caspase-1 (ab286125; 1 μg/mL, Abcam), anti-ASC (ab180799; 1:1000, Abcam), anti-transforming growth factor (TGF-β1; ab215715; 1:1000, Abcam), and anti-GAPDH (ab181602; 1:1000, Abcam). GAPDH was used as an internal reference. Finally, protein bands were visualized with an electrochemical luminescence on a Tanon 5200 machine (Tanon, Shanghai, China). Protein band intensities were detected with the Image J software (NIH, MD, USA).

Statistical analysis

Each assay was repeated at least in triplicate. Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software 8.0. A one-way analysis of variance followed by Tukey’s test was utilized for comparisons of multiple groups. p < 0.05 was regarded as statistically significant.