Article pubs.acs.org/JAFC
Peroxyauraptenol Inhibits Inflammation and NLRP3 Inflammasome Activation by Inhibiting Reactive Oxygen Species Generation and Preserving Mitochondrial Integrity Louis Kuoping Chao,† Cheng-Hsiu Lin,§ Huan-Wen Chiu,§ Wei-Ting Wong,§ Hsiao-Wen Chiu,§ Yu-Ling Tasi,# Yueh-Hsiung Kuo,⊥,Δ,¶ Yi-Chich Chiu,○ May-Lan Liu,⊗ Chen-Lung Ho,∇ and Kuo-Feng Hua*,§ †
Department of Cosmeceutics, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402 Department of Biotechnology and Animal Science, National Ilan University, No. 1, Sec. 1, Shen-Lung Road, Ilan, Taiwan 260 # Graduate Institute of Life Science, National Defense Medical Center, No. 161, Sec. 6, Minquan E. Road, Taipei, Taiwan 114 ⊥ Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402 Δ Tsuzuki Institute for Traditional Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402 ¶ Department of Biotechnology, Asia University, No. 500 Lioufeng Road, Wufeng, Taichung, Taiwan 41354 ○ Department of Biomechatronic Engineering, National Ilan University, No. 1, Sec. 1, Shen-Lung Road, Ilan, Taiwan 260 ⊗ Department of Nutritional Science, Toko University, No. 51, Sec. 2, Xuefu Road, Chiayi, Taiwan 61363 ∇ Division of Wood Cellulose, Taiwan Forestry Research Institute, No. 53 Nanhai Road, Taipei, Taiwan 100 §
ABSTRACT: Peroxyauraptenol (PXT) is a peroxide-containing coumarin compound isolated from the seeds of Cnidium monnieri. PXT exerts anti-inflammatory activities, as it reduces the levels of inducible nitric oxide synthase, nitric oxide, IL-6, and NLRP3 inflammasome-derived IL-1β in lipopolysaccharide-activated macrophages. PXT also exerts anti-inflammatory activity by reducing reactive oxygen species generation (including mitochondrial), mitogen-activated protein kinase, protein kinase C-α/δ phosphorylation, and the release of mitochondrial DNA into the cytosol. In addition, PXT suppresses the phagocytic activity of macrophages and IL-1β secretion by Klebsiella pneumoniae-infected macrophages. The unique peroxide group is important for the anti-inflammatory activity of PXT, as this activity is reduced when the peroxide group is replaced by a hydroxyl group. These findings suggest that PXT may be a candidate for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation-related diseases. KEYWORDS: peroxyauraptenol, inflammation, NLRP3 inflammasome, reactive oxygen species, mitochondria
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diabetes mellitus,11 atherosclerosis,12 obesity,13,14 silicosis,15 Alzheimer’s disease,16 gout,17 and kidney disease.18 Given the importance of inflammation and the inflammasome in the pathogenesis of diseases, anti-inflammatory approaches or biological agents that target inflammasome or specific proinflammatory mediator pathways can be used to improve the control of inflammation-related disorders. The fruit of Cnidium monnieri, a medical food, has been the subject of considerable interest because of its broad spectrum of pharmacological properties. The most thoroughly characterized main constituent from the fruit of C. monnieri is osthole. We have demonstrated that osthole possesses anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated macrophages4 and mitigates renal inflammation in mice.19,20 In the present study, we isolated peroxyauraptenol (PXT), a coumarin compound with a unique peroxide group, from the fruit of C.
INTRODUCTION Inflammation is one of the important host defense mechanisms in mammals; however, uncontrolled inflammation is harmful to health.1 The overproduction of pro-inflammatory cytokines and mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), nitric oxide (NO), and cyclooxygenase-2 (COX-2), by macrophages is a feature of activated innate immunity.2−4 The strong link between inflammation and disease is becoming increasingly evident; for example, an elevation in circulating inflammatory cytokine IL-6 is a risk factor for the pathogenesis of inflammatory bowel diseases.5 NO is produced from L-arginine by inducible nitric oxide synthase (iNOS) and contributes to some pathogeneses by promoting oxidative stress, tissue injury, and even cancer, if generated in excess.6 IL-1β release is controlled by the inflammasome, a caspase-1containing multiprotein complex, and plays important roles in microbial infections and metabolic processes.7 The most thoroughly characterized inflammasome is the NLRP3 inflammasome, which is considered to be a controller of disease development, including pathogen infections,8−10 type 2 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1210
November 14, 2014 December 23, 2014 December 31, 2014 December 31, 2014 DOI: 10.1021/jf5054436 J. Agric. Food Chem. 2015, 63, 1210−1219
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Journal of Agricultural and Food Chemistry monnieri. PXT showed anti-inflammatory activity, but the immune modulation properties of PXT and osthole were found to be different, despite their similar chemical structures. The anti-inflammatory activity of PXT was associated with the unique peroxide group, as the anti-inflammatory activity was reduced when this group was replaced by a hydroxyl group.
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MATERIALS AND METHODS
Materials. LPS (from Escherichia coli 0111:B4), ATP, and mouse antibodies against mouse phospho-ERK1/2, phospho-JNK1/2, phospho-p38, and actin were purchased from Sigma (St. Louis, MO, USA). Rabbit antibodies against mouse phospho-PKC-α/δ, IL-1β, caspase-1, iNOS, and COX-2 and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse IL-1β, IL-6, and TNF-α ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-mouse NLRP3 antibody was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The AlamarBlue assay kit was purchased from AbD Serotec Ltd. (Oxford, UK). All of the inflammasome inducers and the QUANTI-Blue reagent were purchased from InvivoGen (San Diego, CA, USA). Compound Extraction and Purification. C. monnieri seeds (600 g) were purchased from a traditional Chinese medicine dispensary in Taiwan. The seeds were extracted with 15 L of ethanol (95% v/v) at room temperature for 10 days, and the supernatants were then concentrated to produce an alcoholic extract. PXT and auraptenol were purified by semipreparative high-performance liquid chromatography (HPLC) (D-Star DLC-20 Instruments with a UV−vis detector) with a Phenomenex Luna Silica (2) column (250 mm length, 10 mm i.d., particle shape/size = 5.0 μm) (Figure 1A). The separation conditions were as follows: 500 μL was injected for each separation; flow rate, 4 mL/min; mobile phase, acetone/hexane = 1:6. The structures of PXT and auraptenol were identified by physical and spectral data (mp, EIMS, 1H NMR, UV, IR) compared with previous research values.4,21 Cell Cultures. The murine macrophage cell lines RAW 264.7 and J774A.1 were obtained from American Type Culture Collection (Rockville, MD, USA). RAW 264.7 macrophages stably transfected with the NF-κB reporter gene (RAW-Blue cells) were purchased from InvivoGen. Mouse peritoneal macrophages were elicited by intraperitoneal injection of 4% sterile thioglycollate medium (2 mL). After 3 days, the mice were sacrificed by cervical dislocation, and the macrophages were harvested. AlamarBlue Assay for Cell Viability. RAW 264.7 cells were incubated with or without PXT for 24 h. The AlamarBlue assay was used to determine the cytotoxicity of PXT according to the protocol described in the manufacturer’s instructions (AbD Serotec, Oxford, UK).4 Detection of Pro-inflammatory Mediators and Protein Phosphorylation. The levels of NO and cytokines (IL-1β, IL-6, and TNF-α) in the culture medium were measured by the Griess reaction and ELISA, respectively. The levels of iNOS, COX-2, and caspase-1 and the phosphorylation levels of ERK1/2, JNK1/2, p38, and PKC-α/δ in cells were measured by Western blotting. The detailed procedures were described in our previous study.4 The density of the bands of the Western blotting was analyzed by ImageJ software. We selected an identical rectangular area around each lane of the Western blot even there was no band of control lane. The density of the selected area was analyzed by ImageJ, and the relative band density was normalized to actin before fold change was calculated. The densitometry fold change of each group was calculated by comparison to the control group, as the control group had a fold change of 1. The bars accompanying blots are the means of three separate experiments. Detection of ROS. 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 USA), at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a microplate absorbance reader (Bio-Rad Laboratories, Inc.). Mitochondrial ROS generation was measured by detecting the fluorescence intensity of MitoSOX
Figure 1. Effect of PXT on the expression levels of inflammatory mediators. (A) Chromatography of alcoholic extract of C. monnieri seeds by a silica column. (B−D) RAW 264.7 macrophages were incubated for 30 min with or without PXT and then for 24 h with or without 1 μg/mL LPS. The levels of NO (B), IL-6 (C), and TNF-α (D) in the culture medium were measured by the Griess reaction and ELISA, respectively. (E) RAW 264.7 macrophages were incubated for 30 min with or without PXT and then for 24 h with or without 1 μg/ mL LPS. The levels of iNOS and COX-2 were assayed by Western blotting. (F) RAW 264.7 macrophages were incubated for 24 h with or without PXT, and the cell viability was then assayed by the AlamarBlue assay. The data are expressed as the means ± SD of three separate experiments. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ and ∗∗∗ indicate a significant difference at the levels of p < 0.05 and p < 0.001, respectively.
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DOI: 10.1021/jf5054436 J. Agric. Food Chem. 2015, 63, 1210−1219
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Figure 2. Effect of PXT on the phosphorylation levels of MAPKs. (A) RAW 264.7 macrophages were incubated for 30 min with or without 40 μM PXT and then for 0−60 with or without 1 μg/mL LPS. The phosphorylation levels of ERK1/2, JNK1/2, and p38 were assayed by Western blotting. (B) Quantitation of the phosphorylation levels of ERK1/2. (C) Quantitation of the phosphorylation levels of JNK1/2. (D) Quantitation of the phosphorylation levels of p38. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ indicates a significant difference at the level of p < 0.05. Phagocytosis Assay. J774A.1 macrophages were seeded in 96well flat-bottom plates at a density of 5 × 103 cells in 100 μL of RPMI 1640 medium containing 10% FBS per well and incubated with 1 μg/ mL LPS for 6 h. The cells were incubated with or without 40 μM PXT or 10 μM colchicine for 30 min and then incubated for 1 h with or without 100 μg of fluorescence-conjugated Escherichia coli. The fluorescence intensity was measured at an excitation wavelength of 509 nm and an emission wavelength of 533 nm. Net phagocytosis was calculated by subtracting the average fluorescence intensity of the nocell negative-control wells from all of the experimental wells. The percent of phagocytosis was then calculated as a fraction of the net positive control phagocytosis, as follows:
(Invitrogen, Carlsbad, CA, USA) at an excitation wavelength of 514 nm and an emission wavelength of 585 nm using a microplate absorbance reader. Detection of Mitochondrial DNA Release. The detection of mitochondrial DNA (mt-DNA) in the cytosol was performed as described.22 Briefly, after treatment, cells were homogenized in a protease inhibitor-containing buffer (100 mM, pH 7.4, Tricine− NaOH, 0.25 M sucrose, 1 mM EDTA) and then centrifuged for 10 min at 700g and 4 °C. The protein concentration and volume of the supernatant were normalized, and the sample was then centrifuged for 30 min at 10000g and 4 °C. The resulting pellets were identified as the mitochondrial fraction, and the supernatants were identified as the cytosolic fraction. DNA was isolated from 200 μL of the cytosolic fraction. The mt-DNA copy number was measured by quantitative PCR and normalized to nuclear DNA (encoding 18S rRNA) levels using a ratio of cytochrome c oxidase I DNA to nuclear DNA. The results are expressed as fold changes between the sample and control groups. The quality of the mitochondrial and cytosolic fractions was monitored by detecting the protein expression level of voltagedependent anion channel (VDAC, a marker protein for mitochondria). We found that VDAC can be detected only in the mitochondrial fraction and is not present in the cytosolic fraction (data not shown), indicating that the cytosolic fraction is free of mitochondria. The primers used were as follows: mouse 18S, forward, 5′-TAGAGGGACAAGTGGCGTTC-3′, reverse, 5′-CGCTGAGCCAGTCAGTGT-3′; mouse cytochrome c oxidase I, forward, 5′-GCCCCAGATATAGCATTCCC-3′, reverse, 5′-GTTCATCCTGTTCCTGCTCC3′. NF-κB Reporter Assay. The medium (20 μL) from treated RAWBlue cells was mixed with 200 μL of QUANTI-Blue medium (Invitrogen) in 96-well plates and incubated at 37 °C for 15 min. The secreted embryonic alkaline phosphatase activity was assessed by measuring the optical density at 655 nm using a microplate absorbance reader.4
% of phagocytosis =
net experimental phagocytosis × 100% net positive control phagocytosis
Bacterial Infection. J774A.1 macrophages were infected with Klebsiella pneumoniae (NTUH K-2044) at a multiplicity of infection (MOI) of 100. The number of viable bacteria used in each experiment was carefully determined by plate counting. One hour after infection, the cells were incubated with penicillin (250 IU/mL)/streptomycin (250 μg/mL) to limit the growth of residual extracellular bacteria. The culture medium was collected at 24 h after infection, and the levels of IL-1β in the medium were measured by ELISA. Statistical Analysis. All values are the mean ± SD. The data analysis involved one-way ANOVA with a subsequent Scheffé test.
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RESULTS PXT Reduces NO Generation, Inducible NO Synthase Expression, and IL-6 Secretion. The anti-inflammatory activity of PXT was investigated using LPS-activated RAW 264.7 macrophages. We found that NO generation (Figure 1B) and IL-6 secretion (Figure 1C) were inhibited by PXT in a 1212
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Figure 3. Effect of PXT on the phosphorylation levels of PKC. (A) RAW 264.7 macrophages were incubated for 30 min with or without 40 μM PXT and then for 0−60 with or without 1 μg/mL LPS. The phosphorylation levels of PKC-α and PKC-δ were assayed by Western blotting. (B) Quantitation of the phosphorylation levels of PKC-α. (C) Quantitation of the phosphorylation levels of PKC-δ. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ indicates a significant difference at the level of p < 0.05.
occurs via the inhibition of ROS generation, LPS-primed J774A.1 macrophages were incubated with PXT or the antioxidant N-acetylcysteine (NAC) for 30 min before the addition of CPPD, MSU, and ATP. We found that PXT reduced CPPD- and MSU-induced ROS generation; however, PXT was not able to reduce ATP-induced ROS generation (Figure 4B). As damaged and ROS-generating mitochondria activate the NLRP3 inflammasome,22,26,27 to determine whether PXT inhibits the NLRP3 inflammasome by preventing mitochondrial damage, we used the fluorescent probe MitoSOX to detect ROS released from damaged mitochondria. Mitochondrial ROS (mt-ROS) levels were increased by MSU and CPPD treatments, and PXT reduced the mt-ROS levels in these cells (Figure 4C). The overgeneration of mt-ROS induces the translocation of mitochondrial DNA (mt-DNA) into the cytosol, and this cytosolic mt-DNA is a coactivator of the NLRP3 inflammasome.27 Thus, we examined whether the translocation of mt-DNA into the cytosol is decreased by PXT and found that the levels of mt-DNA released into the cytosol were increased in MSU- and CPPD-activated macrophages but were significantly reduced by PXT (Figure 4D). These results indicate that PXT protects mitochondria from damage in macrophages. In addition, caspase-11 has been demonstrated to be required to activate caspase-1 in response to bacteria;28 however, PXT did not affect the expression levels of caspase-11 induced by LPS and interferon-γ (Figure 4E). PXT Reduces IL-1β Precursor Expression. NLRP3 induction is a critical checkpoint for the priming step of NLRP3 inflammasome activation and is regulated by NF-κB.29 Therefore, we investigated whether PXT inhibits the LPSmediated priming step for the NLRP3 inflammasome. We incubated cells with PXT for 30 min before treatment with LPS for 6 h and found that PXT did not inhibit the expression level of NLRP3, although it did reduce the expression level of the IL-
dose-dependent manner, whereas TNF-α secretion was not reduced significantly (Figure 1D). PXT also reduced iNOS expression in a dose-dependent manner, whereas COX-2 expression was not affected (Figure 1E). To examine whether the reduction in pro-inflammatory mediators resulted from reduced cell viability, the toxicity of PXT toward RAW 264.7 cells was examined, and we found that PXT had no significant effect on cell survival at a concentration of 40 μM (Figure 1F). PXT Reduces the Phosphorylation Levels of MitogenActivated Protein Kinases (MAPKs) and PKC-α/δ. MAPKs, including ERK1/2, JNK1/2, and p38, are activated by LPS and regulate inflammatory mediator expression in macrophages.2−4,23 In the present study, we found that LPS induced an increase in the phosphorylation levels of ERK1/2, JNK1/2, and p38 and that these effects, except ERK1/2, were reduced by PXT (Figure 2). In addition, PKC, a component of the TLR4 signaling pathway, regulates macrophage activation in response to LPS.2−4,23 As shown in Figure 3, LPS induced increases in the phosphorylation levels of PKC-α and PKC-δ, and these effects were reduced by PXT. Interestingly, PXT alone increased the phosphorylation levels of PKC-α and PKCδ; however, the detailed mechanism is unclear. PXT Reduces NLRP3 Inflammasome Activation by Reducing Mitochondrial Damage. We examined whether PXT is able to inhibit the IL-1β secretion induced by NLRP3 inflammasome activators such as monosodium urate (MSU) crystals, calcium pyrophosphate dihydrate (CPPD), SiO2 nanoparticles, alum crystals, nigericin, and ATP. We found that IL-1β secretion after treatment with MSU, CPPD, SiO2 nanoparticles, and alum crystals was significantly reduced by PXT, whereas PXT was not able to inhibit IL-1β secretion induced by nigericin and ATP (Figure 4A). The generation of ROS is a crucial element for NLRP3 activation.24,25 To determine whether the inhibition of IL-1β secretion by PXT 1213
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Figure 4. PXT attenuates NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5.5 h with or without 1 μg/mL LPS, then for 30 min with or without PXT in the continued presence or absence of LPS, and then with or without 100 μg/mL MSU (24 h), 100 μg/mL CPPD crystals (24 h), 200 μg/mL nano SiO2 (24 h), 500 μg/mL alum crystals (24 h), 5 mM ATP (0.5 h), or 10 μM nigericin (0.5 h). The levels of IL-1β in the culture medium were measured by ELISA. (B) J774A.1 macrophages were incubated for 5.5 h with 1 μg/mL LPS, then for 30 min with or without 40 μM PXT or 10 mM NAC in the continued presence of LPS, and then with or without 100 μg/mL CPPD crystals, 100 μg/mL MSU, or 5 mM ATP for 10 min. The levels of ROS in the cells were measured by 2′,7′-dichlorofluorescein diacetate. (C) J774A.1 macrophages were incubated for 5.5 h with 1 μg/mL LPS, then for 30 min with or without 40 μM PXT in the continued presence of LPS, and then with or without 100 μg/mL MSU or 100 μg/mL CPPD crystals for 10 min. The levels of ROS in the cells were measured by 2′,7′-dichlorofluorescein diacetate. (D) J774A.1 1214
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Journal of Agricultural and Food Chemistry Figure 4. continued
macrophages were incubated for 5.5 h with 1 μg/mL LPS, then for 30 min with or without 40 μM PXT in the continued presence of LPS, and then with or without 100 μg/mL MSU or 100 μg/mL CPPD crystals for 30 min. Mitochondrial DNA release into the cytosol was measured by the detection of cytochrome c oxidase I DNA in the cytosol. (E) J774A.1 macrophages were incubated for 6 h with 1 μg/mL LPS and 10 ng/mL IFN-γ. The expression level of caspase-11 mRNA was measured by RT-quantitative PCR. The data are expressed as the means ± SD of three separate experiments. ∗ and ∗∗∗ indicate a significant difference at the levels of p < 0.05 and p < 0.001, respectively.
Figure 5. Effect of PXT on the priming step of the NLRP3 inflammasome. (A) J774A.1 macrophages were incubated for 30 min with or without PXT and then for 6 h with or without 1 μg/mL LPS in the continued presence or absence of PXT; the protein expression levels of NLRP3 and proIL-1β in the cells were measured by Western blotting. (B) RAW-Blue cells were incubated for 30 min with or without PXT or 10 mM NAC and then for 24 h with or without 1 μg/mL LPS in the continued presence or absence of PXT or NAC; the activation levels of NF-κB were measured by an NF-κB reporter assay. (C) J774A.1 macrophages were incubated for 30 min with or without 40 μM PXT or 10 mM NAC and then for 10 min with or without 1 μg/mL LPS in the continued presence or absence of PXT or NAC. The levels of ROS in the cells were measured by 2′,7′dichlorofluorescein diacetate. (D) J774A.1 macrophages were incubated for 30 min with or without PXT, then for 5.5 h with or without 1 μg/mL LPS, and 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 blotting, respectively. (E) Mouse primary peritoneal macrophages were incubated for 30 min with or without PXT, then for 5.5 h with or without 1 μg/mL LPS, and then for 30 min with or without 5 mM ATP. The levels of IL-1β in the culture medium were measured by ELISA. (F) J774A.1 macrophages were incubated for 5.5 h with or without 1 μg/mL LPS, then for 30 min with or without PXT in the continued presence or absence of LPS, and then with or without 100 μg/mL FLA-ST or 100 ng/mL MDP for 24 h. 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. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ and ∗∗∗ indicate a significant difference at the levels of p < 0.05 and p < 0.001, respectively.
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Journal of Agricultural and Food Chemistry 1β precursor proIL-1β (Figure 5A). Using NF-κB-dependent alkaline phosphatase reporter cells (RAW-Blue cells), we then examined whether PXT is able to inhibit NF-κB activation. As shown in Figure 5B, we found that NF-κB transcriptional activity in LPS-stimulated macrophages was not affected by PXT but was markedly inhibited by NAC, which was used as a positive control. These results indicated that PXT does not inhibit the priming signal of the NLRP3 inflammasome. In addition, we investigated the effect of PXT on ROS generation, which plays important roles in the LPS-induced expression of proIL-1β,30 and found that LPS-induced ROS generation was reduced by PXT and NAC (Figure 5C). Although PXT was not able to reduce the ATP-induced expression of IL-1β in LPSprimed macrophages (Figure 4A), we assessed whether the secretion levels of IL-1β are reduced by PXT if it is added before LPS priming because we observed decreased expression levels of LPS-induced proIL-1β in PXT-treated cells (Figure 5A). We found that ATP-induced IL-1β secretion was significantly inhibited by PXT when it was added before LPS priming (Figure 5D, left); notably, the activation levels of caspase-1 were also inhibited by PXT under the same condition (Figure 5D, right). PXT also inhibited IL-1β secretion in primary peritoneal macrophages under the same condition (Figure 5E). Furthermore, PXT was not selective for NLRP3dependent inflammasome stimuli, as PXT reduced the IL-1β secretion triggered by FLA-ST (flagellin from S. typhimurium) or muramyl dipeptide, which are dependent on the NLRP1 and NLRC4 inflammasomes,31,32 respectively, and independent of NLRP3 (Figure 5F). PXT Reduces the Phagocytic Efficiency of Macrophages and Decreases the Expression Levels of IL-1β in Bacteria-Infected Macrophages. PXT reduced the IL-1β secretion induced by crystal NLRP3 inflammasome inducers, including alum crystals, CPPD, and SiO2 nanoparticles (Figure 4A). As the activation of the NLRP3 inflammasome by these inducers is dependent on their uptake by phagocytosis, we investigated the hypothesis that PXT inhibits crystal-mediated activation of the NLRP3 inflammasome by inhibiting phagocytic activity. To examine whether PXT affects the phagocytic activity of macrophages, LPS-primed J774A.1 macrophages were treated with PXT or colchicine (a microtubule polymerization inhibitor) for 30 min, and the phagocytosis activity was then assessed by measuring the uptake of fluorescence-conjugated E. coli. We found that both PXT and colchicine decreased the uptake of fluorescenceconjugated E. coli by macrophages (Figure 6A). In addition, we tested whether PXT affects IL-1β secretion by macrophages infected with live K. pneumoniae and found that PXT reduced the secretion level (Figure 6B). These results suggest that PXT may modulate macrophage function at K. pneumoniae-infected sites by impairing phagocytosis and reducing cytokine production. The Peroxide Group of PXT Is Important for Its Antiinflammatory Activity. Because PXT contains a unique peroxide group (−OOH) on its carbon chain, we investigated the hypothesis that this unique group is important for the molecule’s anti-inflammatory activity. To this end, we isolated a PXT analogue, auraptenol, which contains a hydroxyl group (−OH) at the corresponding peroxide group position of PXT (Figure 1A), from the ethanol extract of C. monnieri fruits. We then compared the anti-inflammatory activities of PXT and auraptenol in LPS-activated macrophages and found that auraptenol reduced LPS-induced IL-6 secretion (23.5 ± 1.5
Figure 6. PXT reduces the phagocytic efficiency of macrophages and decreases the expression levels of IL-1β in bacteria-infected macrophages. (A) J774A.1 macrophages were incubated with LPS for 6 h and then for 30 min with or without PXT or colchicine. The cells were incubated with fluorescence-conjugated E. coli for 1 h. The phagocytic efficiency was determined by measuring the fluorescence intensity. (B) J774A.1 macrophages were infected with K. pneumoniae for 24 h at 100 MOI. 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. ∗∗∗ indicates a significant difference at the level of p < 0.001.
ng/mL) to 17.5 ± 0.9 ng/mL at 100 μM, whereas 10 μM PXT reduced it to 7.2 ± 1.0 ng/mL (Figure 7A). In addition, 100 μM auraptenol reduced LPS-induced NO production (39 ± 5 μM) to 25 ± 3 μM, although 10 μM PXT reduced it to 5 ± 2 μM (Figure 7B). Furthermore, whereas auraptenol only slightly inhibited the expression levels of iNOS and proIL-1β in LPS-
Figure 7. The peroxide group of PXT is important for its antiinflammatory activity. (A, B) RAW 264.7 macrophages were incubated for 30 min with or without PXT or auraptenol (aura) and then for 24 h with or without 1 μg/mL LPS. The levels of IL-6 (A) and NO (B) in the culture medium were measured by ELISA and the Griess reaction, respectively. (C) RAW 264.7 macrophages were incubated for 30 min with or without aura and then for 6 h (for proIL-1β) and 24 h (for iNOS) with or without 1 μg/mL LPS. The levels of proIL-1β and iNOS in the cells were measured by Western blotting. The data are expressed as the means ± SD for three separate experiments. The Western blotting results are representative of those obtained in three different experiments. ∗, ∗∗, and ∗∗∗ indicate a significant difference at the levels of p < 0.05, p < 0.01, and p < 0.001, respectively. 1216
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K+ efflux has a role in the regulation of the NLRP3 inflammasome and can be achieved either directly by nigericin or indirectly by the purinergic receptor for ATP.38,39 It has been reported that crystal-induced NALP3 inflammasome activation is inhibited by phagocytosis inhibitors.15 These results indicate that crystal uptake by phagocytosis is a prerequisite for activation of the NARP3 inflammasome. In this study, we demonstrated that PXT reduced phagocytosis, which partially explains why PXT inhibited the NLRP3 inflammasome-mediated IL-1β secretion induced by MSU, CPPD, SiO2 nanoparticles, and alum crystals. In contrast, PXT did not affect IL-1β secretion induced by nigericin and ATP in LPS-primed macrophages, suggesting that PXT might not affect K+ efflux induced by these molecules. PXT is a peroxide (−OOH)-containing coumarin compound. When subjected to acid, base, or temperature increases in the environment, the −OOH group on the compound is likely to degrade into hydroxyl radical (•OH), a most destructive free radical that reacts readily with the metallic core atoms of intracellular enzymes and in turn modulates the secretion of certain cytokines. However, when the concentrations of these free radicals are sufficient, they may partake in important intracellular metabolism processes and become a line of defense in preventing or fighting disease. In this study, we found that freshly extracted ethanolic PXT tended to decrease in concentration during storage, which became notable with more than 6 months at room temperature; the situation was much improved by storage at −20 °C. However, storing the ethanolic solution of auraptenol at room temperature for more than 6 months resulted in an unchanged concentration based on HPLC and NMR analyses, indicating that the compound is highly stable. Although PXT exhibited anti-inflammatory activities in LPSactivated macrophages, it did not cause a general unresponsiveness to LPS, as the expressions of COX-2 and TNF-α were not reduced by PXT. These findings indicate that the administration of PXT might prevent excessive inflammatory response but does not cause immunodeficiency. C. monnieri fruits have been used to treat gout in traditional medicine, although there is no scientific evidence supporting this. Here, we demonstrate that PXT reduces MSU-induced activation of the NLRP3 inflammasome by inhibiting ROS generation and mitochondrial DNA release, explaining why C. monnieri fruits are able to ameliorate gout.17 In summary, we demonstrate that PXT possesses anti-inflammatory activity by reducing the expression levels of NO and IL-6, as well as by reducing the activation of the NLRP3 inflammasome. These results suggest that PXT may have a potential use as a natural antiinflammation agent or nutraceutical for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases.
activated macrophages (Figure 7C), PXT significantly inhibited expression, as shown in Figures 1E and 5A, respectively. These results indicated that the anti-inflammatory activity of auraptenol is lower than that of PXT and suggested that the unique peroxide group is important for the anti-inflammatory activity of PXT.
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DISCUSSION PXT was purified along with osthole from the ethanol extract of C. monnieri seeds. The structure of PXT is similar to that of osthole; however, their immune modulation properties in LPSactivated macrophages are quite different.4 The different biological outcomes between PXT and osthole are as follows: (1) PXT reduces the levels of IL-6, but osthole increases them. (2) PXT does not affect the levels of TNF-α and COX-2, whereas osthole reduces them. (3) PXT does not affect the phosphorylation levels of ERK1/2, yet osthole increases them. (4) PXT does not affect the activation levels of NF-κB, whereas osthole reduces them. (5) PXT reduces the phosphorylation levels of PKC-δ phosphorylation, but osthole does not. The priming step of the NLRP3 inflammasome is regulated by ROS, which are required for NLRP3 expression.25 PXT did not affect the priming step of the NLRP3 inflammasome, although PXT did reduce the levels of ROS in LPS-activated macrophages (Figure 5C). ROS is involved in both the priming and activation steps of the NLRP3 inflammasome,24 and the up-regulation of ROS levels in macrophages results in the activation of PI3K, promoting caspase-1 activation and IL-1β secretion.33 The inhibition of MSU- and CPPD-induced ROS generation may be responsible for the reduced NLRP3 inflammasome-derived IL-1β secretion caused by PXT. Furthermore, we found that PXT reduced mitochondrial ROS generation, which plays an important role in NLRP3 inflammasome activation.26,27 The overproduction of mitochondrial ROS promotes a mitochondrial permeability transition that facilitates the cytosolic release of mitochondrial DNA, which stimulates the activation of the NLRP3 inflammasome.26,27 PXT also reduced the cytosolic release of mitochondrial DNA, indicating that mitochondrial stabilization may be responsible for the reduced activation of the NLRP3 inflammasome mediated by PXT. PXT did not inhibit either ATP-induced IL-1β secretion (Figure 4A) or ROS generation (Figure 4B) in LPS-primed macrophages; PXT also had no effect on LPS-induced NLRP3 expression (Figure 5A). These results indicated that PXT did not affect the priming or activation signal of the NLRP3 inflammasome due to LPS or ATP, respectively. Notably, however, PXT did reduce ATP-induced caspase-1 activation when it was added before LPS or ATP (Figure 5D). One possible mechanism is that PXT might increase the cellular levels of cAMP, which has been demonstrated to inhibit inflammasome assembly by binding to NLRP3 directly.34 Osthole was reported to increase the tissue cAMP content by inhibiting the activity of cAMP phosphodiesterase in guinea pig trachea.35 Another possible mechanism is that PXT might increase autophagy, which limits NLRP3 inflammasome activation by targeting inflammasome components for degradation36,37 and by inhibiting the release of mitochondrial DNA from damaged mitochondria.22 Furthermore, caspase-11 induction by LPS or interferon-γ synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation;8 however, PXT did not affect the expression levels of caspase11 induced by LPS and interferon-γ.
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AUTHOR INFORMATION
Corresponding Author
*(K.-F.H.) Phone: +(886)-3-935-7400, ext. 7626. Fax: +(886)3-931-1526. E-mail: [email protected]. Funding
Contract grant sponsor: Ministry of Science and Technology, Taiwan; Contract grant number: NSC 102-2628-B-197-001MY3; NSC 103-2923-B-197-001-MY3. CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan, and Taiwan Ministry of Health and Welfare Clinical Trial and 1217
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The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat. Med. 2011, 17 (2), 179−188. (15) Hornung, V.; Bauernfeind, F.; Halle, A.; Samstad, E. O.; Kono, H.; Rock, K. L.; Fitzgerald, K. A.; Latz, E. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 2008, 9 (8), 847−856. (16) Heneka, M. T.; Kummer, M. P.; Stutz, A.; Delekate, A.; Schwartz, S.; Vieira-Saecker, A.; Griep, A.; Axt, D.; Remus, A.; Tzeng, T. C.; Gelpi, E.; Halle, A.; Korte, M.; Latz, E.; Golenbock, D. T. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 2013, 493 (7434), 674−678. (17) Martinon, F.; Pétrilli, V.; Mayor, A.; Tardivel, A.; Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006, 440 (7081), 237−241. (18) Anders, H. J.; Muruve, D. A. The inflammasomes in kidney disease. J. Am. Soc. Nephrol. 2011, 22 (6), 1007−1018. (19) Yang, S. M.; Chan, Y. L.; Hua, K. F.; Chang, J. M.; Chen, H. L.; Tsai, Y. J.; Hsu, Y. J.; Chao, L. K.; Yang, F. L.; Tsai, Y. L.; Wu, S. H.; Wang, Y. F.; Tsai, C. L.; Chen, A.; Ka, S. M. Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis. Free Radical Biol. Med. 2014, 73C, 260−269. (20) Hua, K. F.; Yang, S. M.; Kao, T. Y.; Chang, J. M.; Chen, H. L.; Tsai, Y. J.; Chen, A.; Yang, S. S.; Chao, L. K.; Ka, S. M. Osthole mitigates progressive IgA nephropathy by inhibiting reactive oxygen species generation and NF-κB/NLRP3 pathway. PLoS One 2013, 8 (10), No. e77794. (21) Wang, Z. C.; Feng, D. Q.; Ke, C. H. Coumarins from the herb Cnidium monnieri and chemically modified derivatives as antifoulants against Balanus albicostatus and Bugula neritina larvae. Int. J. Mol. Sci. 2013, 14 (1), 1197−1206. (22) Nakahira, K.; Haspel, J. A.; Rathinam, V. A.; Lee, S. J.; Dolinay, T.; Lam, H. C.; Englert, J. A.; Rabinovitch, M.; Cernadas, M.; Kim, H. P.; Fitzgerald, K. A.; Ryter, S. W.; Choi, A. M. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat. Immunol. 2011, 12 (3), 222−230. (23) Su, S. C.; Hua, K. F.; Lee, H.; Chao, L. K.; Tan, S. K.; Lee, H.; Yang, S. F.; Hsu, H. Y. LTA and LPS mediated activation of protein kinases in the regulation of inflammatory cytokines expression in macrophages. Clin. Chim. Acta 2006, 374 (1−2), 106−115. (24) Tschopp, J.; Schroder, K. NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat. Rev. Immunol. 2010, 10 (3), 210−215. (25) Bauernfeind, F.; Bartok, E.; Rieger, A.; Franchi, L.; Núñez, G.; Hornung, V. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J. Immunol. 2011, 187 (2), 613−617. (26) Kepp, O.; Galluzzi, L.; Kroemer, G. Mitochondrial control of the NLRP3 inflammasome. Nat. Immunol. 2011, 12 (3), 199−200. (27) Zhou, R.; Yazdi, A. S.; Menu, P.; Tschopp, J. A role for mitochondria in NLRP3 inflammasome activation. Nature 2011, 469, 221−225. (28) Kayagaki, N.; Warming, S.; Lamkanfi, M.; Vande Walle, L.; Louie, S.; Dong, J.; Newton, K.; Qu, Y.; Liu, J.; Heldens, S.; Zhang, J.; Lee, W. P.; Roose-Girma, M.; Dixit, V. M. Non-canonical inflammasome activation targets caspase-11. Nature 2011, 479 (7371), 117−121. (29) Bauernfeind, F. G.; Horvath, G.; Stutz, A.; Alnemri, E. S.; MacDonald, K.; Speert, D.; Fernandes-Alnemri, T.; Wu, J.; Monks, B. G.; Fitzgerald, K. A.; Hornung, V.; Latz, E. Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 2009, 183 (2), 787−791. (30) Liao, P. C.; Chao, L. K.; Chou, J. C.; Dong, W. C.; Lin, C. N.; Lin, C. Y.; Chen, A.; Ka, S. M.; Ho, C. L.; Hua, K. F. Lipopolysaccharide/adenosine triphosphate-mediated signal transduction in the regulation of NLRP3 protein expression and caspase-
Research Center of Excellence (MOHW103-TDU-B-212113002). Notes
The authors declare no competing financial interest.
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Peroxyauraptenol Inhibits Inflammation and NLRP3 Inflammasome Activation by Inhibiting Reactive Oxygen Species Generation and Preserving Mitochondrial Integrity Louis Kuoping Chao,† Cheng-Hsiu Lin,§ Huan-Wen Chiu,§ Wei-Ting Wong,§ Hsiao-Wen Chiu,§ Yu-Ling Tasi,# Yueh-Hsiung Kuo,⊥,Δ,¶ Yi-Chich Chiu,○ May-Lan Liu,⊗ Chen-Lung Ho,∇ and Kuo-Feng Hua*,§ †
Department of Cosmeceutics, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402 Department of Biotechnology and Animal Science, National Ilan University, No. 1, Sec. 1, Shen-Lung Road, Ilan, Taiwan 260 # Graduate Institute of Life Science, National Defense Medical Center, No. 161, Sec. 6, Minquan E. Road, Taipei, Taiwan 114 ⊥ Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402 Δ Tsuzuki Institute for Traditional Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402 ¶ Department of Biotechnology, Asia University, No. 500 Lioufeng Road, Wufeng, Taichung, Taiwan 41354 ○ Department of Biomechatronic Engineering, National Ilan University, No. 1, Sec. 1, Shen-Lung Road, Ilan, Taiwan 260 ⊗ Department of Nutritional Science, Toko University, No. 51, Sec. 2, Xuefu Road, Chiayi, Taiwan 61363 ∇ Division of Wood Cellulose, Taiwan Forestry Research Institute, No. 53 Nanhai Road, Taipei, Taiwan 100 §
ABSTRACT: Peroxyauraptenol (PXT) is a peroxide-containing coumarin compound isolated from the seeds of Cnidium monnieri. PXT exerts anti-inflammatory activities, as it reduces the levels of inducible nitric oxide synthase, nitric oxide, IL-6, and NLRP3 inflammasome-derived IL-1β in lipopolysaccharide-activated macrophages. PXT also exerts anti-inflammatory activity by reducing reactive oxygen species generation (including mitochondrial), mitogen-activated protein kinase, protein kinase C-α/δ phosphorylation, and the release of mitochondrial DNA into the cytosol. In addition, PXT suppresses the phagocytic activity of macrophages and IL-1β secretion by Klebsiella pneumoniae-infected macrophages. The unique peroxide group is important for the anti-inflammatory activity of PXT, as this activity is reduced when the peroxide group is replaced by a hydroxyl group. These findings suggest that PXT may be a candidate for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation-related diseases. KEYWORDS: peroxyauraptenol, inflammation, NLRP3 inflammasome, reactive oxygen species, mitochondria
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diabetes mellitus,11 atherosclerosis,12 obesity,13,14 silicosis,15 Alzheimer’s disease,16 gout,17 and kidney disease.18 Given the importance of inflammation and the inflammasome in the pathogenesis of diseases, anti-inflammatory approaches or biological agents that target inflammasome or specific proinflammatory mediator pathways can be used to improve the control of inflammation-related disorders. The fruit of Cnidium monnieri, a medical food, has been the subject of considerable interest because of its broad spectrum of pharmacological properties. The most thoroughly characterized main constituent from the fruit of C. monnieri is osthole. We have demonstrated that osthole possesses anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated macrophages4 and mitigates renal inflammation in mice.19,20 In the present study, we isolated peroxyauraptenol (PXT), a coumarin compound with a unique peroxide group, from the fruit of C.
INTRODUCTION Inflammation is one of the important host defense mechanisms in mammals; however, uncontrolled inflammation is harmful to health.1 The overproduction of pro-inflammatory cytokines and mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), nitric oxide (NO), and cyclooxygenase-2 (COX-2), by macrophages is a feature of activated innate immunity.2−4 The strong link between inflammation and disease is becoming increasingly evident; for example, an elevation in circulating inflammatory cytokine IL-6 is a risk factor for the pathogenesis of inflammatory bowel diseases.5 NO is produced from L-arginine by inducible nitric oxide synthase (iNOS) and contributes to some pathogeneses by promoting oxidative stress, tissue injury, and even cancer, if generated in excess.6 IL-1β release is controlled by the inflammasome, a caspase-1containing multiprotein complex, and plays important roles in microbial infections and metabolic processes.7 The most thoroughly characterized inflammasome is the NLRP3 inflammasome, which is considered to be a controller of disease development, including pathogen infections,8−10 type 2 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1210
November 14, 2014 December 23, 2014 December 31, 2014 December 31, 2014 DOI: 10.1021/jf5054436 J. Agric. Food Chem. 2015, 63, 1210−1219
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Journal of Agricultural and Food Chemistry monnieri. PXT showed anti-inflammatory activity, but the immune modulation properties of PXT and osthole were found to be different, despite their similar chemical structures. The anti-inflammatory activity of PXT was associated with the unique peroxide group, as the anti-inflammatory activity was reduced when this group was replaced by a hydroxyl group.
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MATERIALS AND METHODS
Materials. LPS (from Escherichia coli 0111:B4), ATP, and mouse antibodies against mouse phospho-ERK1/2, phospho-JNK1/2, phospho-p38, and actin were purchased from Sigma (St. Louis, MO, USA). Rabbit antibodies against mouse phospho-PKC-α/δ, IL-1β, caspase-1, iNOS, and COX-2 and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse IL-1β, IL-6, and TNF-α ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-mouse NLRP3 antibody was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The AlamarBlue assay kit was purchased from AbD Serotec Ltd. (Oxford, UK). All of the inflammasome inducers and the QUANTI-Blue reagent were purchased from InvivoGen (San Diego, CA, USA). Compound Extraction and Purification. C. monnieri seeds (600 g) were purchased from a traditional Chinese medicine dispensary in Taiwan. The seeds were extracted with 15 L of ethanol (95% v/v) at room temperature for 10 days, and the supernatants were then concentrated to produce an alcoholic extract. PXT and auraptenol were purified by semipreparative high-performance liquid chromatography (HPLC) (D-Star DLC-20 Instruments with a UV−vis detector) with a Phenomenex Luna Silica (2) column (250 mm length, 10 mm i.d., particle shape/size = 5.0 μm) (Figure 1A). The separation conditions were as follows: 500 μL was injected for each separation; flow rate, 4 mL/min; mobile phase, acetone/hexane = 1:6. The structures of PXT and auraptenol were identified by physical and spectral data (mp, EIMS, 1H NMR, UV, IR) compared with previous research values.4,21 Cell Cultures. The murine macrophage cell lines RAW 264.7 and J774A.1 were obtained from American Type Culture Collection (Rockville, MD, USA). RAW 264.7 macrophages stably transfected with the NF-κB reporter gene (RAW-Blue cells) were purchased from InvivoGen. Mouse peritoneal macrophages were elicited by intraperitoneal injection of 4% sterile thioglycollate medium (2 mL). After 3 days, the mice were sacrificed by cervical dislocation, and the macrophages were harvested. AlamarBlue Assay for Cell Viability. RAW 264.7 cells were incubated with or without PXT for 24 h. The AlamarBlue assay was used to determine the cytotoxicity of PXT according to the protocol described in the manufacturer’s instructions (AbD Serotec, Oxford, UK).4 Detection of Pro-inflammatory Mediators and Protein Phosphorylation. The levels of NO and cytokines (IL-1β, IL-6, and TNF-α) in the culture medium were measured by the Griess reaction and ELISA, respectively. The levels of iNOS, COX-2, and caspase-1 and the phosphorylation levels of ERK1/2, JNK1/2, p38, and PKC-α/δ in cells were measured by Western blotting. The detailed procedures were described in our previous study.4 The density of the bands of the Western blotting was analyzed by ImageJ software. We selected an identical rectangular area around each lane of the Western blot even there was no band of control lane. The density of the selected area was analyzed by ImageJ, and the relative band density was normalized to actin before fold change was calculated. The densitometry fold change of each group was calculated by comparison to the control group, as the control group had a fold change of 1. The bars accompanying blots are the means of three separate experiments. Detection of ROS. 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 USA), at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a microplate absorbance reader (Bio-Rad Laboratories, Inc.). Mitochondrial ROS generation was measured by detecting the fluorescence intensity of MitoSOX
Figure 1. Effect of PXT on the expression levels of inflammatory mediators. (A) Chromatography of alcoholic extract of C. monnieri seeds by a silica column. (B−D) RAW 264.7 macrophages were incubated for 30 min with or without PXT and then for 24 h with or without 1 μg/mL LPS. The levels of NO (B), IL-6 (C), and TNF-α (D) in the culture medium were measured by the Griess reaction and ELISA, respectively. (E) RAW 264.7 macrophages were incubated for 30 min with or without PXT and then for 24 h with or without 1 μg/ mL LPS. The levels of iNOS and COX-2 were assayed by Western blotting. (F) RAW 264.7 macrophages were incubated for 24 h with or without PXT, and the cell viability was then assayed by the AlamarBlue assay. The data are expressed as the means ± SD of three separate experiments. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ and ∗∗∗ indicate a significant difference at the levels of p < 0.05 and p < 0.001, respectively.
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Figure 2. Effect of PXT on the phosphorylation levels of MAPKs. (A) RAW 264.7 macrophages were incubated for 30 min with or without 40 μM PXT and then for 0−60 with or without 1 μg/mL LPS. The phosphorylation levels of ERK1/2, JNK1/2, and p38 were assayed by Western blotting. (B) Quantitation of the phosphorylation levels of ERK1/2. (C) Quantitation of the phosphorylation levels of JNK1/2. (D) Quantitation of the phosphorylation levels of p38. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ indicates a significant difference at the level of p < 0.05. Phagocytosis Assay. J774A.1 macrophages were seeded in 96well flat-bottom plates at a density of 5 × 103 cells in 100 μL of RPMI 1640 medium containing 10% FBS per well and incubated with 1 μg/ mL LPS for 6 h. The cells were incubated with or without 40 μM PXT or 10 μM colchicine for 30 min and then incubated for 1 h with or without 100 μg of fluorescence-conjugated Escherichia coli. The fluorescence intensity was measured at an excitation wavelength of 509 nm and an emission wavelength of 533 nm. Net phagocytosis was calculated by subtracting the average fluorescence intensity of the nocell negative-control wells from all of the experimental wells. The percent of phagocytosis was then calculated as a fraction of the net positive control phagocytosis, as follows:
(Invitrogen, Carlsbad, CA, USA) at an excitation wavelength of 514 nm and an emission wavelength of 585 nm using a microplate absorbance reader. Detection of Mitochondrial DNA Release. The detection of mitochondrial DNA (mt-DNA) in the cytosol was performed as described.22 Briefly, after treatment, cells were homogenized in a protease inhibitor-containing buffer (100 mM, pH 7.4, Tricine− NaOH, 0.25 M sucrose, 1 mM EDTA) and then centrifuged for 10 min at 700g and 4 °C. The protein concentration and volume of the supernatant were normalized, and the sample was then centrifuged for 30 min at 10000g and 4 °C. The resulting pellets were identified as the mitochondrial fraction, and the supernatants were identified as the cytosolic fraction. DNA was isolated from 200 μL of the cytosolic fraction. The mt-DNA copy number was measured by quantitative PCR and normalized to nuclear DNA (encoding 18S rRNA) levels using a ratio of cytochrome c oxidase I DNA to nuclear DNA. The results are expressed as fold changes between the sample and control groups. The quality of the mitochondrial and cytosolic fractions was monitored by detecting the protein expression level of voltagedependent anion channel (VDAC, a marker protein for mitochondria). We found that VDAC can be detected only in the mitochondrial fraction and is not present in the cytosolic fraction (data not shown), indicating that the cytosolic fraction is free of mitochondria. The primers used were as follows: mouse 18S, forward, 5′-TAGAGGGACAAGTGGCGTTC-3′, reverse, 5′-CGCTGAGCCAGTCAGTGT-3′; mouse cytochrome c oxidase I, forward, 5′-GCCCCAGATATAGCATTCCC-3′, reverse, 5′-GTTCATCCTGTTCCTGCTCC3′. NF-κB Reporter Assay. The medium (20 μL) from treated RAWBlue cells was mixed with 200 μL of QUANTI-Blue medium (Invitrogen) in 96-well plates and incubated at 37 °C for 15 min. The secreted embryonic alkaline phosphatase activity was assessed by measuring the optical density at 655 nm using a microplate absorbance reader.4
% of phagocytosis =
net experimental phagocytosis × 100% net positive control phagocytosis
Bacterial Infection. J774A.1 macrophages were infected with Klebsiella pneumoniae (NTUH K-2044) at a multiplicity of infection (MOI) of 100. The number of viable bacteria used in each experiment was carefully determined by plate counting. One hour after infection, the cells were incubated with penicillin (250 IU/mL)/streptomycin (250 μg/mL) to limit the growth of residual extracellular bacteria. The culture medium was collected at 24 h after infection, and the levels of IL-1β in the medium were measured by ELISA. Statistical Analysis. All values are the mean ± SD. The data analysis involved one-way ANOVA with a subsequent Scheffé test.
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RESULTS PXT Reduces NO Generation, Inducible NO Synthase Expression, and IL-6 Secretion. The anti-inflammatory activity of PXT was investigated using LPS-activated RAW 264.7 macrophages. We found that NO generation (Figure 1B) and IL-6 secretion (Figure 1C) were inhibited by PXT in a 1212
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Figure 3. Effect of PXT on the phosphorylation levels of PKC. (A) RAW 264.7 macrophages were incubated for 30 min with or without 40 μM PXT and then for 0−60 with or without 1 μg/mL LPS. The phosphorylation levels of PKC-α and PKC-δ were assayed by Western blotting. (B) Quantitation of the phosphorylation levels of PKC-α. (C) Quantitation of the phosphorylation levels of PKC-δ. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ indicates a significant difference at the level of p < 0.05.
occurs via the inhibition of ROS generation, LPS-primed J774A.1 macrophages were incubated with PXT or the antioxidant N-acetylcysteine (NAC) for 30 min before the addition of CPPD, MSU, and ATP. We found that PXT reduced CPPD- and MSU-induced ROS generation; however, PXT was not able to reduce ATP-induced ROS generation (Figure 4B). As damaged and ROS-generating mitochondria activate the NLRP3 inflammasome,22,26,27 to determine whether PXT inhibits the NLRP3 inflammasome by preventing mitochondrial damage, we used the fluorescent probe MitoSOX to detect ROS released from damaged mitochondria. Mitochondrial ROS (mt-ROS) levels were increased by MSU and CPPD treatments, and PXT reduced the mt-ROS levels in these cells (Figure 4C). The overgeneration of mt-ROS induces the translocation of mitochondrial DNA (mt-DNA) into the cytosol, and this cytosolic mt-DNA is a coactivator of the NLRP3 inflammasome.27 Thus, we examined whether the translocation of mt-DNA into the cytosol is decreased by PXT and found that the levels of mt-DNA released into the cytosol were increased in MSU- and CPPD-activated macrophages but were significantly reduced by PXT (Figure 4D). These results indicate that PXT protects mitochondria from damage in macrophages. In addition, caspase-11 has been demonstrated to be required to activate caspase-1 in response to bacteria;28 however, PXT did not affect the expression levels of caspase-11 induced by LPS and interferon-γ (Figure 4E). PXT Reduces IL-1β Precursor Expression. NLRP3 induction is a critical checkpoint for the priming step of NLRP3 inflammasome activation and is regulated by NF-κB.29 Therefore, we investigated whether PXT inhibits the LPSmediated priming step for the NLRP3 inflammasome. We incubated cells with PXT for 30 min before treatment with LPS for 6 h and found that PXT did not inhibit the expression level of NLRP3, although it did reduce the expression level of the IL-
dose-dependent manner, whereas TNF-α secretion was not reduced significantly (Figure 1D). PXT also reduced iNOS expression in a dose-dependent manner, whereas COX-2 expression was not affected (Figure 1E). To examine whether the reduction in pro-inflammatory mediators resulted from reduced cell viability, the toxicity of PXT toward RAW 264.7 cells was examined, and we found that PXT had no significant effect on cell survival at a concentration of 40 μM (Figure 1F). PXT Reduces the Phosphorylation Levels of MitogenActivated Protein Kinases (MAPKs) and PKC-α/δ. MAPKs, including ERK1/2, JNK1/2, and p38, are activated by LPS and regulate inflammatory mediator expression in macrophages.2−4,23 In the present study, we found that LPS induced an increase in the phosphorylation levels of ERK1/2, JNK1/2, and p38 and that these effects, except ERK1/2, were reduced by PXT (Figure 2). In addition, PKC, a component of the TLR4 signaling pathway, regulates macrophage activation in response to LPS.2−4,23 As shown in Figure 3, LPS induced increases in the phosphorylation levels of PKC-α and PKC-δ, and these effects were reduced by PXT. Interestingly, PXT alone increased the phosphorylation levels of PKC-α and PKCδ; however, the detailed mechanism is unclear. PXT Reduces NLRP3 Inflammasome Activation by Reducing Mitochondrial Damage. We examined whether PXT is able to inhibit the IL-1β secretion induced by NLRP3 inflammasome activators such as monosodium urate (MSU) crystals, calcium pyrophosphate dihydrate (CPPD), SiO2 nanoparticles, alum crystals, nigericin, and ATP. We found that IL-1β secretion after treatment with MSU, CPPD, SiO2 nanoparticles, and alum crystals was significantly reduced by PXT, whereas PXT was not able to inhibit IL-1β secretion induced by nigericin and ATP (Figure 4A). The generation of ROS is a crucial element for NLRP3 activation.24,25 To determine whether the inhibition of IL-1β secretion by PXT 1213
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Figure 4. PXT attenuates NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5.5 h with or without 1 μg/mL LPS, then for 30 min with or without PXT in the continued presence or absence of LPS, and then with or without 100 μg/mL MSU (24 h), 100 μg/mL CPPD crystals (24 h), 200 μg/mL nano SiO2 (24 h), 500 μg/mL alum crystals (24 h), 5 mM ATP (0.5 h), or 10 μM nigericin (0.5 h). The levels of IL-1β in the culture medium were measured by ELISA. (B) J774A.1 macrophages were incubated for 5.5 h with 1 μg/mL LPS, then for 30 min with or without 40 μM PXT or 10 mM NAC in the continued presence of LPS, and then with or without 100 μg/mL CPPD crystals, 100 μg/mL MSU, or 5 mM ATP for 10 min. The levels of ROS in the cells were measured by 2′,7′-dichlorofluorescein diacetate. (C) J774A.1 macrophages were incubated for 5.5 h with 1 μg/mL LPS, then for 30 min with or without 40 μM PXT in the continued presence of LPS, and then with or without 100 μg/mL MSU or 100 μg/mL CPPD crystals for 10 min. The levels of ROS in the cells were measured by 2′,7′-dichlorofluorescein diacetate. (D) J774A.1 1214
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Journal of Agricultural and Food Chemistry Figure 4. continued
macrophages were incubated for 5.5 h with 1 μg/mL LPS, then for 30 min with or without 40 μM PXT in the continued presence of LPS, and then with or without 100 μg/mL MSU or 100 μg/mL CPPD crystals for 30 min. Mitochondrial DNA release into the cytosol was measured by the detection of cytochrome c oxidase I DNA in the cytosol. (E) J774A.1 macrophages were incubated for 6 h with 1 μg/mL LPS and 10 ng/mL IFN-γ. The expression level of caspase-11 mRNA was measured by RT-quantitative PCR. The data are expressed as the means ± SD of three separate experiments. ∗ and ∗∗∗ indicate a significant difference at the levels of p < 0.05 and p < 0.001, respectively.
Figure 5. Effect of PXT on the priming step of the NLRP3 inflammasome. (A) J774A.1 macrophages were incubated for 30 min with or without PXT and then for 6 h with or without 1 μg/mL LPS in the continued presence or absence of PXT; the protein expression levels of NLRP3 and proIL-1β in the cells were measured by Western blotting. (B) RAW-Blue cells were incubated for 30 min with or without PXT or 10 mM NAC and then for 24 h with or without 1 μg/mL LPS in the continued presence or absence of PXT or NAC; the activation levels of NF-κB were measured by an NF-κB reporter assay. (C) J774A.1 macrophages were incubated for 30 min with or without 40 μM PXT or 10 mM NAC and then for 10 min with or without 1 μg/mL LPS in the continued presence or absence of PXT or NAC. The levels of ROS in the cells were measured by 2′,7′dichlorofluorescein diacetate. (D) J774A.1 macrophages were incubated for 30 min with or without PXT, then for 5.5 h with or without 1 μg/mL LPS, and 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 blotting, respectively. (E) Mouse primary peritoneal macrophages were incubated for 30 min with or without PXT, then for 5.5 h with or without 1 μg/mL LPS, and then for 30 min with or without 5 mM ATP. The levels of IL-1β in the culture medium were measured by ELISA. (F) J774A.1 macrophages were incubated for 5.5 h with or without 1 μg/mL LPS, then for 30 min with or without PXT in the continued presence or absence of LPS, and then with or without 100 μg/mL FLA-ST or 100 ng/mL MDP for 24 h. 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. The Western blotting results are representative of those obtained in three different experiments, and the histograms represent the change in the ratio relative to actin compared to the control group. ∗ and ∗∗∗ indicate a significant difference at the levels of p < 0.05 and p < 0.001, respectively.
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Journal of Agricultural and Food Chemistry 1β precursor proIL-1β (Figure 5A). Using NF-κB-dependent alkaline phosphatase reporter cells (RAW-Blue cells), we then examined whether PXT is able to inhibit NF-κB activation. As shown in Figure 5B, we found that NF-κB transcriptional activity in LPS-stimulated macrophages was not affected by PXT but was markedly inhibited by NAC, which was used as a positive control. These results indicated that PXT does not inhibit the priming signal of the NLRP3 inflammasome. In addition, we investigated the effect of PXT on ROS generation, which plays important roles in the LPS-induced expression of proIL-1β,30 and found that LPS-induced ROS generation was reduced by PXT and NAC (Figure 5C). Although PXT was not able to reduce the ATP-induced expression of IL-1β in LPSprimed macrophages (Figure 4A), we assessed whether the secretion levels of IL-1β are reduced by PXT if it is added before LPS priming because we observed decreased expression levels of LPS-induced proIL-1β in PXT-treated cells (Figure 5A). We found that ATP-induced IL-1β secretion was significantly inhibited by PXT when it was added before LPS priming (Figure 5D, left); notably, the activation levels of caspase-1 were also inhibited by PXT under the same condition (Figure 5D, right). PXT also inhibited IL-1β secretion in primary peritoneal macrophages under the same condition (Figure 5E). Furthermore, PXT was not selective for NLRP3dependent inflammasome stimuli, as PXT reduced the IL-1β secretion triggered by FLA-ST (flagellin from S. typhimurium) or muramyl dipeptide, which are dependent on the NLRP1 and NLRC4 inflammasomes,31,32 respectively, and independent of NLRP3 (Figure 5F). PXT Reduces the Phagocytic Efficiency of Macrophages and Decreases the Expression Levels of IL-1β in Bacteria-Infected Macrophages. PXT reduced the IL-1β secretion induced by crystal NLRP3 inflammasome inducers, including alum crystals, CPPD, and SiO2 nanoparticles (Figure 4A). As the activation of the NLRP3 inflammasome by these inducers is dependent on their uptake by phagocytosis, we investigated the hypothesis that PXT inhibits crystal-mediated activation of the NLRP3 inflammasome by inhibiting phagocytic activity. To examine whether PXT affects the phagocytic activity of macrophages, LPS-primed J774A.1 macrophages were treated with PXT or colchicine (a microtubule polymerization inhibitor) for 30 min, and the phagocytosis activity was then assessed by measuring the uptake of fluorescence-conjugated E. coli. We found that both PXT and colchicine decreased the uptake of fluorescenceconjugated E. coli by macrophages (Figure 6A). In addition, we tested whether PXT affects IL-1β secretion by macrophages infected with live K. pneumoniae and found that PXT reduced the secretion level (Figure 6B). These results suggest that PXT may modulate macrophage function at K. pneumoniae-infected sites by impairing phagocytosis and reducing cytokine production. The Peroxide Group of PXT Is Important for Its Antiinflammatory Activity. Because PXT contains a unique peroxide group (−OOH) on its carbon chain, we investigated the hypothesis that this unique group is important for the molecule’s anti-inflammatory activity. To this end, we isolated a PXT analogue, auraptenol, which contains a hydroxyl group (−OH) at the corresponding peroxide group position of PXT (Figure 1A), from the ethanol extract of C. monnieri fruits. We then compared the anti-inflammatory activities of PXT and auraptenol in LPS-activated macrophages and found that auraptenol reduced LPS-induced IL-6 secretion (23.5 ± 1.5
Figure 6. PXT reduces the phagocytic efficiency of macrophages and decreases the expression levels of IL-1β in bacteria-infected macrophages. (A) J774A.1 macrophages were incubated with LPS for 6 h and then for 30 min with or without PXT or colchicine. The cells were incubated with fluorescence-conjugated E. coli for 1 h. The phagocytic efficiency was determined by measuring the fluorescence intensity. (B) J774A.1 macrophages were infected with K. pneumoniae for 24 h at 100 MOI. 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. ∗∗∗ indicates a significant difference at the level of p < 0.001.
ng/mL) to 17.5 ± 0.9 ng/mL at 100 μM, whereas 10 μM PXT reduced it to 7.2 ± 1.0 ng/mL (Figure 7A). In addition, 100 μM auraptenol reduced LPS-induced NO production (39 ± 5 μM) to 25 ± 3 μM, although 10 μM PXT reduced it to 5 ± 2 μM (Figure 7B). Furthermore, whereas auraptenol only slightly inhibited the expression levels of iNOS and proIL-1β in LPS-
Figure 7. The peroxide group of PXT is important for its antiinflammatory activity. (A, B) RAW 264.7 macrophages were incubated for 30 min with or without PXT or auraptenol (aura) and then for 24 h with or without 1 μg/mL LPS. The levels of IL-6 (A) and NO (B) in the culture medium were measured by ELISA and the Griess reaction, respectively. (C) RAW 264.7 macrophages were incubated for 30 min with or without aura and then for 6 h (for proIL-1β) and 24 h (for iNOS) with or without 1 μg/mL LPS. The levels of proIL-1β and iNOS in the cells were measured by Western blotting. The data are expressed as the means ± SD for three separate experiments. The Western blotting results are representative of those obtained in three different experiments. ∗, ∗∗, and ∗∗∗ indicate a significant difference at the levels of p < 0.05, p < 0.01, and p < 0.001, respectively. 1216
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K+ efflux has a role in the regulation of the NLRP3 inflammasome and can be achieved either directly by nigericin or indirectly by the purinergic receptor for ATP.38,39 It has been reported that crystal-induced NALP3 inflammasome activation is inhibited by phagocytosis inhibitors.15 These results indicate that crystal uptake by phagocytosis is a prerequisite for activation of the NARP3 inflammasome. In this study, we demonstrated that PXT reduced phagocytosis, which partially explains why PXT inhibited the NLRP3 inflammasome-mediated IL-1β secretion induced by MSU, CPPD, SiO2 nanoparticles, and alum crystals. In contrast, PXT did not affect IL-1β secretion induced by nigericin and ATP in LPS-primed macrophages, suggesting that PXT might not affect K+ efflux induced by these molecules. PXT is a peroxide (−OOH)-containing coumarin compound. When subjected to acid, base, or temperature increases in the environment, the −OOH group on the compound is likely to degrade into hydroxyl radical (•OH), a most destructive free radical that reacts readily with the metallic core atoms of intracellular enzymes and in turn modulates the secretion of certain cytokines. However, when the concentrations of these free radicals are sufficient, they may partake in important intracellular metabolism processes and become a line of defense in preventing or fighting disease. In this study, we found that freshly extracted ethanolic PXT tended to decrease in concentration during storage, which became notable with more than 6 months at room temperature; the situation was much improved by storage at −20 °C. However, storing the ethanolic solution of auraptenol at room temperature for more than 6 months resulted in an unchanged concentration based on HPLC and NMR analyses, indicating that the compound is highly stable. Although PXT exhibited anti-inflammatory activities in LPSactivated macrophages, it did not cause a general unresponsiveness to LPS, as the expressions of COX-2 and TNF-α were not reduced by PXT. These findings indicate that the administration of PXT might prevent excessive inflammatory response but does not cause immunodeficiency. C. monnieri fruits have been used to treat gout in traditional medicine, although there is no scientific evidence supporting this. Here, we demonstrate that PXT reduces MSU-induced activation of the NLRP3 inflammasome by inhibiting ROS generation and mitochondrial DNA release, explaining why C. monnieri fruits are able to ameliorate gout.17 In summary, we demonstrate that PXT possesses anti-inflammatory activity by reducing the expression levels of NO and IL-6, as well as by reducing the activation of the NLRP3 inflammasome. These results suggest that PXT may have a potential use as a natural antiinflammation agent or nutraceutical for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases.
activated macrophages (Figure 7C), PXT significantly inhibited expression, as shown in Figures 1E and 5A, respectively. These results indicated that the anti-inflammatory activity of auraptenol is lower than that of PXT and suggested that the unique peroxide group is important for the anti-inflammatory activity of PXT.
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DISCUSSION PXT was purified along with osthole from the ethanol extract of C. monnieri seeds. The structure of PXT is similar to that of osthole; however, their immune modulation properties in LPSactivated macrophages are quite different.4 The different biological outcomes between PXT and osthole are as follows: (1) PXT reduces the levels of IL-6, but osthole increases them. (2) PXT does not affect the levels of TNF-α and COX-2, whereas osthole reduces them. (3) PXT does not affect the phosphorylation levels of ERK1/2, yet osthole increases them. (4) PXT does not affect the activation levels of NF-κB, whereas osthole reduces them. (5) PXT reduces the phosphorylation levels of PKC-δ phosphorylation, but osthole does not. The priming step of the NLRP3 inflammasome is regulated by ROS, which are required for NLRP3 expression.25 PXT did not affect the priming step of the NLRP3 inflammasome, although PXT did reduce the levels of ROS in LPS-activated macrophages (Figure 5C). ROS is involved in both the priming and activation steps of the NLRP3 inflammasome,24 and the up-regulation of ROS levels in macrophages results in the activation of PI3K, promoting caspase-1 activation and IL-1β secretion.33 The inhibition of MSU- and CPPD-induced ROS generation may be responsible for the reduced NLRP3 inflammasome-derived IL-1β secretion caused by PXT. Furthermore, we found that PXT reduced mitochondrial ROS generation, which plays an important role in NLRP3 inflammasome activation.26,27 The overproduction of mitochondrial ROS promotes a mitochondrial permeability transition that facilitates the cytosolic release of mitochondrial DNA, which stimulates the activation of the NLRP3 inflammasome.26,27 PXT also reduced the cytosolic release of mitochondrial DNA, indicating that mitochondrial stabilization may be responsible for the reduced activation of the NLRP3 inflammasome mediated by PXT. PXT did not inhibit either ATP-induced IL-1β secretion (Figure 4A) or ROS generation (Figure 4B) in LPS-primed macrophages; PXT also had no effect on LPS-induced NLRP3 expression (Figure 5A). These results indicated that PXT did not affect the priming or activation signal of the NLRP3 inflammasome due to LPS or ATP, respectively. Notably, however, PXT did reduce ATP-induced caspase-1 activation when it was added before LPS or ATP (Figure 5D). One possible mechanism is that PXT might increase the cellular levels of cAMP, which has been demonstrated to inhibit inflammasome assembly by binding to NLRP3 directly.34 Osthole was reported to increase the tissue cAMP content by inhibiting the activity of cAMP phosphodiesterase in guinea pig trachea.35 Another possible mechanism is that PXT might increase autophagy, which limits NLRP3 inflammasome activation by targeting inflammasome components for degradation36,37 and by inhibiting the release of mitochondrial DNA from damaged mitochondria.22 Furthermore, caspase-11 induction by LPS or interferon-γ synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation;8 however, PXT did not affect the expression levels of caspase11 induced by LPS and interferon-γ.
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AUTHOR INFORMATION
Corresponding Author
*(K.-F.H.) Phone: +(886)-3-935-7400, ext. 7626. Fax: +(886)3-931-1526. E-mail: [email protected]. Funding
Contract grant sponsor: Ministry of Science and Technology, Taiwan; Contract grant number: NSC 102-2628-B-197-001MY3; NSC 103-2923-B-197-001-MY3. CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan, and Taiwan Ministry of Health and Welfare Clinical Trial and 1217
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Research Center of Excellence (MOHW103-TDU-B-212113002). Notes
The authors declare no competing financial interest.
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DOI: 10.1021/jf5054436 J. Agric. Food Chem. 2015, 63, 1210−1219
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DOI: 10.1021/jf5054436 J. Agric. Food Chem. 2015, 63, 1210−1219
