The Chinese Academy of Sciences (Shanghai, China) provided the human dopaminergic neuroblastoma SH-SY5Y cell line, while Gibco (Thermo Scientific, Shanghai, China) supplied the fetal bovine serum (FBS). Invitrogen (Thermo Scientific, Shanghai, China) supplied the Minimum Essential Medium (MEM), F12, GlutaMAX, sodium pyruvate, non-essential amino acid and penicillin/streptomycin. Antibodies against NLRP3 (1:1000; Proteinch; 00080435); ASC (1:1000; Proteinch; A16672); IL-1β (1:1000; ABclonal; A16288); IL-18 (1:1000; ABclonal; A16737); GSDMD/N-GSDMD (1:1000; Abcam; ab209845); GSDME/N-GSDME (1:1000; Abcam; ab215191); cleaved caspase-3 (1:1000; CST; 9664S), cleaved caspase-1 (1:1000; CST; 4199T) and β-actin (1:1000; CST; 4970S) were separately obtained from attached commercial company. Additionally, dimethyl sulfoxide (DMSO), 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), and N-Acetyl-L-cysteine (NAC) were acquired from Sigma-Aldrich (Shanghai, China). All related chemical agents were of analytical grade.
Human dopaminergic neuroblastoma SH-SY5Y cell lines were cultured in MEM/F12 medium supplemented with 10% FBS, 1% GlutaMAX, 1% sodium pyruvate, 1% non-essential amino acid, and 1% penicillin/streptomycin. A humidified incubator with air levels at 95% and a CO2 level of 5% was used to cultivate the cells at 37°C. The growth medium was changed at intervals of 2 to 3 d.
Thereafter, the cells were placed into 96-, 24- or 6-well plates and left to incubate for 24–72 h. The existing media were then replaced with new media comprising varying concentrations of ACR and left to incubate for a further 24–72 h.
The viability of the cells was determined using MTT evaluation . Briefly, a 96-well plate was used to seed SH-SY5Y cells at 8×103 cells per well, after which they were treated for 24 h, 48 h and 72 h, with different ACR concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5 mM each. Next, 10 µL of MTT (5 mg/mL) was added to each well and they were incubated for 2 h at 37°C. The media was replaced by 100 µL DMSO, after which a TECAN Infinite M200 PRO microplate reader was used to determine the absorbance at 570 nm. The control cell formazan density was regarded as 100%, while the cytotoxicities of the different groups were presented as proportions of the control group.
LDH assay and cell morphological examination
Cell pyroptosis was determined via LDH release using a CytoTox 96 cytotoxicity assay kit (Promega, Shanghai, China), according to the manufacturer’s instructions. Briefly, the cells were seeded into a transparent 6-well plate. When the cells had grown approximately 80%, they were treated with ACR prepared with serum-free medium for 24 h. The cell supernatant was subsequently collected and transferred into a new 96-well plate and left to react for 30 min in the dark with the LDH detection working solution. The quantitative LDH levels were measured using a TECAN Infinite M200 PRO microplate reader (Tecan Trading AG, Switzerland) at 490 nm.
Cell morphology was observed using a Nikon Eclipse TI microscope (Nikon, Tokyo, Japan) at 100×/250× magnification. Briefly, cells were cultured in a 24-well plate at a density of 105 cells/well, after which they were treated for 24 h with different concentrations of ACR. Cellular ballooning was denoted with black arrows.
Measurement of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) contents
For NAC treatment, the cells were pre-treated with 500 µM NAC for 1 h, and then co-treated with ACR for 24 h. A 2,7-dichlorofluorescin diacetate (DCFH-DA) staining kit (E004-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to measure the ROS formation. Meanwhile, the cellular GSH (A006-2-1) and MDA (A003-1-2) concentrations were measured according to the directions of the supplier (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). A bicinchoninic acid (BCA) protein assay kit (A045-4-2, provided by the Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used for cell lysis, employing an ultrasonic cell disruptor for protein concentration determination.
Western blot analysis
Radioimmunoprecipitation (RIPA) assay buffer (Beyotime, Shanghai, China) was used to extract cell pellets for 30 min, after which the lysates were subjected to centrifugation at 4°C for 10 min at 12,000 g to remove the insoluble substances. Thereafter, the protein was extracted from the supernatant and kept at 95°C for 5 min. The proteins (20–50 µg) were then separated using SDS-PAGE and moved to poly (vinylidene fluoride) (PVDF) membranes (GE Healthcare, IL, USA). The membranes were blocked with 5% fat-free milk (Sangon Biotech, Shanghai, China) or 5% bovine serum albumin (BSA) (Bio-Sharp, Hefei, China) for 1 h at room temperature, after which they were incubated overnight at 4°C with specific primary antibodies. The secondary HRP-conjugated antibodies were subsequently washed three times with tris-buffered saline with Tween 20 (TBST), then used to incubate the membranes at room temperature for 1 h. Protein detection was accomplished using an enhanced chemiluminescence (ECL) assay, obtained from Bio-Rad (Shanghai, China), while a Tanon 4200 imaging system (Tanon, Shanghai, China) was used for scanning.
The expression levels of IL-1β, IL-18 and tumor necrosis factor-α (TNF-α) in the cell supernatant were measured using an ELISA kit (Elabscience, Houston, TX), according to the manufacturer’s instructions. Briefly, 50 µL cell supernatant of each sample was added into a pre-coated 96-well microplate and left for 2 h at room temperature. The wells were then washed four times with the provided washing solution, biotinylated antibody was added and the cells were cultured at room temperature for 1 h. Thereafter, the washing process was repeated before HRP-streptavidin solution was added and the cells were then incubated at room temperature for 15–30 min, after which time a stop solution was added, and absorbance was measured at 450 nm immediately on a microplate reader.
Chemical inhibitor treatment
Doses of Ac-DEVD-CHO (Beyotime, Shanghai, China) and Ac-YVAD-CMK (Santa Cruz Biotechnology, Shanghai, China) were chosen via an MTT cell viability assay. For chemical inhibitor treatment, the SH-SY5Y cells were pre-treated for 24 h with either the Ac-DEVD-CHO or Ac-YVAD-CMK, and then treated either with or without ACR for 24 h. After the inhibition, the levels of LDH in the different treatment groups were measured in cell supernatant to check the inhibition efficiency.
Short interfering RNA (siRNA) silencing
The SH-SY5Y cells were transfected with a negative control siRNA (Santa Cruz Biotechnology, Shanghai, China) or caspase-1 siRNA/caspase-3 siRNA (Santa Cruz Biotechnology, Shanghai, China) using a Lipofectamine 3000 Kit (Thermo Fisher Scientific, USA), according to the manufacturer’s instructions. Briefly, cells were seeded onto 6-well plates and transfected with a mixture comprising the siRNA duplexes, Lipofectamine 3000, and Opti-MEM reduced serum medium (Gibco, USA). Following incubation for 48 h, the medium was replaced with a fresh cell cultural medium containing 10% FBS. Cells were then treated with either ACR or a vehicle (phosphate buffered saline (PBS)) and left for 24 h, after which they were harvested for LDH levels measurement and protein expression analyses.
Each assay was conducted at least in triplicate. Data was analyzed using the GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA), and the results were introduced as mean ± SD. Statistical comparisons between the experimental groups were evaluated by one-way analysis of variance (ANOVA) with Duncan's multiple comparison test using SPSS software v. 21. Consequently, p < 0.05 values were regarded as statistically significant and were denoted by different letters. Western blotting gray analysis was performed using the Image J imaging processing program (Wayne Rasband, National Institutes of Health, USA).