Nob (>99.05%) was purchased from Selleck Chemicals (S2333; Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The final concentration of DMSO was maintained at <0.1% in all experiments and treatments. PA (P0500; Sigma-Aldrich; Merck KGaA) was dissolved in 0.1 M NaOH at 70°C and then complexed with 10% bovine serum albumin (BSA) at 55°C for 10 min to achieve the final palmitate concentration (100 mM). The antibodies against SIRT1 (1:2,000; cat. no. ab110304), pro-Caspase-1 (1:1,000; cat. no. ab179515), Caspase-1 (1:5,000; cat. no. ab201476), IL-18 (1:500; cat. no. ab71495), IL-1β (1:1,000; cat. no. ab9722) and β-actin (1:5,000; cat. no. ab8226) were purchased from Abcam (Cambridge, UK), and the antibody against NLRP3 (1:1,000; cat. no. 15101) was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-mouse and anti-rabbit IgG (1:5,000, cat. nos. ab205719 or ab205718) secondary antibodies were also purchased from Abcam. The small interfering RNA (siRNA) of SIRT1 (sc-40987) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

AML-12 cells, an immortalized normal mouse hepatocyte cell line, were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Science, Inc.) and insulin-transferrin-selenium (Sigma-Aldrich; Merck KGaA) at 37°C under 5% CO₂.

SIRT1-siRNA transfection was performed according to the manufacturer's protocol of the transfection reagent. Prior to transfection, the cells were seeded in 6-well plates at a concentration of 5×10⁵ cells/ml. Cells were transfected with 50 nM SIRT1-siRNA or 50 nM negative control using X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) for 36 h at 37°C.

AML-12 cells were suspended in DMEM/F12 and plated at a density of 1×10⁴ cells/well in 96-well plates. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit (Dojindo Co., Kumamoto, Japan). Cells were treated with various concentrations (0, 10, 20, 50, 100, 200, 400 or 800 µM) of Nob for 12 h; or with 100 µM Nob for various times intervals (0, 2, 4, 8, 16, 24, 48 or 72 h), or different concentrations (0, 50, 100 or 200 µM) of Nob with 400 µM PA for 12 h. All cells were incubated at 37°C. Subsequent to the indicated treatments, 10 µl CCK-8 solution was added to each well and then incubated for 4 h at 37°C in 5% CO₂. The absorbance at 450 nm was measured with a spectrophotometer (Thermo Fisher Scientific, Inc., Shanghai, China).

Cells were treated with various interventions, following which the culture medium was collected and the supernatant was obtained by centrifugation (3,000 × g for 10 min) at 4°C. The concentrations of IL-1β and IL-18 in the supernatant were assayed with ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. The intra- and inter-assay coefficients of variation were 5.0 and 8% for IL-1β, and 6.8 and 7% for IL-18, respectively. Every sample was analyzed in triplicate, and absorbance values were read at 450 nm using a spectrophotometer.

Western blotting was performed as previously described (2,22). Total protein in hepatocytes was extracted using a protein extraction kit (cat. no. C510003; Sangon Biotech Co., Ltd., Shanghai, China). Briefly, the protein concentration in hepatocyte extracts was determined using a protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China). A total of 30 µg of protein from each sample was separated by 12% SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride membrane. Next, the membranes were blocked in 3% BSA/Tris-buffered saline-Tween 20 (TBS-T) buffer at room temperature for 4 h. The blocked membranes were then incubated overnight at 4°C with primary antibodies. The membranes were then washed with TBS-T and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG at room temperature for 45 min. Immunoreactive bands were visualized by enhanced chemiluminescence solution (EMD Millipore, Temecula, CA, USA). All bands were analyzed using Image-pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The total RNA was extracted from AML-12 cells using RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. The RNA concentration and quality were measured using a K5500 micro-spectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China) and by electrophoresis on 1% agarose gels, respectively. Next, 1 µg total RNA in each sample was reverse-transcribed into cDNA using an RT kit (Takara Biotechnology Co., Ltd.) according to the supplier's protocol. The mRNA expression levels were then evaluated using qPCR technology with the SYBR Green QuantiTect RT-PCR kit (Takara Biotechnology Co., Ltd.) and a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was conducted under the following conditions: Initial denaturation at 94°C for 2 min; 35 cycles of amplification, including denaturation at 94°C for 10 sec, annealing at 60°C for 15 sec and extension at 72°C for 30 sec; and final extension at 72°C for 5 min. The primers used are shown in Table I. The relative expression of each target gene was normalized to β-actin and calculated using the 2^−∆∆Cq method (23).

Data are expressed as the mean ± standard deviation. Statistical analyses were conducted using IBM SPSS version 19.0 software (IBM Corp., Armonk, NY, USA). Statistical significance was calculated using Student's t-test in cases of comparisons between two groups, or using one-way ANOVA with subsequent Bonferroni correction in cases of comparisons among more than two groups. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of Nob is presented in Fig. 1A. The direct cytotoxicity of Nob was assessed by CCK-8 assay. The results revealed that Nob at concentrations up to 400 µM did not affect cell viability, while cell viability was significantly reduced at 800 µM (Fig. 1B). Furthermore, as shown in Fig. 1C, exposing AML-12 cells to Nob for 72 h at a concentration of 100 µM did not influence the cell viability (Fig. 1C). In addition, PA (400 µM) alone caused significant cytotoxicity, while treatment with Nob exerted dose-dependently protective effects on the cell viability following PA stimulation (Fig. 1D). The expression of apoptosis-associated molecules was also examined. The results demonstrated that PA exposure alone significantly increased the mRNA expression levels of apoptosis-associated molecules, including Caspase-3, Caspase-9 and B-cell lymphoma 2-associated X protein, whereas co-treatment with Nob reversed the effects of PA on mRNA expression of these molecules (Fig. 1E-G). Taken together, the results indicated that Nob alleviated PA-induced cytotoxicity in AML-12 cells. Consequently, the present study selected the concentrations of 50, 100 and 200 µM Nob for treatments in subsequent experiments.

Initially, the effects of PA on NLRP3 inflammasome activation were measured, and it was revealed that PA significantly increased NLRP3 inflammasome activation, and IL-1β and IL-18 secretion in AML-12 cells (Fig. 2A-G). Notably, Nob decreased the mRNA and protein expression levels of NLRP3, Caspase-1, IL-1β and IL-18 in a dose-dependent manner in PA-treated AML-12 cells (Fig. 2A-E). Furthermore, Nob also suppressed the secretion of IL-1β and IL-18 cytokines in a dose-dependent manner in PA-treated AML-12 cells (Fig. 2F and G). Taken together, these results indicated that Nob inhibits PA-induced NLRP3 inflammasome activation in AML-12 cells.

As shown in Fig. 3A, Nob increased SIRT1 protein expression in a dose-dependent manner. PA treatment alone evidently decreased the protein expression of SIRT1; however, Nob reversed the inhibitory effect of PA on SIRT1 expression (Fig. 3B). These results demonstrated that Nob upregulates SIRT1 expression in AML-12 cells.

To investigate whether SIRT1 mediated the protective effect of Nob, the present study used SIRT1-siRNA to knockdown SIRT1. NC siRNA did not affect SIRT1 expression (Fig. 4A) or cell viability (data not shown) compared with the blank control. The results revealed that SIRT1-siRNA significantly reduced the mRNA and protein expression levels of SIRT1 compared with with NC group in AML-12 cells (Fig. 4A and B). Notably, Nob was unable to suppress the PA-induced NLRP3 inflammasome activation in the presence of SIRT1-siRNA (Fig. 4C-J). Specifically, Nob decreased the mRNA and protein expression levels of NLRP3, Caspase-1, IL-1β and IL-18 in the presence of PA, while this effect was reversed by SIRT1-siRNA (Fig. 4D-H). The concentrations of IL-1β and IL-18 exhibited the same pattern (Fig. 4I and J). Furthermore, Nob was unable to reverse the PA-induced reduction in cell viability in the presence of SIRT1-siRNA (Fig. 4K). In summary, these results demonstrated that Nob suppresses PA-induced NLRP3 inflammasome activation in a SIRT1-dependent manner.