Curcumin suppresses NLRP3 inflammasome activation and protects ...

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Jul 9, 2015 - Scope: The NLRP3 inflammasome responds to various pathogen-derived factors and danger- associated molecules, mediating IL-1 maturation ...
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DOI 10.1002/mnfr.201500316

Mol. Nutr. Food Res. 2015, 59, 2132–2142

RESEARCH ARTICLE

Curcumin suppresses NLRP3 inflammasome activation and protects against LPS-induced septic shock Zizhen Gong1,2,3 , Jiefei Zhou1,2,3 , Hui Li4 , Yanhong Gao5 , Congfeng Xu6 , Shengnan Zhao1,2,3 , Yingwei Chen2,3 , Wei Cai1,2,3∗ and Jin Wu1,2,3 1

Department of pediatric Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, P. R. China 2 Shanghai Institute for Pediatric Research, Shanghai Jiaotong University School of Medicine, Shanghai, P. R. China 3 Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, Shanghai, P. R. China 4 Department of Pathology, Shanghai institute of Health Science, Shanghai, P. R. China 5 Department of Geriatrics, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, P. R. China 6 Shanghai Institute of Immunology, Institutes of Medical Sciences, Shanghai Jiaotong University School of Medicine, Shanghai, P. R. China Scope: The NLRP3 inflammasome responds to various pathogen-derived factors and dangerassociated molecules, mediating IL-1␤ maturation, therefore is involved in multiple inflammatory diseases. Curcumin has been shown to possess strong anti-inflammatory activity, but the underlying mechanism is not fully understood. Here, we sought to investigate the role and mechanism of curcumin on the inhibition of mature IL-1␤ production via the regulation of NLRP3 inflammasome. Methods and results: Curcumin dramatically inhibited the production of mature IL-1␤ in LPSprimed macrophages triggered by multiple NLRP3 inflammasome activators, and also reduced the level of cleaved caspase-1 as measured by western blot and ELISA. Curcumin prevented K+ efflux, the common trigger for NLRP3 inflammasome activation, and attenuated lysosomes disruption and intracellular ROS formation as well. The inhibition of NLRP3 inflammasome by curcumin was in part mediated via the suppression of extracellular regulated protein kinases phosphorylation. Furthermore, administration of curcumin significantly reduced peritoneal IL-1␤ and HMGB-1 concentration induced by LPS and improved the survival of mice suffering from lethal endotoxic shock. Conclusion: Curcumin potently inhibits the activation of NLRP3 inflammasome which may contribute to its anti-inflammatory activity. Our finding offers a mechanistic basis for the therapeutic potential of curcumin in septic shock and other NLRP3 inflammasome-driven diseases.

Received: April 22, 2015 Revised: July 9, 2015 Accepted: July 24, 2015

Keywords: Anti-inflammation / Curcumin / IL-1␤ / Inflammasome / MAPK



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Correspondence: Jin Wu E-mail: [email protected] Abbreviations: ASC, apoptosis associated speck-like protein; ATP, adenosine triphosphate; BMDM, Bone marrow derived macrophage; DAMP, damage-associated molecular pattern; HMGB-1, high mobility group box-1 protein; IBD, inflammatory bowel disease; IL-1␤, Interleukin-1␤; LPS, Lipopolysaccharide; MAPK, motigen-activated protein kinase; MSU, monosodium urate; NAC, N-acetyl-L-cysteine; NLR, nucleotide-binding and oligomerization domain (NOD)-like receptor; PAMP, pathogenassociated molecular pattern; PRR, pathogen-recognition receptors; ROS, reactive oxygen species; TLR, Toll-like receptor  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction

Inflammation is a crucial host response triggered by invading pathogens and injured tissue. Moderate inflammatory response contributes to host defense which helps to remove pathogens and repair damaged tissue. However, uncontrolled inflammation may promote further tissue damage and lead to serious disorders. Recent studies have highlighted the ∗ Additional correspondence author: Wei Cai, E-mail: [email protected] Colour Online: See the article online to view Figs. 1, 3, 4, 6 and 7 in colour.

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critical role of NLR3 inflammasome in the inflammatory response [1, 2]. NLRP3 inflammasome can be activated by a broad range of stimuli, including bacterial RNA [3], viruses [4], fungi [5], pore-forming toxins (such as nigericin, listeriolysin) [6,7], extracellular ATP as well as crystal particles (such as uric acid and cholesterol crystals) [8, 9]. Upon stimulation, NLRP3 recruits apoptosis associated speck-like protein (ASC) and caspase-1 (known as interleukin-1 converting enzyme, ICE) and assembles into a large cytoplasmic complexes, then eventually leads to the maturation and secretion of potent proinflammatory cytokines, such as IL-1␤ [10,11], therefore facilitates to recruit immune cells to the infection site and activate adaptive immune response. However, dysregulated NLRP3 inflammasome activation is involved in diverse inflammatory diseases, including type 2 diabetes, arthritis, Crohn’s disease as well as septic shock. The pivotal role of NLRP3 inflammasome in LPS-induced endotoxic shock has been established by a large number of studies [12–14]. High plasma level of IL-1␤ correlates well with the severity of shock [15]. Nitric oxide, which inhibits NLRP3 inflammasome activation could protect against LPS-induced septic shock [12]. Mice deficient in any component of NLRP3 inflammasome exhibit defective production of mature IL-1␤ and are resistant to LPS-induced endotoxic shock. Given the pro-inflammatory role of NLRP3 inflammasome, strategies aimed at modulation of its activation may represent an appealing treatment for septic shock and other inflammatory diseases it contributes to. Curcumin, a phenolic product derived from perennial herb Curcuma longa, possess multiple biological activities and is proved its pharmacological safety in numerous preclinical and early-phase clinical studies [16, 17]. Growing evidence has suggested that curcumin was effective in both acute and chronic models of inflammation. Administration of curcumin attenuates acute inflammatory damage in experimental traumatic brain injury and protects mice from LPSinduced hepatic inflammation [18]. Curcumin can also interfere with LPS-stimulated expression and secretion of MIP2, IL-1␤, KC and MIP-1␣ in colonic epithelial cells (CEC) and macrophages and therefore has the implication potential for inflammatory bowel disease (IBD) [19]. So far, the anti-inflammatory activity of curcumin has been primarily ascribed to the inhibition of NF-␬B [18, 20]. However, whether the effects of curcumin involve suppressing NLRP3 inflammasome activation has never been addressed. In the current study, we focused our investigation on the role and mechanism of curcumin in the inhibition of NLRP3 inflammasome activation, as well as its effect on the prevention of LPS-induced septic shock in vivo.

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Sigma-Aldrich (St. Louis, MO); MSU (monosodium urate) crystal and poly (dA:dT) were obtained from Invivogen (San Diego, CA). Nigericin was purchased from Cayman Chemical (Ann Arbor, MI). RPMI 1640, DMEM and antibiotics were obtained from Invitrogen (Carlsbad, CA). 2 , 7 dichlorofluorescein diacetate (DCF-DA) and MitoSOX Red reagent were from Invitrogen/Molecular Probes. Antibodies against mouse IL-1␤, caspase-1, HMGB-1 were obtained from Cell Signaling Technologies (Beverly, MA), Santa cruz Biotechnology (Santa cruz, CA) and Abcam (Cambridge, MA), respectively. IL-1␤ ELISA Kit was purchased from eBioscience (San Diego, CA).

2.2 Mice 6- to 8-wk-old C57BL/6 mice were purchased from Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China) and maintained in a specific pathogen free (SPF) facility. The animal use and all the experiment procedures complied with national guidelines and approved by the Shanghai Laboratory Animal Care and Use Committee.

2.3 Cells The murine macrophage cell line J774A.1 was obtained from Type Culture Collection of the Institutes of Biomedical Sciences, Fudan University (Shanghai, China). Cells were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Invitrogen) at 37⬚C with 5% CO2. Peritoneal macrophages were isolated by peritoneal lavage 3 days after intraperitoneal injection of 2 mL thioglycollate solution (4%, Sigma). Cells were then incubated at 37⬚C for 3 h and non-adherent cells were removed. The remaining adherent cells were used in the following experiments. Bone marrow derived macrophages (BMDMs) were obtained and cultured as described elsewhere [21].

2.4 Activation of NLRP3 inflammasome J774A.1 cells or mouse peritoneal macropahges were primed with 1 ␮g/mL LPS for 5 h before stimulation with ATP (5 mM, 30 min), nigericin (20 ␮M, 30 min), aluminum crystal (500 ␮g/mL, 6 h) or MSU crystal (250 ␮g/mL, 3 h). To evaluate the effect of curcumin, indicated concentration of curcumin was added to the cell culture medium 30 min prior to the stimulation with ATP, nigericin, aluminum or MSU crystals.

Materials and methods 2.5 Salmonella infection

2.1 Reagents Lipopolysaccharide (LPS), curcumin, adenosine triphosphate (ATP) and N-acetyl-L-cysteine (NAC) were purchased from  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

J774A.1 cells (1 × 106 ) were infected for 1 h with Salmonella (1:100) and then cultured in fresh medium supplemented with gentamicin (100 ␮g/mL). www.mnf-journal.com

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2.6 Enzyme-linked immunosorbent assay (ELISA) Supernatants of J774A.1 cells or peritoneal macrophages were harvested, and the IL-1␤ level was determined by Mouse IL-1 beta (IL-1␤) ELISA Kit (eBioscience) according to the manufacturer’s instructions. 2.7 Western blot Cells were lysed by protein lysis buffer (Sigma) supplemented with protease and phosphatase inhibitors (Theromo), meanwhile, the supernatant was concentrated by acetone precipitation. Cell lysates (50 ␮g of total protein) and concentrated supernatant proteins were resolved on SDS-polyacrylamide gels and transferred to 0.2 ␮m polyvinylidene difluoride membranes. The membranes were blocked with 5% milk before incubated with specific antibodies against IL1-␤ (Cell Signaling Technologies), Caspase-1 (Santa Cruz), NLRP3 (R&D), HMGB-1 (Abcam) and ␤-actin (Sigma). For the detection of phosphorylated motigen-activated protein kinase (MAPK), cell lysates containing 50 ␮g protein were resolved by SDSPAGE, transferred to PVDF membranes and probed with specific antibodies against phospho-ERK (p-ERK), p-JNK or pp38 (Cell Signaling, Beverly, MA, USA). After the incubation with a stripping buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min, membranes were re-probed with antibodies against total ERK, JNK and p38. Reactive signals were detected with ECL Western Blotting Substrate (Thermo Fisher Scientific) and ChemiDocTM XRS+ System (Bio-Rad). The density of target bands was analyzed by Image Lab 3.0 (Bio-Rad). 2.8 Lysosome and cathepsin B imaging LPS primed macrophages (1 × 106 ) in 6-well plates were pre-incubated with or without curcumin (50 ␮M, 30 min) or U0126 (10 ␮M, 30 min) and then stimulated by nigericin (20 ␮M, 30 min). Cells were stained with Lyso Tracker Green DND-26 (Invitrogen) or Cathepsin B fluorogenic substrate (Neuromics) for 1 h at 37⬚C, followed by Hoechst staining for half an hour. Fluorogenic signals were captured by inverted fluorescence microscopy (Leica). 2.9 Reactive oxygen species (ROS) measurement LPS primed J774A.1 cells or BMDMs were incubated with or without curcumin (50 ␮M, 30 min) or U0126 (10 ␮M, 30 min) before stimulated by nigericin (20 ␮M, 30 min) or aluminum crystal (data not shown). ROS production was measured by using 2 , 7 -dichlorofluorescein diacetate (DCF-DA) probes or MitoSOX Red mitochondrial superoxide indicator (Invitrogen) according to the manufacturer’s instructions. Briefly, cells were incubated with DCF-DA (15 ␮M) for 1 h or MitoSOX (10 ␮M) for 10 min at 37⬚C after nigericin treat C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Mol. Nutr. Food Res. 2015, 59, 2132–2142

ment. Fluorescence was visualized directly under a fluorescence microscope. 2.10 Apoptosis assay J774A.1 macrophages (1 × 106 ) were cultured in 6-well plates and were either left untreated or treated with 50 ␮M curcumin for 1 h at 37⬚C. Apoptosis was determined using an Annexin V Apoptosis kit (BD Biosciences). Data were acquired with a FACSCanto II flow cytometer (BD Biosciences). 2.11 ICP-OES assay LPS primed J774A.1 cells (1 × 107 ) in petri-dishes were incubated with or without curcumin (50 ␮M, 30 min) and followed by nigericin stimulation (20 ␮M, 30 min). The cells were lysed in ultra pure nitric acid before microwave digestion. The digested samples were then diluted to 5% HNO3 and intracellular K+ was analyzed by Perkin Elmer Optima 8000 ICP-OES Spectrometer. External K calibration was performed between 0 and 10 ppm. Data were averaged from three individual experiments. 2.12 In vivo mouse septic shock model Mice were injected intraperitoneally with LPS (1.5 mg/kg) alone or LPS plus curcumin (2 mg). After 6 h, mice were sacrificed and peritoneal lavage fluid was harvested for the analysis of IL-1␤ secretion and HMGB-1 release by ELISA, meanwhile, peritoneal macrophages were isolated for the measurement of intracellular ROS level and cathepsin B leakage by fluorescent staining. To induce septic shock, mice were injected intraperitoneally with LPS (25 mg/kg) in the presence or absence of curcumin (2 mg), blood was then collected and analyzed for ALT, BUN and Creatinine 6 h after injection, and the mortality was monitored at regular intervals.

2.13 Statistics All data were expressed as mean ± standard error of the mean (s.e.m.). Statistical significance was assessed by 2-tailed Student’s t-test or one-way ANOVA. Significance was defined as p