The animal experimental protocol was approved by Air Force Medical University's Institutional Animal Care and Use Committee (approval no. 2017-01347). Healthy neonatal male Sprague-Dawley rats (male; age, 6–24 h; weight, 5–6 g) were obtained from the Institute of Zoology, Chinese Academy of Medical Sciences. The rats were maintained in specific pathogen-free conditions at 22±2°C and 60% humidity, with a 12-h light/dark cycle and free access to food and water. The primary cardiomyocytes were prepared from ventricles of neonatal rats according to a previously described protocol with slight modifications (27,28). In brief, rats were euthanized with an intraperitoneal injection of sodium pentobarbital (200 mg/kg) under sterile conditions. Subsequently, hearts were collected from rats and cut into pieces in Ca²⁺- and Mg²⁺-free D-Hanks balanced salt solution (Beyotime Institute of Biotechnology) supplemented with 0.1% trypsin. Then, 0.1% type II collagenase was added to digest the cells four times. Cells were centrifuged at 4°C and 120 × g for 5 min. The pellets were resuspended in DMEM (Gibco; Thermo Fisher, Scientific, Inc.) supplemented with 15% FBS (Gibco; Thermo Fisher, Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were transferred into 60-mm primary culture dishes (Corning Inc.) precoated with 1% gelatin at 37°C for 1.5 h to remove non-cardiomyocytes. Non-adherent cardiomyocytes were plated (1×10⁶ cells/dish) into 60-mm gelatin-precoated primary culture dishes. After 24 h, cardiomyocytes were washed with PBS (Beyotime Institute of Biotechnology) thrice and cultured in serum-free maintenance medium [80% DMEM, 20% M199 (Gibco; Thermo Fisher, Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin]. To validate cardiomyocytes, striated muscle-specific sarcomeric α-actin monoclonal antibody was employed.

Cardiomyocytes were cultured in serum-free maintenance medium at 37°C with 5% CO₂. At 80% confluence, cells were transfected with 100 nM non-targeting small interfering RNA (si)-negative control (NC; siNC), siHMGB1 or siPPARγ (all purchased from Shanghai GenePharma Co., Ltd.) for 48 h at 37°C using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following 48 h of transfection, the cells were used for subsequent experiments. The sequences of each siRNA were as follows: siNC, 5′-GCTCTGGAGCAGTTCCGATAT-3′; siHMGB1, 5′-CCATCACAGTGTTGTTAA-3′; and siPPARγ, 5′-TAACGAATGGGATTTGTCTG-3′.

Cardiomyocytes were divided into the following groups: i) Control, cardiomyocytes cultured in serum-free maintenance medium; ii) propofol, cardiomyocytes treated with propofol (Sigma-Aldrich; Merck KGaA; 12.5, 25, 50 or 100 µM) for 6, 12, 24 or 48 h; iii) LPS, cardiomyocytes stimulated with 1 µg/ml LPS (Escherichia coli 055:B5; Sigma-Aldrich; Merck KGaA) for 4 h; iv) propofol + LPS, cardiomyocytes treated with propofol (12.5, 25, 50 or 100 µM) for 6, 12, 24 or 48 h then stimulated with 1 µg/ml LPS for 4 h; v) propofol + LPS + siRNA, cardiomyocytes transfected with 100 nM siNC, siHBGB1 or siPPARγ for 24 h, treated with propofol (50 µM) for 24 h and then stimulated with 1 µg/ml LPS for 4 h; vi) propofol + LPS + inhibitor, cardiomyocytes pretreated with recombinant rat HMGB1 (rHMGB1; Chimerigen Laboratories; 100 ng/ml) or GW9662 (a PPARγ activation inhibitor; MedChemExpress; 10 µM) for 30 min followed by treatment with propofol (50 µM) for 24 h and then stimulation with 1 µg/ml LPS for 4 h. All the treatments were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

An MTT assay (Dojindo Molecular Technologies, Inc.) was performed to measure cell viability. Cardiomyocytes were seeded (2×10⁴ cells/well) into a 96-well plate. Following culture for 24 h, cells were treated as aforementioned. Subsequently, 10 µl MTT (0.5 mg/ml) was added to each well and incubated at 37°C for 4 h. Cell culture medium was removed and DMSO was added to dissolve the purple formazan crystals. The absorbance of each well was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad Laboratories, Inc.). Cell viability is presented as a percentage of the control.

Following treatment, the culture media from each group was collected. LDH release in the culture media was measured using the LDH assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

Cardiomyocyte apoptosis was determined using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences). Following treatment, cells were harvested, washed twice with ice-cold PBS (pH 7.4) and centrifuged at 300 × g at 4°C for 5 min. Cells were resuspended in 1X binding buffer to a final concentration of 5×10⁵ cells/ml. Subsequently, 5 µl Annexin V-FITC and 10 µl PI were added to the cells, gently vortexed and incubated in the dark at 37°C for 15 min. Apoptotic cardiomyocytes were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software (version 3.3; BD Biosciences). Early (positive Annexin V-FITC staining) plus late apoptotic cells (positive PI staining) were counted to determine the levels of cardiomyocyte apoptosis.

Cardiomyocyte MMP was measured using a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) detection kit (Nanjing Jiancheng Bioengineering Institute). JC-1 is a dual-emission MMP sensing dye that selectively aggregates in polarized (healthy) mitochondria and aggregated JC-1 emits red fluorescence. Mitochondrial depolarization (loss of MMP) prevents JC-1 from entering the mitochondria, thus monomeric JC-1 remains in the cytosol and emits green fluorescence. Following treatment, cardiomyocytes were washed with PBS thrice, incubated with 200 µM JC-1 at 37°C for 15 min and washed twice with PBS. The fluorescence of aggregated JC-1 (red) was visualized at an emission wavelength of 590 nm and the fluorescence of monomeric JC-1 (green) was visualized at a wavelength of 529 nm using a fluorescence microscope (magnification, ×400; Carl Zeiss AG). The ratio of red to green fluorescence intensity was calculated to determine the MMP.

Caspase-3 activity was detected to evaluate cell apoptosis using a Caspase-3 activity assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Caspase-3 catalyzes the conversion of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into pNA, which displays a strong absorption peak at a wavelength of 405 nm. Following treatment, cardiomyocytes were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 1,000 × g at 4°C for 15 min. The supernatants were harvested to evaluate caspase-3 activity. The supernatant (50 µl) was added to 2X reaction buffer (50 µl) and Ac-DEVD-pNA (50 µl) and incubated at 37°C for 4 h. Absorbance was measured at a wavelength of 405 nm using a microplate reader.

Following treatment, total RNA was extracted from cardiomyocytes using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using PrimeScript RT master mix (Takara Bio, Inc.). The temperature protocol for the reverse transcription was 5 min at 25°C, 60 min at 42°C and 15 min at 70°C. Subsequently, qPCR was performed using SYBR-Green Premix-Ex Tag kit (Takara Bio, Inc.) on an ABI 7500 Fast Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation for 10 min at 95°C; followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. The following primers were used for qPCR: TNF-α forward, 5′-TGGAACTGGCAGAAGAGGCACT-3′ and reverse, 5′-GTTCAGTAGACAGAAGAGCGTGGTG-3′; IL-6 forward, 5′-AGAAAAGAGTTGTGCAATGGCA-3′ and reverse, 5′-GGCAAATTTCCTGGTTATATCC-3′; IL-1β forward, 5′-CACTACAGGCTCCGAGATGAACAAC-3′ and reverse, 5′-TTGTCGTTGCTTGGTTCTCCTTGT-3′; IL-18 forward, 5′-GACTGGCTGTGACCCTATCTGTGA-3′ and reverse, 5′-TTGTGTCCTGGCACACGTTTC-3′; and GAPDH forward, 5′-TCCATGACAACTTTGGTATCG-3′ and reverse, 5′-TGTAGCCAAATTCGTTGTCA-3′. mRNA expression levels were quantified using the 2^−ΔΔCq method (29) and normalized to the internal reference gene GAPDH.

Following treatment, cardiomyocyte culture media was harvested and centrifuged at 500 × g at room temperature for 5 min. The supernatants were pooled to measure TNF-α, IL-6, IL-1β and IL-18 levels using rat TNF-α (cat. no. 560479; BD Biosciences), IL-6 (cat. no. 550319; BD Biosciences), IL-1β (cat. no. ab255730; Abcam) and IL-18 (cat. no. ab213909; Abcam) ELISA kits, respectively, according to the manufacturer's protocols.

Following treatment, cardiomyocytes were harvested, lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 1,000 × g at 4°C for 10 min. Protein concentrations in the supernatant were quantified using a BCA assay kit (Beyotime Institute of Biotechnology). Proteins (30 µg) were separated via 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% skimmed milk in TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Subsequently, the membranes were incubated at 4°C overnight with primary antibodies targeted against: NLRP3 (Santa Cruz Biotechnology, Inc.; cat. no. sc-134306; 1:1,000), ASC (Santa Cruz Biotechnology, Inc.; cat. no. sc-514414; 1:1,000), pro-caspase-1 (Santa Cruz Biotechnology, Inc.; cat. no. sc-56036; 1:800), caspase-1 p10 (Santa Cruz Biotechnology, Inc.; cat. no. sc-514; 1:500), pro-IL-1β (Santa Cruz Biotechnology, Inc.; cat. no. sc-12742; 1:500), IL-1β (Santa Cruz Biotechnology, Inc.; cat. no. sc-515598; 1:1,000), pro-IL-18 (Abcam; cat. no. ab191860; 1:100), IL-18 (Abcam; cat. no. ab223293; 1:1,000), HMGB1 (Abcam; cat. no. ab79823; 1:10,000), PPARγ (Abcam; cat. no. ab272718; 1:1,000) and β-actin (Abcam; cat. no. ab6276; 1:5,000). Following primary antibody incubation, the membranes were washed three times with TBST and incubated with an HRP-conjugated goat anti-rabbit IgG (Abcam; cat. no. ab6721; 1:5,000), HRP-conjugated rabbit anti-mouse IgG (Abcam; cat. no. ab6728; 1:5,000) or HRP-conjugated goat anti-Armenian hamster IgG (Abnova; cat. no. PAB9133; 1:3,000) secondary antibodies for 1 h at room temperature. The membranes were washed three times with TBST. Protein bands were visualized using an ECL kit (Pierce; Thermo Fisher Scientific, Inc.) followed by exposure to X-ray films. β-actin was used as the loading control.

Following treatment, cardiomyocytes were harvested, lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 1,000 × g at 4°C for 10 min. Protein concentrations in the supernatant were quantified using a BCA assay kit. Cell lysates (500 µg) were precleared with 20 µl of protein A/G-agarose beads (Santa Cruz Biotechnology, Inc.), according to the manufacturer's protocol, and incubated with anti-ASC antibody (Santa Cruz Biotechnology, Inc. cat. no. sc-514414) or normal IgG at 4°C overnight with gentle agitation. Subsequently, 20 µl protein A/G-agarose beads were added to the immune complexes and incubated for 6 h at 4°C. The resultant mixtures were centrifuged at 250 × g at 4°C for 5 min and the supernatants were removed. The pellets were washed three times with PBS and western blotting was performed according to the aforementioned protocol. Proteins were separated via SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies targeted against: NLRP3, ASC and caspase-1. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using an ECL kit.

Data are presented as the mean ± SD from three independent experiments. Statistical analyses were performed using SPSS software (version 17.0; SPSS, Inc.). Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Propofol has been reported to attenuate LPS-induced myocardial injury (22,23). The MTT and LDH release assays were performed to determine the effects of propofol on LPS-induced cardiomyocyte damage. Compared with the control group, LPS stimulation significantly reduced cardiomyocyte viability and enhanced LDH release, whereas propofol (25, 50, or 100 µM) pretreatment for 24 h significantly increased cell viability and decreased LDH release in LPS-stimulated cardiomyocytes in a concentration-dependent manner; however, there were no significant differences observed between the 50 and 100 µM propofol groups (Fig. 1A and B). Compared with the LPS group, LPS-stimulated cardiomyocytes that were pretreated with 50 µM propofol for different durations (12, 24, and 48 h) displayed significantly increased cell viability and decreased LDH release in a time-dependent manner; however, there was no significant difference observed between the 24 and 48 h groups (Fig. 1C and D). Therefore, 50 µM propofol treatment for 24 h was selected for subsequent experiments due to its protective effects against LPS-induced injury in cardiomyocytes. The results indicated that propofol ameliorated LPS-induced cardiomyocyte injury.

The effect of propofol on LPS-induced cardiomyocyte apoptosis and inflammation was assessed. The flow cytometry results indicated that compared with the control group, LPS significantly increased cell apoptosis, which was significantly reduced by pretreatment with propofol (Fig. 2A and B). Propofol also significantly abolished LPS-induced loss of MMP (Fig. 2C). Moreover, LPS-induced increases in caspase-3 activity were significantly reduced by pretreatment with propofol (Fig. 2D). The mRNA expression levels of proinflammatory cytokines, including TNF-α and IL-6, were significantly increased by LPS treatment compared with the control group. However, pretreatment with propofol significantly decreased TNF-α and IL-6 expression levels in LPS-treated cardiomyocytes (Fig. 2E and F). Similarly, LPS-injured cardiomyocytes that were pretreated with propofol released significantly less TNF-α and IL-6 compared with the LPS group (Fig. 2G and H). The results demonstrated that propofol alleviated LPS-induced cardiomyocyte apoptosis and inflammation.

The NLRP3 inflammasome is a prominent and early mediator of inflammatory responses in myocardial injury (30). NLRP3 inflammasome activation serves an important role in high glucose-induced H9c2 cell toxicity (31). Therefore, whether propofol prevented NLRP3 inflammasome activation in LPS-induced cardiomyocytes was investigated. Propofol markedly reduced LPS-induced increases in the protein expression levels of NLRP3, ASC, caspase-1 p10, IL-1β and IL-18 in cardiomyocytes (Fig. 3A). The Co-IP assay results demonstrated that the formation of the NLRP3-ASC-pro-caspase-1 complex was notably inhibited by pretreatment with propofol in LPS-injured cardiomyocytes (Fig. 3B). LPS-induced upregulation of IL-1β and IL-18 mRNA expression levels was significantly decreased by pretreatment with propofol (Fig. 3C and D). Propofol also significantly inhibited IL-1β and IL-18 release in LPS-injured cardiomyocytes (Fig. 3E and F). Collectively, the results suggested that propofol inhibited LPS-induced NLRP3 inflammasome activation in cardiomyocytes.

HMGB1 produced by cardiomyocytes contributes to LPS-induced myocardial dysfunction (14). To assess whether propofol reduced LPS-induced HMGB1 expression in cardiomyocytes, western blotting was performed. Compared with the control group, LPS notably increased HMGB1 expression levels, which were obviously reduced by pretreatment with propofol (Fig. 4A). HMGB1 activates the NLRP3 inflammasome in vascular smooth muscle cells (13). Subsequently, whether propofol-mediated inactivation of the NLRP3 inflammasome was HMGB1-dependent was investigated. siHMGB1 transfection markedly decreased HMGB1 protein expression levels in control and LPS-stimulated cardiomyocytes compared with siNC transfection (Fig. 4B). In LPS-stimulated cardiomyocytes, propofol-mediated downregulation of NLRP3, ASC, pro-caspase-1 and caspase-1 p10 protein expression levels were obviously counteracted by rHMGB1 pretreatment, but notably enhanced by siHMGB1 transfection (Fig. 4C). Similarly, rHMGB1 pretreatment significantly attenuated propofol-mediated inhibition of IL-1β and IL-18 release in LPS-stimulated cardiomyocytes, whereas HMGB1 knockdown enhanced the inhibitory effects of propofol on IL-1β and IL-18 release (Fig. 4D and E). The results suggested that propofol inhibited NLRP3 inflammasome activation by reducing HMGB1 expression in LPS-injured cardiomyocytes.

PPARγ inhibits LPS-induced HMGB1 release in RAW 264.7 cells (15). Previous studies have revealed that propofol represses inflammation by activating PPARγ in the renal tissues of sepsis model mice (32) and by upregulating PPARγ expression in THP-1 macrophage-derived foam cells (33). Compared with the control group, PPARγ expression was notably downregulated in LPS-injured cardiomyocytes, which was reversed by pretreatment with propofol (Fig. 5A). Subsequently, whether PPARγ was involved in propofol-mediated inactivation of the HMGB1-dependent NLRP3 inflammasome in LPS-injured cardiomyocytes was assessed. In control and LPS-stimulated cardiomyocytes, PPARγ protein expression levels were obviously lower in siPPARγ-transfected cardiomyocytes compared with control or siNC-transfected cardiomyocytes (Fig. 5B). siPPARγ transfection or GW9662 treatment reversed propofol-mediated downregulation of HMGB1, NLRP3, ASC, pro-caspase-1 and caspase-1 p10 protein expression levels in LPS-injured cardiomyocytes (Fig. 5C). Moreover, propofol-mediated inhibition of IL-1β and IL-18 release was significantly reversed by siPPARγ transfection or GW9662 treatment in LPS-injured cardiomyocytes (Fig. 5D and E). The results indicated that PPARγ was involved in propofol-mediated inactivation of the HMGB1-dependent NLRP3 inflammasome in LPS-injured cardiomyocytes.