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Original Research Article Resveratrol

inhibits

NLRP3

inflammasome

activation

by

preserving

mitochondrial integrity and augmenting autophagy† Ya-Ping Chang1, Shuk-Man Ka2, Wan-Han Hsu3, Ann Chen4, Louis Kuoping Chao5, Chai-Ching Lin1, Cho-Chen Hsieh1, Ming-Cheng Chen1, Huan-Wen Chiu1, Chen-Lung Ho6, Yi-Chich Chiu7, May-Lan Liu8, Kuo-Feng Hua1

1

Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan; 2Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan; 3Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan; 4Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; 5Department of Cosmeceutics, China Medical University, Taichung, Taiwan; 6Division of Wood Cellulose, Taiwan Forestry Research Institute, Taipei, Taiwan; 7Department of Biomechatronic Engineering, National Ilan University, Ilan, Taiwan; 8Department of Nutritional Science, Toko University, Chiayi, Taiwan *To whom correspondence should be addressed: Kuo-Feng Hua, Department of Biotechnology and Animal Science, National Ilan University, No. 1, Sec. 1, Shen-Lung road, Ilan, 260, Taiwan, Tel: +(886)-3-935-7400, ext. 7626; Fax: +(886)-3-931-1526; E-mail: [email protected]

†This

article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.24903] Additional Supporting Information may be found in the online version of this article. Received 25 June 2014; Revised 12 December 2014; Accepted 18 December 2014 Journal of Cellular Physiology

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DOI 10.1002/jcp.24903

Accepted Article

Running title: Resveratrol inhibits NLRP3 inflammasome Keywords: Resveratrol; NLRP3 inflammasome; mitochondria; autophagy

Contract grant sponsor: Ministry of Science and Technology, Taiwan; Contract grant number: NSC 102-2628-B-197-001-MY3; NSC 103-2923-B-197-001-MY3

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Abstract The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that controls the release of IL-1β and plays important roles in the development of inflammatory disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome-derived IL-1β secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. In addition, resveratrol administration mitigates

glomerular proliferation, glomerular

sclerosis, and

glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy. This article is protected by copyright. All rights reserved

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Introduction The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that

controls caspase-1 activity and the release of IL-1β and IL-18 in the innate immune system (Latz et al., 2013). A strong link between the NLRP3 inflammasome and the development of inflammatory disease is becoming increasingly evident. Recent studies using NLRP3 inflammasome knockout mice have shown that the NLRP3 inflammasome controls the pathogenesis of type II diabetes mellitus (Wen et al., 2011), atherosclerosis (Duewell et al., 2010), Alzheimer's disease (Halle et al., 2008.), gout (Martinon et al., 2006), cancers (Bruchard et al., 2013; Chow et al., 2012), and renal disease (Kim et al., 2013). Although clinical application of this knowledge has not yet been achieved due to a lack of a specific NLRP3 inflammasome inhibitor, drug-like antagonists targeting NLRP3-inflammasome-associated pathways are under development (López-Castejón et al., 2012). Chronic kidney disease (CKD) is one of the leading causes of mortality in the

world. Renal inflammation is one of the major pathways underlying the progression of CKD into the later stages of renal disease, chronic renal failure and uremia. However, there is no specific treatment for most types of CKD. We previously established a progressive IgA nephropathy (Prg-IgAN) model in mice that is suitable for investigating the progression of IgA nephropathy, the most common type of

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primary glomerulonephritis (Hua et al., 2013; Yang et al., 2013; Ka et al., 2011; Chao et al., 2006). Our previous studies showed that the NLRP3 inflammasome is activated in the Prg-IgAN model, and inhibiting the NLRP3 inflammasome ameliorates renal function and reduces glomerular proliferation and periglomerular mononuclear leukocyte infiltration (Hua et al., 2013; Yang et al., 2013). Resveratrol is a polyphenolic compound that is naturally produced by several

plants, especially the skin of red grapes. The biological functions of resveratrol were widely investigated and include anti-inflammation (Zong et al., 2012), anti-oxidation (Hao et al., 2013), anti-cancer (Yang et al., 2013), cardiovascular protection (Danz et al., 2009), anti-diabetes (Jimenez-Gomez et al., 2013), renal protection (Wen et al., 2013),

protection

against

Alzheimer's

disease

(Feng

et

al.,

2013)

and

antihyperuricemic activities (Shi et al., 2012). Recent studies showed that resveratrol reduces NLRP3 inflammasome activation induced by ionizing irradiation (Fu et al., 2013), LPS with ATP (Huang et al., 2013), a high-fat diet (Yang and Lim, 2014), and isoflurane (Li et al., 2014); however, the detailed mechanisms are unclear. In this study, we demonstrate that resveratrol inhibits NLRP3 inflammasome activation in macrophages by reducing both the priming and activating signal of the NLRP3 inflammasome. Resveratrol reduced mitochondrial damage and increased autophagy, leading to decreased NLRP3 inflammasome activation. We also demonstrate that

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resveratrol has a beneficial effect in a mouse Prg-IgAN model by reducing superoxide anion production and inhibiting NLRP3 inflammasome activation.

Materials and Methods Materials Resveratrol (cis form) was purchased from the Cayman Chemical Company

(Ann Arbor, MI, USA). LPS (from Escherichia coli 0111:B4), ATP, actinomycin D and

mouse

antibodies

against

mouse

phospho-ERK1/2,

phospho-JNK1/2,

phospho-p38, and actin were purchased from Sigma (St. Louis, MO). Rabbit antibodies against mouse phospho-PKC-, phospho-PKC-δ, IL-1, and caspase-1 and horseradish peroxidase-labeled secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against mouse LC3 and p62 were obtained from Cell Signaling Technology (Beverly, MA). Mouse IL-1, IL-6, and TNF- ELISA kits were purchased from R&D Systems (Minneapolis, MN). A mouse anti-mouse NLRP3 antibody was purchased from Enzo Life Sciences Inc. (Exeter, UK). The AlamarBlue® assay kit was purchased from AbD Serotec Ltd (Oxford, UK).

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Cell cultures The murine macrophage cell line J774A.1 was obtained from the American Type

Culture Collection (Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine

(all from Life Technologies, Carlsbad, CA) at 37C in a 5% CO2

incubator. Peritoneal macrophages were elicited by intraperitoneal injection of 4% sterile thioglycollate medium (2 ml). After 3 days, the mice were killed by cervical dislocation and the macrophages were harvested. The peritoneal macrophages were grown in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 2.5 g/ml amphotericin B, and 10% FBS. Stable PKCδ knockdown J774A.1 cells (sh-PKCδ cells) and control cells (sh-SC cells) were established by stable transfection using shRNA plasmids targeting PKCδ and a scrambled sequence, respectively (OriGene Technologies, Inc., Rockville, MD).

IL-1β secretion and caspase-1 activation For IL-1β secretion and caspase-1 activation in Fig. 1A, cells were incubated with

or without the addition of resveratrol for 30 min, then for 5.5 h with or without 1 g/ml LPS, then with or without the addition of 5 mM ATP for 0.5 h. Then, IL-1 in the culture medium was measured by ELISA and activated caspase-1 (p10) and

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pro-caspase-1 (p45) in cells was measured by Western blot. For IL-1β secretion in Fig. 1B, 2 and 3, cells were incubated for 5.5 h with or without 1 g/ml LPS, then with or without resveratrol for 30 min, then with or without ATP (5 mM, 0.5 h), nigericin (10 µM, 0.5 h), MSU (500 µg/ml, 24 h), CPPD crystals (100 µg/ml, 24 h), nano SiO2 (200 µg/ml, 24 h), alum crystals (500 µg/ml, 24 h), FLA-ST (100 g/ml, 24 h), or MDP (100 ng/ml, 24 h). IL-1 in the culture medium was measured by ELISA. For caspase-1 activation, the cells shown in Fig. 1B were incubated for 5.5 h with or without 1 g/ml LPS, then with or without addition of resveratrol for 30 min, then for 0.5 h with or without 5 mM ATP. Then, the levels of p10 and p45 in the cells were measured by Western blot. For the IL-1β secretion shown in Fig. 7D, cells were incubated with or without 1 g/ml LPS for 5.5 h, then with or without SB203580 for 30 min, then with or without 60 µM resveratrol for 30 min, and then with or without 5 mM ATP for 30 min. The IL-1 level in the culture medium was measured by ELISA. For the IL-1β secretion shown in Fig. 7, J774A.1 cells (Fig. 7D) or sh-SC and sh-PKCδ cells (Fig. 7E) were incubated with or without 1 g/ml LPS for 5.5 h, then with or without SB203580 (1 µM in Fig. 7E) for 30 min, then with or without 60 µM resveratrol for 30 min, and then with or without 5 mM ATP for 30 min. The IL-1 level in the culture medium was measured by ELISA.

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Phosphorylation levels of MAPK and PKC and protein expression of proIL-1β, NLRP3, LC3 and p62 Cells were treated as indicated, and the protein phosphorylation and expression

levels were measured by Western blot. The detailed procedures for Western blotting were described in a previous study (Liao et al., 2013).

ROS detection General ROS production was measured by detecting the fluorescence intensity of

2’,7’-dichlorofluorescein, the oxidation product of 2’,7’-dichlorofluorescein diacetate (Molecular Probes, Eugene, OR). For ATP-induced ROS generation, J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml LPS, then with or without 60 µM resveratrol, 10 mM N-acetyl cysteine (NAC), or DMSO (vehicle) for 30 min, then incubated with 2 µM 2’,7’-dichlorofluorescein diacetate for an additional 30 min, and then stimulated with 5 mM ATP for 0-80 min. The fluorescence intensity of 2’,7’-dichlorofluorescein was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm on a microplate absorbance reader (Bio-Rad Laboratories, Inc.).

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Mitochondrial function For measurement of the inner transmembrane potential and mitochondrial mass,

mitochondrial

ROS

generation, and mitochondrial

DNA release,

J774A.1

macrophages were incubated for 5.5 h with or without 1 μg/ml LPS, then incubated with or without 60 μM resveratrol or DMSO for 30 min, then for 30 min with or without 5 mM ATP, 500 µg/ml MSU, 100 µg/ml CPPD crystals, or 200 µg/ml nano SiO2. To measure the inner transmembrane potential and mitochondrial mass, cells were stained for 15 min at 37°C with 25 nM MitoTracker Deep Red and MitoTracker Green (Invitrogen), washed with PBS, treated with trypsin and resuspended in PBS containing 1% heat-inactivated FBS. Data were acquired by flow cytometry. For mitochondrial ROS generation, cells were stained with 5 µM MitoSOX (Invitrogen) for 20 min, and then the fluorescence intensity of MitoSOX was detected at an excitation wavelength of 514 nm and an emission wavelength of 585 nm on a microplate absorbance reader. For mitochondrial DNA release, the cells were homogenized in a buffer containing 100 mM pH 7.4 Tricine-NaOH, 0.25 M sucrose, 1 mM EDTA and protease inhibitors and then centrifuged for 10 min at 700 x g at 4°C. The protein concentration and volume of the supernatant were normalized, followed by centrifugation for 30 min at 10,000 g at 4°C. The resulting pellets were identified as the mitochondrial fraction, and the supernatants were identified as the

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cytosolic fraction. DNA was isolated from 200 μl of the cytosolic fraction, and the mtDNA copy number was measured by quantitative PCR and normalized to the nuclear DNA (represented by 18S ribosomal RNA) levels as the ratio of cytochrome c oxidase I DNA over nuclear DNA. The results were expressed as the fold change between the sample and the control group, so the control group would have a fold change of one. The quality of the mitochondrial fraction and the cytosolic fraction was monitored by detecting the expression level of the voltage-dependent anion channel protein (VDAC, a marker protein for mitochondria). We found that VDAC could only be detected in the mitochondrial fraction, but not in the cytosolic fraction (data not shown). These results indicate that the cytosolic fraction is free from mitochondria.

Primers

were

designed

as

follows:

5’-TAGAGGGACAAGTGGCGTTC-3’,

mouse

18S,

forward, reverse,

5’-CGCTGAGCCAGTCAGTGT-3’; mouse cytochrome c oxidase I, forward, 5’-GCCCCAGATATAGCATTCCC-3’,

reverse,

5’-GTTCATCCTGTTCCTGCTCC-3’.

Pyroptosis detection J774A.1 macrophages were incubated for 5.5 h with or without 1 g/ml LPS,

then with or without resveratrol for 30 min, then with or without 5 mM ATP for 0.5 h.

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For the LDH release assay, the supernatants were evaluated for the presence of the cytoplasmic enzyme LDH using the CytoTox 96® Non-radioactive Cytotoxicity Assay kit according to the manufacturer’s instructions (Promega, Madison, WI). The percentage of cytotoxicity was calculated as 100 × (experimental LDH - spontaneous LDH)/(maximum LDH release - spontaneous LDH). For the cell viability assay, AlamarBlue® assay kits were used to measure proliferation using the protocol described by the manufacturer (AbD Serotec Ltd). For the cell size assay, cell size was determined by drawing circles around representative cells from 20 fields and calculating the area using ImageJ software. For the PI uptake assay, 40 µg/ml PI was added to the cell cultures after treatment. The fluorescence intensity of the retained PI was detected at an excitation wavelength of 536 nm and an emission wavelength of 617 nm on a microplate absorbance reader.

Intracellular cAMP detection Intracellular cAMP levels were measured in cell lysates by an ELISA-based

competitive immunoassay (KGE002B, R&D Systems). Briefly, J774A.1 macrophages were incubated for 5.5 h with or without 1 μg/ml LPS, then incubated with or without 60 μM resveratrol for 30 min, then for 30 min with or without 5 mM ATP.

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Intracellular cAMP levels were measured according to the protocol described by the manufacturer.

Detection of LC3 by confocal microscopy J774A.1 macrophages were seeded onto cover slides and incubated for 30 min

with or without 5 mM 3-MA and then for 4 h with or without 60 μM resveratrol. After removal of the medium, the cells were washed twice with phosphate-buffered saline and fixed with ice-cold methanol at -20°C followed by permeabilization with 0.25% Triton X-100 at 4°C. The primary antibody to LC3 was incubated at 4°C overnight before incubation with the FITC-conjugated secondary antibody. Analysis was performed using a confocal microscope at National Ilan University (FV1000-IX81, Olympus) equipped with the appropriate excitation and emission filters.

Prg-IgAN mouse model BCD mice (B6.129S2-Igh-6tm1Cgn/J) were obtained from Professor John T.

Kung (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan) and maintained at the animal center of the National Defense Medical Center (Taipei, Taiwan). Prg-IgAN was induced by 28 daily injections of purified IgA anti-phosphorylcholine antibodies and pneumococcal C-polysaccharide (PnC), as

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described previously (Chao et al., 2006). The mice (n =7 each group) were treated daily with resveratrol (100 mg/kg body weight) or vehicle (a mixture of DMF, Tween-80, and saline) by oral gavage throughout the experiment, the first dose being given 3 days before disease induction. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of The National Defense Medical Center, Taiwan and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Pathological evaluation For histopathology, the tissues were fixed in 10% buffered formalin and

embedded in paraffin, and then sections (3 μm) were prepared and stained with hematoxylin

and

eosin

(H&E).

For

immunohistochemistry,

formalin-fixed,

paraffin-embedded tissue sections (3 μm) were stained with anti-CD3 antibodies or anti-F4/80 antibodies (Dako, Carpinteria, CA, USA). One hundred glomeruli were examined in at least two renal tissue sections per slide by light microscopy at a magnification of ×400. The severity of the renal lesions was scored as described previously (Ka et al., 2007). Renal ROS levels were measured by dihydroethidium (DHE) binding, and the fluorescence was quantified by counting the percentage of the total nuclei that were positive per kidney in each section.

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Results

Resveratrol reduces NLRP3-inflammasome-derived IL-1β secretion Incubation of J774A.1 macrophages with resveratrol before treatment with LPS

and ATP significantly inhibited IL-1 secretion (Fig. 1A, left panel) and caspase-1 activation (Fig. 1A, right panel). These results indicate that resveratrol was able to inhibit NLRP3 inflammasome activation; however, these results do not reveal how resveratrol inhibits the NLRP3 inflammasome. We were interested in whether resveratrol influences the priming signal from LPS or the activation signal from ATP. We therefore examined the effect of resveratrol on both signaling cascades. We incubated J774A.1 cells with LPS for 5.5 h (LPS priming), followed incubation with resveratrol for 30 min before ATP stimulation. We found that resveratrol inhibited IL-1 secretion (Fig. 1B, left panel) and caspase-1 activation (Fig. 1B, right panel),

but, as shown in Fig. 1C, it had no significant effect on the secretion of TNF-α and IL-6, which are independent of the NLRP3 inflammasome. These results indicate that resveratrol inhibits the ATP-mediated activation signal of the NLRP3 inflammasome. We further examined whether resveratrol could inhibit the activation signal induced by other NLRP3 inflammasome activators, including nigericin, monosodium urate (MSU), calcium pyrophosphate dihydrate (CPPD), SiO2 nanoparticles, and alum

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crystals. We found that IL-1β secretion after treatment with nigericin, MSU, CPPD, and SiO2 nanoparticles was significantly reduced by resveratrol, and IL-1β secretion induced by alum crystals was inhibited slightly, but not significantly, by resveratrol (Fig. 2A). Under the same conditions, resveratrol also significantly inhibited IL-1β

secretion in mouse primary peritoneal macrophages induced by ATP, SiO2

nanoparticles, and CPPD (Fig. 2B). NLRP3 induction is a critical checkpoint for the priming step of NLRP3 activation (Bauernfeind et al., 2009). We investigated whether resveratrol inhibits the LPS-mediated priming signal for the NLRP3 inflammasome. We incubated J774A.1 macrophages with resveratrol for 30 min before treatment for 6 h with LPS and found that resveratrol did not inhibit the expression level of NLRP3 (Fig. 2C), although it reduced the expression level of proIL-1β (Fig. 2D). These results indicated that resveratrol does not inhibit the priming signal of the NLRP3 inflammasome.

Resveratrol reduces NLRP1 and NLRC4 inflammasome-derived IL-1β secretion We next determined whether the effect of resveratrol on IL-1β secretion is

selective for the NLRP3-dependent inflammasome. We found that resveratrol is not selective for NLRP3-dependent inflammasome stimuli, as resveratrol reduced IL-1 secretion triggered by FLA-ST (flagellin from S. typhimurium) (Fig. 3A) or muramyl

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dipeptide (MDP) (Fig. 3B), which is dependent on the NLRP1 and NLRC4 inflammasomes, respectively (Latz et al., 2013), and independent of NLRP3. IL-1 secretion triggered by FLA-ST and MDP was also reduced by resveratrol in mouse primary peritoneal macrophages (Fig. 3C).

Resveratrol reduces caspase-1-dependent pyroptosis Pyroptosis is a type of caspase-1-dependent cell death characterized by a loss of

membrane integrity that results from caspase-1-dependent insertion of a pore into the

membrane, leading to fluid influx, cell swelling, and lysis (Bergsbaken et al., 2009). To evaluate the effect of resveratrol on pyroptosis, J774A.1 macrophages were treated with resveratrol before LPS and ATP treatment, and the samples were assayed for LDH release. Fig. 4A shows that the LDH release induced by LPS and ATP was significantly reduced by resveratrol. To confirm cell death with an alternative assay, an AlamarBlue® cell viability assay was used. The results showed that cell viability was reduced by LPS and ATP and that resveratrol reversed this effect (Fig. 4B). The LDH and AlamarBlue® assays suggest that exposure to LPS and ATP disrupts the plasma membrane, and this effect is rescued by resveratrol. We used uptake of a fluorescent membrane-impermeant dye, propidium iodide (PI), to quantitatively examine membrane damage in individual cells during LPS and ATP exposure (Fink

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and Cookson, 2006). Incubation of J774A.1 macrophages with LPS and ATP resulted in an increase in PI uptake, and resveratrol reduced the PI uptake significantly (Fig. 4C). In addition, to determine whether this reduced plasma membrane integrity affected cell size, cell size was quantified after exposure to LPS and ATP. Fig. 4D depicts the increase in cell size after exposure to LPS and ATP in J774A.1 macrophages (mean of 78 ± 3 μm2 for control cells compared with 127 ± 4 μm2 for LPS and ATP exposed cells). Notably, resveratrol significantly reduced the cell size increase induced by LPS and ATP.

Resveratrol prevents mitochondrial damage Damaged and ROS-generating mitochondria activate the NLRP3 inflammasome (Kepp et al., 2011; Nakahira et al., 2011; Zhou et al., 2011). To determine whether resveratrol inhibited the NLRP3 inflammasome by preventing mitochondrial damage, we used the fluorescent probes MitoTracker Deep Red and MitoTracker Deep Green to stain the active and total mitochondria, respectively. We observed an increase in the percentage of cells with damaged mitochondria (less staining with MitoTracker Deep Red) in LPS-primed macrophages treated with ATP, MSU, CPPD, and SiO2; resveratrol reduced the percentage of cells with damaged mitochondria (Fig. 5A). ROS will be released from damaged mitochondria and promote NLRP3

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inflammasome activation (Kepp et al., 2011; Nakahira et al., 2011; Zhou et al., 2011). Mitochondrial ROS (mt-ROS) levels were increased when LPS-primed macrophages

were treated with ATP, MSU, CPPD, and SiO2. Treatment with resveratrol reduced the mt-ROS levels in these cells (Fig. 5B), indicating that resveratrol protects mitochondria from damage in macrophages. Damaged mitochondria and mt-ROS induce the translocation of mitochondrial DNA (mt-DNA) into the cytosol, and this cytosolic mt-DNA is a coactivator of the NLRP3 inflammasome (Zhou et al., 2011). We examined whether the translocation of mt-DNA into the cytosol was decreased by resveratrol. We found that the levels of mt-DNA release into the cytosol were increased in ATP-, MSU-, CPPD-, and SiO2-activated macrophages, but they were

significantly reduced by resveratrol (Fig. 5C). These results indicate that there was less mitochondrial injury in resveratrol-treated cells than in control cells, suggesting that resveratrol protects cells from NLRP3 inflammasome stimulation. In addition, cAMP acts as a negative regulator of inflammatory responses (Peters-Golden, 2009), and inhibits NLRP3 inflammasome activation (Lee et al., 2012). The addition of ATP reduced the LPS-mediated cAMP increase in J774A.1 macrophages; notably, resveratrol increased cellular cAMP level (Fig. 5D).

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Resveratrol suppresses the NLRP3 inflammasome by augmenting autophagy Activation of autophagy limits IL-1β secretion by inhibiting NLRP3 inflammasome activation (Shi et al., 2012; Nakahira et al., 2011) and reducing proIL-1β protein expression (Harris et al., 2011). These findings prompted us to test whether resveratrol inhibits NLRP3 inflammasome-derived IL-1β secretion through autophagy induction. Western blot analysis showed that the autophagy marker LC3B-II, which is increased during autophagy, was significantly increased in resveratrol- or rapamycin (positive control)-activated J774A.1 macrophages (Fig. 6A, upper panel). The effect of resveratrol on LC3 was confirmed because the number of cells with punctate FITC-LC3 localization was increased following treatment with resveratrol (Fig. 6A, bottom panel). In addition, the level of p62, another autophagy marker, is regulated by the

balance

between

its

transcriptional

regulation

(incoming

flux)

and

post-translational autophagic degradation (outgoing flux) (Puissant et al., 2012). We found that p62 expression was increased by resveratrol (Fig. 6B, upper panel), but resveratrol decreased p62 expression in the presence of actinomycin D, a transcription inhibitor (Fig. 6B, bottom panel). These results indicate that resveratrol exerts its effect at the level of both the synthesis and degradation of p62. To confirm the autophagy-inducing activity of resveratrol, acridine orange, a cell-permeable fluorescent dye, was used to detect lysosomes, which are present in increased

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numbers during autophagy. J774A.1 macrophages incubated with resveratrol had a significantly higher fluorescent signal than control cells when detected by spectrophotometry. Resveratrol also resulted in higher acridine orange staining, analyzed by flow cytometry (Fig. 6C), indicating that resveratrol-treated J774A.1 macrophages had a larger quantity of acidic compartments, a characteristic of cells with increased autophagic activity. LPS induces autophagy in macrophages (Waltz et al., 2011). We found that resveratrol further enhanced LPS-induced autophagy, evidenced by an increase in LC3-II levels and acridine orange staining (Fig. 6D), indicating an enhancement of the autophagic response. To assess the potential role of autophagy in the resveratrol-mediated inhibition of the NLRP3 inflammasome, J774A.1 macrophages were pretreated with the autophagy inhibitor 3-MA followed by LPS and ATP stimulation. We found that 3-MA treatment reversed the inhibitory effect of resveratrol on caspase-1 activation and IL-1β secretion (Fig. 6E), indicating that autophagy mediates the suppressive effect of resveratrol on the NLRP3 inflammasome.

Effect of resveratrol on ATP-mediated ROS generation, PKC phosphorylation, and MAPK phosphorylation In macrophages, ATP-induced ROS production by NADPH oxidase is required for

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caspase-1 activation and IL-1β secretion (Moore and MacKenzie, 2009; Cruz et al., 2007). To determine whether the inhibition of IL-1β secretion by resveratrol occurred

via inhibition of ATP-induced ROS production, LPS-primed J774A.1 macrophages were incubated with resveratrol or the anti-oxidant N-acetyl-cysteine (NAC) for 30 min before the addition of ATP. Our results showed that both resveratrol and NAC significantly reduced ATP-induced ROS production (Fig. 7A). In addition, we investigated whether resveratrol influences ATP-mediated activation of PKC and MAPK. We found that resveratrol reduced the phosphorylation levels of PKC-δ, but not PKC-α (Fig. 7B). Resveratrol also reduced the phosphorylation levels of JNK1/2 in ATP-activated J774A.1 macrophages (Fig. 7C). In contrast, resveratrol significantly increased the phosphorylation levels of p38 in ATP-activated J774A.1 macrophages (Fig. 7C), but it did not significantly increase the phosphorylation levels of ERK1/2 (Fig. 7C). Notably, inhibition of p38 by SB203580 reduced the resveratrol-mediated increase in autophagy (Fig. 7D, upper panel) and restored the inhibitory effect of resveratrol on IL-1β secretion (Fig. 7D, bottom panel). These

results suggest that resveratrol induces autophagy via p38. Furthermore, PKCδ is known to regulate autophagy (Singh et al., 2012). We tested the role of PKCδ in the function of resveratrol by knocking down PKCδ expression using shRNA. We found that IL-1β secretion was reduced in PKCδ knockdown cells and that resveratrol

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further reduced IL-1β secretion (Fig. 7E). Inhibition of p38 by SB203580 increased IL-1β secretion in both control and PKCδ knockdown cells (Fig. 7E).

Resveratrol ameliorates renal inflammation in mice We have previously demonstrated that inhibition of NLRP3 mitigates progressive IgA nephropathy (IgAN) (Hua et al., 2013; Yang et al., 2013). We next evaluated the ability of resveratrol to prevent the development of IgAN in a mouse model. We administered resveratrol daily for 30 consecutive days; the first dose of resveratrol was given two days before disease induction. Vehicle+IgAN mice showed glomerular proliferation and interstitial mononuclear leukocyte infiltration in a peri-glomerular pattern, as well as focal regeneration of the renal tubules at day 28 (Fig. 8A), as measured by light microscopy. However, these abnormalities were greatly inhibited in resveratrol+IgAN mice (Fig. 8A) at day 28. Abnormal T and B cell interactions may play a crucial role in the pathogenesis of renal inflammation and fibrosis (Hua et al., 2013; Yang et al., 2013). We further examined whether inhibition of T or B cell activity may be involved in the beneficial effects of resveratrol in resveratrol+IgAN mice. As seen in Fig. 8B, significant renal periglomerular infiltration of macrophages (F4/80+) and T cells (CD3+) was observed in vehicle+IgAN mice compared to normal control mice, but these effects were markedly reduced in resveratrol+IgAN

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mice. Next, we evaluated the effects of resveratrol on the activation of the NLRP3 inflammasome in IgAN mice. Although the protein expression levels of renal NLRP3 and IL-1β were all significantly increased in the vehicle+IgAN mice compared to normal controls, these effects were inhibited in resveratrol+IgAN mice (Fig. 8C). We then detected the production of ROS in renal tissues. As shown in Fig. 8D, significantly increased levels of ROS were seen in the renal tissues of vehicle+IgAN mice compared with normal control mice, but this effect was greatly suppressed in resveratrol+IgAN mice, as demonstrated by in situ staining of DHE. The full-length western blot images of all figures are shown in supplementary figure 1.

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Discussion The NLRP3 inflammasome is involved in the pathogenesis of many diseases. Full

activation of the NLRP3 inflammasome requires both a priming signal as a result of stimulation of pathogen recognition receptors, e.g., toll-like receptors, and an activation signal that can be induced by various activators, e.g., environmental irritants (alum, silica, and asbestos), endogenous danger signals (ATP, MSU, amyloid-β), and pathogens (Latz et al., 2013; Chen and Sun, 2013; Tschopp and Schroder, 2010). The priming signal leads to NLRP3 protein induction, and the activation signal leads to caspase-1 activation. In this study, we demonstrated that resveratrol inhibited NLRP3 inflammasome activation by suppressing the activation signal. We also demonstrated that resveratrol mitigated progressive IgA nephropathy in mice, which is a NLRP3-inflammasome-associated renal inflammatory disease (Hua et al., 2013; Yang et al., 2013). The priming step of the NLRP3 inflammasome is regulated by ROS, which are

required for NLRP3 expression (Bauernfeind et al., 2011). Resveratrol did not affect the priming step of the NLRP3 inflammasome, although the antioxidant activity of resveratrol is well documented (Huang et al., 2013; Leiro et al., 2004; He et al., 2010). ROS is involved in both the priming step of the NLRP3 inflammasome and in the activation step (Tschopp and Schroder, 2010). The up-regulation of ROS levels in

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ATP-treated macrophages resulted in the activation of PI3K, promoting caspase-1 activation and IL-1β secretion (Cruz et al., 2007). The inhibition of ATP-induced ROS generation may be responsible for the reduced caspase-1 activation and IL-1β secretion by resveratrol. Furthermore, we found that resveratrol reduced mitochondrial ROS generation, which plays an important role in NLRP3 inflammasome activation (Zhou et al., 2011; Kepp et al., 2011). Overproduction of mitochondrial ROS promotes a mitochondrial permeability transition and facilitates the cytosolic release of mitochondrial DNA, which stimulates activation of the NLRP3 inflammasome (Zhou et al., 2011; Kepp et al., 2011). Resveratrol also reduced the mitochondrial permeability transition and the cytosolic release of mitochondrial DNA, indicating that mitochondrial stabilization may be responsible for the reduced activation of NLRP3 inflammasome by resveratrol. Activation of SIRT1 by resveratrol may be associated with its mitochondria protection activity (Xu et al., 2012; Danz et al., 2009). Autophagy is a cellular quality-control system triggered by starvation and

immune signaling that results in the delivery of damaged proteins and organelles to the lysosomes for degradation (Kuballa et al., 2012; Levine et al., 2011). More recently, autophagy has been demonstrated to have multiple immunological functions that influence inflammation and immune responses. For example, autophagy can limit

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NLRP3 inflammasome activation by targeting inflammasome components for degradation (Shi et al., 2012; Harris et al., 2011) and by inhibiting the release of mitochondrial DNA from damaged mitochondria (Nakahira et al., 2011). Accumulating evidence suggests that autophagy induction is associated with the anti-cancer effect of resveratrol (Andreadi et al., 2014; Ge et al., 2013). In contrast, resveratrol has also been shown to increase drug-induced apoptosis by inhibiting autophagy (Alayev et al., 2014; Xu et al., 2012; Lin et al., 2012). In this study, we found that resveratrol reduced mitochondrial DNA release into the cytosol by preserving mitochondrial integrity. This effect was associated with resveratrol’s autophagy induction activity. We further found that resveratrol induced autophagy by activating p38, which also mediated autophagy induction in human T cells and in rat heart cells (Ge et al., 2013; Lv and Zhou, 2012). Furthermore, resveratrol inhibits LPS-induced NO generation (Zong et al., 2012) and increases autophagic responses, suggesting that there may be a relationship between NO generation and autophagy. In this regard, it should be noted that NO inhibits macrophage autophagy during LPS stimulation (Zhang et al., 2012), and reduced NO generation may explain the increased autophagic response induced by resveratrol. Resveratrol has been reported to have health benefits; however, its mechanism of

action remains controversial. One of the most important findings is that resveratrol

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directly inhibits cAMP-dependent phosphodiesterases, leading to elevated cAMP levels (Park et al., 2012). Elevated cAMP levels trigger a cascade of events that ameliorate the symptoms of metabolic diseases associated with aging (Park et al., 2012). Lee et al. demonstrated that cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition (Lee et al., 2012). In this study, we found that resveratrol increased cellular cAMP levels and suppressed activation of the NLRP3 inflammasome. In this regard, it should be noted that increased cAMP levels by resveratrol may be responsible for the decreased activation of the NLRP3 inflammasome by resveratrol. Autophagy induction by resveratrol in umbilical vein endothelial cells is dependent on increased cAMP levels and downstream signaling pathways, including protein kinase A, AMP-activated protein kinase, and SIRT1 (Chen et al., 2013). We suggest that cAMP induced by resveratrol reduced NLRP3 inflammasome activation by directly binding to NLRP3 and also by increasing autophagy. In this study, we demonstrated that resveratrol inhibited the activation of the

NLRP3 inflammasome induced not only by microbial toxins (e.g., nigericin) and environmental irritants (e.g., silica) but also by endogenous danger signals (e.g., monosodium urate and calcium pyrophosphate dihydrate). These results suggest that resveratrol has the potential to prevent and mitigate not only IgAN and

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infection-induced inflammatory disease but also diseases such as silicosis and gout (Martinon et al., 2006; Hornung et al., 2008).

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Figure Legends Figure 1. Resveratrol attenuates NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 30 min with or without resveratrol, then for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without 5 mM ATP. The levels of IL-1β in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blot, respectively. (B) J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without resveratrol in the continued presence or absence of LPS, and then with or without the addition of 5 mM ATP for 30 min. The levels of IL-1β in the culture medium and activated caspase-1 (p10) in the cells were measured by ELISA and Western blot, respectively. (C) J774A.1 macrophages were incubated for 5.5 h with or without 1

µg/ml of LPS, then for 30 min with or without resveratrol in the continued presence or absence of LPS, and then for 30 min with or without 5 mM ATP. TNF-α and IL-6

in the culture medium were measured by ELISA. The data are expressed as the means ± SD for three separate experiments. The Western blot results are representative of those obtained in three different experiments, and the histograms are presented as the change in the ratio relative to p45 compared to the control group. *** indicates a significant difference at the level of p < 0.001.

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Figure 2. Resveratrol attenuates NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without resveratrol in the continued presence or absence of LPS, and then with or without 10 µM nigericin (0.5 h), 100 µg/ml MSU (24 h), 100 µg/ml CPPD crystals (24 h), 200 µg/ml nano SiO2 (24 h), or 500 µg/ml alum crystals (24 h). IL-1β

in the culture medium was measured by ELISA. (B) Mouse primary peritoneal macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without 60 µM resveratrol in the continued presence or absence of LPS, and then with or without 5 mM ATP (0.5 h), 200 µg/ml nano SiO2 (24 h), or 100 µg/ml CPPD crystals (24 h). IL-1β in the culture medium was measured by ELISA. (C and D) J774A.1 macrophages were incubated for 30 min with or without

resveratrol and then for 6 h with or without 1 µg/ml of LPS in the continued presence or absence of resveratrol, and then the protein expression levels of NLRP3 (C) and

proIL-1β (D) in the cells were measured by Western blot. The data are expressed as

the means ± SD for three separate experiments. In Western blots, the results are representative of those obtained in three different experiments and the histograms are presented as the change in the ratio relative to actin compared to the control group. *** indicates a significant difference at the level of p < 0.001.

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Figure 3. Resveratrol attenuates NLRP1 and NLRC3 inflammasome activation. (A and B) J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without resveratrol in the continued presence or absence of LPS, and then for 24 h with or without 100 g/ml FLA-ST (A) or 100 ng/ml MDP (B). IL-1β in the culture medium was measured by ELISA. (C) Mouse primary peritoneal macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without resveratrol in the continued presence or absence of LPS, and then for 24 h with or without 100 g/ml FLA-ST or 100 ng/ml MDP. IL-1β in the culture medium was measured by ELISA. The data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Figure 4. Resveratrol attenuates pyroptosis. J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without resveratrol in the continued presence or absence of LPS, and then for 30 min with or without 5 mM ATP. (A) LDH release was assayed by the CytoTox 96® Non-radioactive Cytotoxicity assay kit. (B) Cell viability was assayed with the AlamarBlue® assay kit. (C) The fluorescence intensity of PI was detected by a microplate absorbance reader.

(D) Cell size was determined by drawing circles around representative cells from 20

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fields and calculating the area using Image J software. The data are expressed as the means ± SD for three separate experiments. * and *** indicates a significant difference at the level of p < 0.05 and p < 0.001, respectively.

Figure 5. Resveratrol attenuates mitochondrial damage. J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without 60 µM resveratrol in the continued presence or absence of LPS, then for 15 min with 25 nM MitoTracker Deep Red and MitoTracker Green, then for 30 min with or without 5 mM ATP, 100 µg/ml MSU, 100 µg/ml CPPD crystals, or 200 µg/ml nano SiO2. (A) The mitochondrial inner transmembrane potential and mitochondrial mass were measured by flow cytometry. (B) Mitochondrial ROS generation in the cells was

measured by MitoSOX. (C) Mitochondrial DNA release into the cytosol was measured by detection of cytochrome c oxidase I DNA in the cytosol. (D) Cellular cAMP levels were measured by a cAMP assay kit. The data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Figure 6. Resveratrol attenuates NLRP3 inflammasome activation by enhancing autophagy. (A) Upper panel: J774A.1 macrophages were incubated for 30 min with

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or without 100 nM bafilomycin A, then for 4 or 6 h with or without 60 μM resveratrol or 4 h with 100 nM rapamycin in the continued presence or absence of bafilomycin A. The protein expression levels of LC3 were measured by Western blot. Bottom panel: J774A.1 macrophages were incubated for 30 min with or without 5 mM 3-MA and then for 4 h with or without 60 μM resveratrol. The localization of LC3 was measured by staining cells with FITC-LC3 antibody and imaging via confocal microscopy. (B) Upper panel: J774A.1 macrophages were treated as indicated in (A), upper panel. The protein expression levels of p62 were measured by Western blot. Bottom panel: J774A.1 macrophages were incubated for 30 min with or without 5 nM actinomycin

D and then for 4 or 6 h with or without 60 μM resveratrol. The protein expression levels of p62 were measured by Western blot. (C) J774A.1 macrophages were incubated for 4 h with or without 60 μM resveratrol or 100 nM rapamycin, then with 1 μg/ml acridine orange for 30 min. The fluorescent signal of acridine orange was measured by spectrophotometry (left) and flow cytometry (right). (D) J774A.1

macrophages were incubated for 30 min with or without 5 mM 3-MA, then for 30 min with or without 60 μM resveratrol, then for 4 h with or without 1 μg/ml LPS in the continued presence or absence of 3-MA and resveratrol. The expression levels of the LC3 protein and the fluorescent signal of acridine orange were measured by Western

blot and fluorescent microscopy, respectively. (E) J774A.1 macrophages were

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incubated for 30 min with or without 5 mM 3-MA, then for 30 min with or without 60

μM resveratrol, then for 5.5 h with or without 1 μg/ml LPS in the continued presence or absence of 3-MA and resveratrol, then for 30 min with or without 5 mM ATP. Activated caspase-1 (p10) in the cells and IL-1β in the culture medium were measured by Western blot and ELISA, respectively. The data are expressed as the mean ± SD for three separate experiments. For Western blots, flow cytometry and fluorescent microscopy, the results are representative of three different experiments, and the histograms show the change in the ratio relative to p45 compared to the control group. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively.

Figure 7. Effect of resveratrol on ATP-induced ROS generation and signaling pathways. (A) J774A.1 macrophages were incubated for 5.5 h with 1 µg/ml of LPS, then for 30 min with or without 60 µM resveratrol or 10 mM NAC in the continued presence of LPS, and then with or without 5 mM ATP for 0-80 min. ROS generation in the cells was measured by 2’,7’-dichlorofluorescein diacetate. (B and C) J774A.1 macrophages were incubated for 5.5 h with 1 µg/ml of LPS, then for 30 min with or without 60 µM resveratrol in the continued presence of LPS, and then with or without 5 mM ATP for 0-60 min. The phosphorylation levels of PKC (B) and MAPK (C)

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were measured by Western blot. (D) Upper panel: J774A.1 macrophages were incubated for 30 min with or without 1 μM SB203580, then for 4 h with or without 60 μM resveratrol, then for 30 min with 1 μg/ml acridine orange. The fluorescent signal of acridine orange was measured by flow cytometry. Bottom panel: J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without SB203580, then for 30 min with or without 60 µM resveratrol in the continued presence or absence of LPS and SB203580, and then for 30 min with or without the addition of 5 mM ATP. The levels of IL-1β in the culture medium were measured by ELISA. (E) Control J774A.1 macrophages (sh-SC) and PKCδ knockdown J774A.1 macrophages (sh-PKCδ) were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without 1 µM SB203580, then for 30 min with or without 60 µM resveratrol in the continued presence or absence of LPS and SB203580, and then for 30 min with or without the addition of 5 mM ATP. The levels of IL-1β in the culture medium were measured by ELISA. The data are

expressed as the means ± SD for three separate experiments. For Western blots and flow cytometry, the results are representative of three different experiments.

Figure 8. Resveratrol ameliorates renal inflammation in mice. (A) Histopathology of the kidney, H&E stain, original magnification 400×. The arrowheads indicate

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periglomerular mononuclear leukocyte infiltration. Histograms represent the scoring of renal pathology. (B) Staining of renal tissues for F4/80+ macrophages and CD3+ T cells, original magnification, 400×. The arrowheads indicate positively stained cells. Histograms represent the scoring of positively stained cells in the glomerulus and perglomerului. (C) Protein expression levels of NLRP3 and IL-1β in renal tissue

assayed by Western blot. The histograms represent the change in the ratio relative to actin compared to the control group. (D) ROS production by in situ staining of DHE, original magnification, 400×. Histograms represent the scoring for DHE staining. *, ** and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively.

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Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and augmenting autophagy.

The NLRP3 inflammasome is a caspase-1-containing multi-protein complex that controls the release of IL-1β and plays important roles in the development...
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