Resveratrol Suppresses Lung Cancer through the NLRP3/IL-1β Signaling Axis to Improve Immune Microenvironment​

Summary

Resveratrol suppresses lung cancer by inhibiting the NLRP3/IL-1β axis, thereby remodeling the immune microenvironment.

Abstract

The incidence and mortality rates of lung cancer remain high, posing a serious threat to human health. This study aims to investigate the role of resveratrol in lung cancer and its regulatory mechanism on the immune microenvironment. Human lung cancer A549 cells were treated with different concentrations of resveratrol, and a syngeneic mouse model of lung cancer was established to evaluate the effects of resveratrol and the NLRP3 inhibitor MCC950 on cell growth and the expression of NLRP3/IL-1β. Meanwhile, tumor growth, cell apoptosis, T cell infiltration, and the expression of key signaling proteins were examined in vivo. The results showed that resveratrol significantly inhibited the proliferation of A549 cells, induced cell cycle arrest and apoptosis, and was accompanied by reduced expression of NLRP3 and IL-1β. In the syngeneic mouse model of lung cancer, resveratrol treatment suppressed tumor cell proliferation, improved tumor morphology, enhanced T lymphocyte infiltration, and concurrently decreased the expression of NLRP3, IL-1β, ASC, and cleaved-caspase-1. These findings indicate that resveratrol inhibits tumor growth and remodels the immune microenvironment by targeting the NLRP3/IL-1β signaling axis, offering new insights and potential strategies for lung cancer immunotherapy.

Introduction

Lung cancer ranks first worldwide in both incidence and mortality among malignancies, responsible for 11.4% of all cancers and 18.0% of cancer-related deaths¹. The five-year survival rate of less than 20%² poses a serious threat to human life and health. The tumor immune microenvironment (TIME) importantly affects malignant phenotypes such as local infiltration, invasion, distant colonization, drug resistance, and immune evasion³. Composed of tumor cells, stromal cells, and immune cells, TIME is characterized by rapid proliferation of tumor cells, hypoxia, and chronic inflammation, which induce oxidative stress with an immunosuppressive milieu, and facilitate tumor progression and immune escape^4,5. The NOD-like receptor family protein 3 (NLRP3)/interleukin-1β (IL-1β) signaling pathway is a key mediator in many physiological and pathological processes⁶. NLRP3, together with apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase protease-1 (Caspase-1), constitutes the inflammasome, a crucial regulator of chronic inflammation⁷. It can sense endogenous danger signals in multiple organs, including the heart, liver, kidney, and lungs⁸. Upon activation by Toll-like receptors, the nuclear factor kappa-B (NF-κB) pathway is triggered, leading to increased pro-IL-1β and inflammasome components. Danger signals like extracellular ATP further promote the assembly of NLRP3 with ASC and pro-Caspase-1, activating Caspase-1 and cleaving pro-IL-1β into IL-1β and releases it into the extracellular environment. Additionally, Caspase-1 cleaves Gasdermin D to form membrane pores, inducing pyroptosis and amplifying the inflammatory response^9,10.

Experimental studies have demonstrated that NLRP3 knockout inhibits chronic intermittent hypoxia-induced upregulation of programmed cell death protein 1 (PD-1) / programmed death-ligand 1 (PD-L1) in tumors and significantly delay lung cancer progression¹¹. IL-1β activates NF-κB and mitogen-activated protein kinase (MAPK) pathways, enhances inflammation and Caspase-1 activity, promotes release of mature IL-1β, and induces pyroptosis, leading to inflammation-mediated cell death¹². Clinically, blocking IL-1α/IL-1β signaling can elevate the one-year and overall survival rates of non-small cell lung cancer (NSCLC) patients, as well as enhance the immune microenvironment¹³. Furthermore, NLRP3 and IL-1β is markedly elevated in NSCLC tissues and correlates with tumor cell migration and invasion¹⁴. Collectively, these findings highlight the NLRP3/IL-1β signaling axis as a promising therapeutic target in lung cancer.

Resveratrol, a natural polyphenolic compound, possesses anti-inflammatory, antioxidant, and antitumor properties¹⁵. In the study of Li S et al.¹⁶, resveratrol not only inhibited the assembly and activation of NLRP3 inflammasome to a certain extent, but also hindered secretion of IL-1β, thereby alleviating the progression of atherosclerotic lesions. Najafiyan B et al.¹⁷ believed that resveratrol effectively induced apoptosis of lung cancer cells and inhibited their proliferation by increasing pro-apoptotic proteins and decreasing anti-apoptotic proteins. However, direct evidence is still lacking regarding whether resveratrol influences the lung cancer immune microenvironment by regulating the NLRP3/IL-1β signaling axis. To date, no study has directly investigated the role of the NLRP3/IL-1β pathway in the modulation of lung cancer immune responses by resveratrol.

Therefore, we propose the following hypothesis: Resveratrol inhibits tumor growth and remodels the immune microenvironment by targeting the NLRP3/IL-1β signaling axis. Specifically, we hypothesize that resveratrol suppresses NLRP3 inflammasome activation, reduces IL-1β secretion, and subsequently enhances T cell infiltration and anti-tumor immune responses. To test this hypothesis, we conducted in vitro experiments and in vivo studies. By comparing the effects of resveratrol with those of the specific NLRP3 inhibitor MCC950, we aim to elucidate the mechanistic role of the NLRP3/IL-1β pathway in the anti-tumor activity of resveratrol. This study may provide new insights into the immunomodulatory mechanisms of resveratrol and offer potential therapeutic strategies for lung cancer immunotherapy.

Protocol

All animal experimental procedures were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Qiqihar Medical University and were conducted in strict accordance with the National Laboratory Animal Administration Regulations of the People's Republic of China and relevant ethical guidelines. The research protocol is provided in Supplementary Figure 1. The main experimental materials and instruments used in this study are listed in the Table of Materials.

Cell Culture
Human lung cancer A549 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL penicillin, 100 µg/mL streptomycin) in a 37 °C incubator with 5% CO₂. When cells reached 80%–90% confluence, they were digested with 0.25% EDTA-containing trypsin for subculture at a ratio of 1:3. All cell experiments were performed using cells in the logarithmic growth phase.

Cell Grouping and Intervention
Resveratrol was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mmol/L stock solution, which was aliquoted and stored at -20 °C protected from light until use. Prior to experimentation, the stock solution was diluted with complete medium to the desired working concentrations, ensuring that the final concentration of DMSO in each treatment group did not exceed 0.1% to exclude solvent effects on cell viability.

Each group had three replicate wells, and the experiment was repeated three times: blank control group (Control): complete medium containing 0.1% DMSO (consistent with the solvent concentration in drug-treated groups); low-concentration resveratrol group (30 µmol/L): complete medium containing 30 µmol/L resveratrol (final DMSO concentration ≤0.1%); medium-concentration resveratrol group (50 µmol/L): complete medium containing 50 µmol/L resveratrol (final DMSO concentration ≤0.1%); high-concentration resveratrol group (100 µmol/L): complete medium containing 100 µmol/L resveratrol (final DMSO concentration ≤0.1%).

Intervention procedure: A549 cells in the logarithmic growth phase were seeded into culture plates at an appropriate density (cell density was adjusted after counting to achieve 70%–80% confluence at the time of intervention). After 24 h of culture to allow cell attachment, the old medium was aspirated, and the drug-containing media from each group described above were added, respectively. Following an additional 24 h of culture, cells were collected for subsequent assays.

Construction of a homologous mouse model of lung cancer
Forty Mus musculus C57BL/6 (C57BL/6) mice, aged 7.23 ± 1.27 weeks and weighed 19.32 ± 1.32 g, were housed under normal conditions with free access to food and water. LLC cells were cultured in DMEM high-glucose medium. Cells in the logarithmic growth phase were collected, centrifuged, and resuspended in PBS. Digestion was performed using 0.25% EDTA-containing trypsin for 5 min, immediately terminated by adding complete medium to prepare a single-cell suspension, followed by centrifugation at 200 x g. for 5 min. The cell pellet was resuspended to a density of 1 x 10⁷ cells/mL. A 0.1 mL aliquot of the cell suspension was injected subcutaneously into the unilateral dorsal shoulder region of C57BL/6 mice using a 1 mL insulin syringe with a 27G needle to establish a syngeneic lung cancer transplantation model. Tumor formation was observed daily, and drug intervention was initiated when tumor volume reached 100 mm³ (calculated using the formula V = (L x W²)/2, where L is the long diameter and W is the short diameter).

Animal Group
Tumor-bearing mice from the syngeneic lung cancer model were randomly divided into 4 groups (n = 10 per group): Model group: intraperitoneal injection of an equal volume of normal saline; MCC950 group: MCC950 was freshly dissolved in sterile normal saline to a concentration of 2 mg/mL on each day of administration. The solution was freshly prepared on each day of administration. Mice received intraperitoneal injection of 10 mg/kg MCC950 once daily, with a dosing volume of 5 mL/kg; Resveratrol group: 50 mg/kg resveratrol dissolved in 0.5% sodium carboxymethyl cellulose solution, administered once daily by gavage (volume 10 mL/kg); Resveratrol + MCC950 group: resveratrol (50 mg/kg by gavage) and MCC950 (10 mg/kg by intraperitoneal injection) were administered concurrently via separate injections, once daily for 21 days. All groups were treated consecutively for 21 days. Starting from the first day of administration (recorded as day 0), tumor size was measured every 3 days using an electronic digital caliper, and tumor volume was calculated until the end of the experiment (day 21). Tumor growth curves for each group of mice were plotted based on these measurement data.

Observation of Cellular Experimental Indicators
Cell Counting Kit-8 (CCK-8) Assay for Cell Proliferation
A549 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 5 x 10³ cells per well in a volume of 100 µL. The plates were incubated overnight at 37 °C with 5% CO₂ to allow cell attachment. The old culture medium was aspirated, and complete media containing different concentrations of resveratrol (0, 30, 50, 100 µmol/L) were added, with five replicate wells set for each concentration. After incubation for 24, 48, and 72 h respectively, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 2 h in the incubator. The optical density (OD) value at a wavelength of 450 nm was measured using a microplate reader. Cell proliferation curves were plotted with time on the horizontal axis and OD values on the vertical axis. The experiment was repeated three times.

Flow Cytometry Analysis of Cell Cycle and Apoptosis
Cell Cycle Analysis: Approximately 1 x 10⁶ A549 cells were collected, washed with pre-cooled PBS, and fixed by adding 1 mL of pre-cooled 70% ethanol dropwise, followed by overnight incubation at -20 °C. After fixation, cells were washed twice with PBS and stained with 500 µL of PI/RNase A staining solution (containing 50 µg/mL PI and 100 µg/mL RNase A) for 30 min at room temperature in the dark. The stained cells were filtered through a 40 µm mesh and then analyzed by flow cytometry.

Apoptosis Analysis: Approximately 1 x 10⁵ A549 cells (including suspended dead cells) were collected, washed with PBS, and resuspended in 300 µL of 1x binding buffer. Then, 5 µL of Annexin V-FITC and 5 µL of PI staining solution were added. The cells were incubated for 15 min at room temperature in the dark and immediately analyzed by flow cytometry.

Detection Parameters: A flow cytometer with a 488 nm excitation wavelength was used. Fluorescein isothiocyanate (FITC) fluorescence signals were collected in the FL1 channel (515 nm), and PI fluorescence signals were collected in the FL2/FL3 channel (560 nm). A total of 20 000 events were collected per sample.

Gating Strategy (representative gating plots are shown in Supplementary Figure 2): First, the target cell population (P1) was gated in the FSC-A vs SSC-A scatter plot to exclude debris. Then, single cells (P2) were gated in the FSC-H vs FSC-W scatter plot to exclude doublets. Unstained, Annexin V-FITC single-stained, and PI single-stained tubes were prepared. The fluorescence compensation matrix was automatically calculated by the software and manually fine-tuned to ensure no spectral overlap between channels. Cell cycle distribution (G0/G1, S, and G2/M phases) was analyzed using ModFit software. For apoptosis analysis, Annexin V⁻/PI⁻ cells were defined as viable cells, Annexin V⁺/PI⁻ cells as early apoptotic cells, Annexin V⁺/PI⁺ cells as late apoptotic cells, and Annexin V⁻/PI⁺ cells as necrotic cells.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from cells and tumor tissues using the Trizol method: 1 mL of Trizol reagent was added to each sample for homogenization, followed by incubation at room temperature for 5 min. Then, 200 µL of chloroform was added, vigorously shaken for 15 s, and incubated at room temperature for 3 min. The mixture was centrifuged at 12 000 x g for 15 min at 4 °C. The upper aqueous phase was transferred to a new tube, mixed with an equal volume of isopropanol, incubated at room temperature for 10 min, and centrifuged at 12 000 x g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed with 1 mL of 75% ethanol, followed by centrifugation at 7 500 x g. for 5 min at 4 °C. After air-drying at room temperature, the RNA pellet was dissolved in 20–50 µL of DEPC-treated water. RNA purity was assessed using a UV spectrophotometer (OD260/OD280 ratio between 1.8–2.0), and the concentration was adjusted to a consistent level.

For cDNA synthesis, 1 µg of total RNA was reverse transcribed according to the instructions of the reverse transcription kit, with reaction conditions: 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min. Amplification was performed using the SYBR Green Premix Ex Taq kit on an HM-P16 real-time quantitative PCR system. The 20 µL reaction system contained: 10 µL of SYBR Green Premix Ex Taq (2x), 0.4 µL each of forward and reverse primers (10 µmol/L), 2 µL of cDNA template, and RNase-free ddH₂O to a final volume of 20 µL. The PCR reaction conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 34 s. Immediately after cycling, melting curve analysis was performed (95 °C for 15 s, 60 °C for 1 min, then ramping to 95 °C at 0.3 °C/s) to verify the specificity of the amplification products. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. Each sample was run in triplicate, and the average Ct value was calculated. The relative expression of target genes was determined using the 2^⁻ΔΔCt method. Primer sequences are listed in Supplementary Table 1. Prior to use, all primers were validated for amplification efficiency (90%–110%) through standard curves, and single peaks in melting curve analysis confirmed amplification specificity.

Western Blot
Cells or tumor tissues were collected and lysed in RIPA lysis buffer containing PMSF and phosphatase inhibitors on ice for 30 min. The lysates were centrifuged at 12,000 x g. for 10 min at 4 °C, and the supernatants were collected. Protein concentrations were determined using the bicinchoninic acid (BCA) method and normalized to a consistent level. Samples were mixed with 5x sodium dodecyl sulfate (SDS) loading buffer and denatured by boiling at 100 °C for 5 min. Proteins were separated by SDS-PAGE using 12% separating gels and 5% stacking gels. A total of 30 µg of protein per lane was loaded. Electrophoresis was performed at a constant voltage of 80 V for 30 min through the stacking gel, followed by 120 V for 60–90 min through the separating gel. Proteins were transferred onto methanol-activated polyvinylidene difluoride (PVDF) membranes using a wet transfer system at a constant current of 200 mA for 90 min in an ice bath. After transfer, membranes were blocked with 5% non-fat milk at room temperature for 1 h. Membranes were then incubated overnight at 4 °C with primary antibodies against NLRP3, IL-1β, ASC, Caspase-1, Cleaved-caspase-1, and GAPDH, all diluted 1:1000 in 5% non-fat milk in TBST. Following three 10-min washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted 1:3000 in 5% non-fat milk in TBST at room temperature for 1 h. After three additional TBST washes, enhanced chemiluminescence (ECL) chemiluminescent substrate was applied, and signals were detected using a gel imaging system. Band intensities were quantified using ImageJ software, and relative protein expression levels were calculated normalized to GAPDH.

Observation of Animal Experimental Indicators
Preparation of Single-Cell Suspension from Tumor Tissue and Cell Proliferation Assay (CCK-8 Method)
After the completion of drug administration, mice were euthanized by cervical dislocation, and tumor tissues were aseptically dissected. The tumor tissues were washed in pre-cooled PBS, cut into approximately 1 mm³ pieces, and digested with an appropriate amount of 0.25% trypsin-EDTA at 37 °C with constant shaking for 30 min. Digestion was terminated by adding DMEM medium containing 10% FBS. The cell suspension was filtered through a 70 µm cell strainer, and the filtrate was collected and centrifuged at 100 x g. for 5 min. The supernatant was discarded. The cell pellet was resuspended in complete medium, counted, and adjusted to a density of 1 x 10⁵ cells/mL. The cell suspension was seeded into 96-well plates at 100 µL per well. After 24 h of culture, 10 µL of CCK-8 reagent was added to each well, incubated for 2–4 h, and the OD value at 450 nm was measured.

Hematoxylin and eosin (HE) staining was used to observe tumor tissues
Fresh tumor tissues were fixed in 4% paraformaldehyde for 24 h. After dehydration through a graded ethanol series and clearing with xylene, the tissues were embedded in paraffin. Continuous sections of 4 µm thickness were cut using a microtome. The sections were routinely deparaffinized and rehydrated, then stained with hematoxylin for 5 min, differentiated in 1% hydrochloric acid ethanol for a few seconds, and blued in running tap water. Subsequently, the sections were stained with 0.5% eosin for 2 min. After dehydration and clearing again, the sections were mounted with neutral balsam. Tumor cell morphology, nuclear-to-cytoplasmic ratio, pathological mitotic figures, and inflammatory cell infiltration were observed under a light microscope and photographed for documentation.

Immunohistochemical Detection of T Lymphocyte Infiltration
Paraffin sections (4 µm) were routinely deparaffinized and rehydrated. Antigen retrieval was performed by placing the sections in citrate buffer (pH 6.0) in a microwave. After cooling, endogenous peroxidase activity was blocked by incubating with 3% H₂O₂ for 10 min at room temperature. Sections were then blocked with 5% BSA for 30 min at room temperature. Primary antibodies (rabbit anti-mouse CD4 monoclonal antibody, 1:500; rabbit anti-mouse CD8 monoclonal antibody, 1:200) were applied and incubated overnight at 4 °C. The next day, HRP-conjugated goat anti-rabbit secondary antibody was applied and incubated for 30 min at 37 °C. Diaminobenzidine (DAB) chromogen was used for visualization, followed by counterstaining with hematoxylin. After dehydration and clearing, sections were mounted. For each section, five non-overlapping fields were randomly selected under high magnification (x400), and CD4⁺ and CD8⁺ T cells were counted. Results were expressed as the number of positive cells/mm².

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay for Apoptosis Detection
Paraffin sections (4 µm) were deparaffinized and rehydrated, followed by antigen retrieval with 20 µg/mL DNase-free proteinase K at 37 °C for 20 min. After washing with PBS, the TUNEL reaction mixture was prepared according to the manufacturer's instructions. Then, 50 µL of TUNEL reaction mixture was applied to each section and incubated at 37 °C for 60 min in the dark. After PBS washing, nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min in the dark. The sections were mounted and observed under a fluorescence microscope. TUNEL-positive cells exhibited green fluorescence with an excitation wavelength of 450–500 nm and emission wavelength of 515–565 nm. Five high-power fields (x200) were randomly selected per section to count the number of TUNEL-positive cells and total cells. The apoptotic index was calculated as (number of TUNEL-positive cells / total number of DAPI-positive cells) x 100%.

Quantitative Real-Time PCR Detection of NLRP3 and IL-1β mRNA Expression in Tumor Tissues
Approximately 50 mg of frozen tumor tissue was ground into powder in liquid nitrogen. Subsequent total RNA extraction, reverse transcription, and qRT-PCR procedures and reaction conditions were identical to those described in the "Observation of Cellular Experimental Indicators" section. GAPDH was used as the internal reference gene, and the relative expression levels of target genes were calculated using the 2^⁻ΔΔCt method.

Western Blot Detection of NLRP3/IL-1β Pathway-Related Protein Expression in Tumor Tissues
Approximately 50 mg of frozen tumor tissue was homogenized on ice in RIPA lysis buffer containing a protease inhibitor cocktail. Subsequent protein extraction, quantification, electrophoresis, transfer, and antibody incubation steps were performed as described in the "Observation of Cellular Experimental Indicators" section. Band intensities were analyzed using ImageJ software and normalized to GAPDH as the internal reference.

Statistical Analysis
Measurement data were expressed as mean ± standard deviation (Mean ± SD). Before inter-group comparisons, the normality of data distribution was tested using the Shapiro-Wilk test, and homogeneity of variances was assessed using Levene's test. For data that followed a normal distribution and had homogeneous variances, comparisons between two groups were performed using the independent samples t-test; comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), followed by pairwise comparisons using the least significant difference t-test (LSD-t) test (if variances were homogeneous) or Tamhane's T2 test (if variances were not homogeneous). A P-value < 0.05 was considered statistically significant.

Results

Resveratrol treatment suppresses proliferation of A549 lung cancer cells, altered their cell cycle distribution, and promoted apoptosis.
CCK-8 assays showed that resveratrol at 30, 50, and 100 µmol/L markedly suppressed A549 cell proliferation compared with the control group (P<0.01), with the most pronounced effect observed in the 100 µmol/L group (Figure 1A). Resveratrol treatment caused cell cycle arrest, with the majority of A549 cells accumulating in the G0/G1 ph...

Discussion

The data indicate that the incidence of lung cancer in China has been increasing annually, accounting for 12.4% of global cancer cases¹⁸. Resveratrol, as a natural polyphenolic compound, has been proven to have anti-inflammatory, antioxidant and anti-tumor activities^{19, 20.} However, whether resveratrol exerts its anti-tumor effects by modulating the immune microenvironment has not yet been elucidated. This study is the first to reveal a novel mechanism by which resveratrol remodels the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
0.25% Trypsin-EDTA	Gibco (USA)	25200056	Cell dissociation
5-Fluorouracil	Solarbio (Beijing, China)	51-21-8	Positive control drug
Annexin V-FITC/PI Apoptosis Kit	BD Biosciences	556547	Apoptosis detection by flow cytometry
Anti-ASC antibody	Hubei Keyi Pharmaceuticals Ltd.	Ab155970	WB (1:1000)
Anti-Caspase-1 antibody	Hubei Keyi Pharmaceuticals Ltd.	Ab179515	WB (1:1000)
Anti-CD4 antibody	Abcam	Ab183685	IHC (1:500)
Anti-CD8 antibody	Abcam	Ab217344	IHC (1:200)
Anti-Cleaved-caspase-1 antibody	Hubei Keyi Pharmaceuticals Ltd.	Ab207802	WB (1:1000)
Anti-GAPDH antibody	Proteintech	60004-1-Ig	WB loading control (1:5000)
Anti-IL-1β antibody	Hubei Keyi Pharmaceuticals Ltd.	Ab9722	WB (1:1000)
Anti-NLRP3 antibody	Hubei Keyi Pharmaceuticals Ltd.	Ab214185	WB (1:1000)
BCA Protein Assay Kit	Thermo Fisher Scientific	23225	Protein quantification
C57BL/6 mice	WuXi AppTec	N/A	7-week old, male, for tumor model
CCK-8 Kit	Dojindo	CK04	Cell proliferation assay
CO? cell incubator	Shandong Bosheng Biotechnology Ltd.	BC-J160	Cell culture
Constant temperature water bath	Hangzhou Junsheng	HHS11-1	Sample incubation
Cryo-embedding machine	Jinhua Jili	LP-500	Tissue embedding
DMEM high-glucose medium	Gibco (USA)	11965092	Cell culture medium
ECL Chemiluminescent Substrate	Millipore	WBKLS0100	Western blot detection
Fetal Bovine Serum (FBS)	Gibco (USA)	10099141	Cell culture supplement
Flow cytometer	BD Biosciences	FACSCanto II	Cell cycle, apoptosis
Gel imaging system	Bio-Rad	ChemiDoc XRS+	Western blot detection
HRP-conjugated secondary antibody	Abcam	Ab205718	WB (1:3000)
Human lung adenocarcinoma A549 cells	Guangzhou Research Technology Ltd.	N/A	Cell line for in vitro study
Inverted fluorescence microscope	Aono Optics	AN-620	Cell imaging, TUNEL
MCC950	Hongxun Biotechnology Co., Ltd (Suzhou, China)	HY-12815	NLRP3 inhibitor
Microplate reader	Thermo Fisher Scientific	Multiskan FC	CCK-8, BCA assay
Murine lung carcinoma LLC cells	Shanghai Haicai Youshi Industrial Co., Ltd.	N/A	Cell line for in vivo model
Penicillin-Streptomycin	Gibco (USA)	15140122	Antibiotics for cell culture
Phosphatase Inhibitor Cocktail	Bimake	B14001	Phosphatase inhibitor
PI/RNase Staining Buffer	BD Biosciences	550825	Cell cycle detection by flow cytometry
PMSF	Beyotime	ST506	Protease inhibitor
PrimeScript RT reagent Kit	TaKaRa (Japan)	RR047A	Reverse transcription
Real-time PCR instrument (qRT-PCR)	Shenzhen Liangyi	HM-P16	Gene expression analysis
Resveratrol (purity 99%)	Jiangsu Duoyang Biotechnology Co., Ltd.	N/A	Drug intervention
RIPA Lysis Buffer	Beyotime	P0013C	Protein extraction
SYBR Green Premix Ex Taq	TaKaRa (Japan)	RR420A	qRT-PCR
Tissue spreading machine	Shenyang Hengsong	HS-TP-G	Section mounting
Trizol Reagent	Thermo Fisher Scientific	15596026	RNA extraction
TUNEL Assay Kit	Roche	11684817910	Apoptosis detection in tissue
UV analyzer	Xiaoxiao Shanghai Photon	E80100049	Nucleic acid analysis