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Original Article

  1. Title

Peroxyauraptenol inhibits inflammation and NLRP3 inflammasome activation by inhibiting reactive oxygen species generation and preserving mitochondrial integrity

  1. Names and affiliations of all authors

Louis Kuoping Chao1, Cheng-Hsiu Lin2, Huan-Wen Chiu2, Wei-Ting Wong2, Hsiao-Wen Chiu2, Yu-Ling Tasi3, Yueh-Hsiung Kuo4,5, Yi-Chich Chiu6, May-Lan Liu7, Chen-Lung Ho8, Kuo-Feng Hua2,*

1Department of Cosmeceutics, China Medical University, Taichung, Taiwan; 2Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan; 3Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan; 4Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung, Taiwan; 5Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung, Taiwan; 6Department of Biomechatronic Engineering, National Ilan University, Ilan, Taiwan; 7Department of Nutritional Science, Toko University, Chiayi, Taiwan; 8Division of Wood Cellulose, Taiwan Forestry Research Institute, Taipei, Taiwan.

  1. Corresponding author information

* To whom correspondence should be addressed: Dr. Kuo-Feng Hua, No. 1, Sec. 1, Shen-Lung Road, Ilan, 260, Taiwan. Tel: +(886)-3-935-7400, ext. 7626; Fax: +(886)-3-931-1526; E-mail: kuofenghua@gmail.com

  1. Running title

Anti-inflammatory activity of peroxyauraptenol

  1. Keywords

Peroxyauraptenol; Inflammation; NLRP3 Inflammasome; Reactive oxygen species; Mitochondria

  1. Total number of figures

7 figures

  1. Grant information

Contract grant sponsor: National Science Council, Taiwan; Contract grant number: NSC 102-2628-B-197-001-MY3.
Abstract

Peroxyauraptenol (PXT) is a peroxide-containing coumarin compound with anti-inflammatory activities, which was isolated from the fruits of Cnidium monnieri. PXT reduced inducible nitrite oxide synthase expression and nitrite oxide levels in, and interleukin-6 secretion by, lipopolysaccharide (LPS)-activated macrophages without affecting tumor necrosis factor- secretion and cyclooxygenase-2 expression. PXT reduced NLRP3 inflammasome-derived IL-1 secretion. The underlying mechanisms for the PXT-mediated anti-inflammatory activity were (1) reduction of LPS-induced reactive oxygen species (ROS) generation; (2) reduction of the LPS-induced phosphorylation of mitogen-activated protein kinases and protein kinase C-/; (3) reduction of mitochondrial ROS generation and release of mitochondrial DNA into cytosol in NLRP3 inflammasome activated macrophages. In addition, PXT was found to suppress phagocytic activity of macrophages and IL-1 secretion in Klebsiella pneumoniae-infected macrophages. Furthermore, the unique peroxide group is important for the anti-inflammatory activity of PXT, as the anti-inflammatory activity is reduced when the peroxide group is replaced by 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- and inflammasome-related diseases.



Introduction

Inflammation is one of the important host defenses mechanisms in mammals; however, uncontrolled inflammation is harmful to health (Goldszmid and Trinchieri, 2012). Over-production of pro-inflammatory cytokines and mediators, such as tumor necrosis factor- (TNF-), interleukin-1β (IL-1β), interleukin-6 (IL-6), nitric oxide (NO), and cyclooxygenase-2 (COX-2) by macrophages is the feature of activated innate immunity (Ho et al., 2013; Hua et al., 2013a; Liao et al., 2010). A strong link between inflammation and disease is becoming increasingly evident. Elevated circulating inflammatory cytokines, in particular, an elevation of IL-6 is a risk factor for the pathogenesis of inflammatory bowel diseases (Allocca et al., 2013). NO is produced from L-arginine by inducible nitric oxide synthase (iNOS) and contributes to some pathogenesis by promoting oxidative stress, tissue injury and, even, cancer if it is generated in excess (Olson et al., 2011).

IL-1 release is controlled by inflammasome, a caspase-1-containing multi-protein complex, and plays important roles in microbial infections and metabolic processes (Strowig et al., 2012). The most thoroughly characterized inflammasome is the NLRP3 inflammasome, which is considered to be a controller for disease development including pathogen infections (Rathinam et al., 2012; Allen et al., 2009; Gross et al., 2009), type 2 diabetes mellitus (Masters et al., 2010), atherosclerosis (Duewell et al., 2010), obesity (Henao-Mejia et al., 2012; Vandanmagsar et al., 2011), silicosis (Hornung et al., 2008), Alzheimer's disease (Heneka et al., 2013), gout (Martinon et al., 2006), and kidney disease (Anders and Muruve, 2011). Given the importance of inflammation and inflammasome in the pathogenesis of diseases, the anti-inflammatory approaches or biological agents that target inflammasome or specific pro-inflammatory mediator pathways can be used to improve the control of inflammation-related disorders.

The fruit of Cnidium monnieri, a Chinese herbal medicine has been the subject of considerable interest because of its broad spectrum of pharmacological properties. The most thoroughly characterized main constituents from the fruit of Cnidium monnieri is osthole. In our previous study we demonstrated that osthole showed anti-inflammatory activity in LPS-stimulated macrophages (Liao et al., 2010), and mitigated renal inflammation in mice (Yang et al., 2014; Hua et al., 2013b). In this study we isolated peroxyauraptenol (PXT), a coumarin compound with a unique peroxide group, from the fruits of Cnidium monnieri. PXT showed anti-inflammatory activity, but the immune modulation properties of PXT and osthole are different, although their chemical structure is similar. The anti-inflammatory activity of PXT is associated with the unique peroxide group, as the anti-inflammatory activity is reduced when the peroxide group is replaced by hydroxyl group.



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). Rabbit antibodies against mouse phospho-PKC-, phospho-PKC-δ, IL-1, caspase-1, iNOS, COX-2, and horseradish peroxidase-labeled second antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse IL-1, IL-6, and TNF- ELISA kits were purchased from R&D Systems (Minneapolis, MN). 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). All the inflammasome inducers and the QUANTI-Blue™ reagent were purchased from InvivoGen (San Diego, CA). PXT was isolated from the fruits of C. monnieri as described in our previously report and its structure has been verified by 1H-NMR and 13C-NMR spectroscopy (Liao et al., 2010). Extraction and purification

600 grams of seeds of C. monnieri were purchased from a traditional Chinese medicine dispensary in Taiwan. They were treated with 15 L of ethanol (95% v/v, 10 days, repeated 3 times) at room temperature. The extracts were then concentrated to produce alcoholic extracts and then 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). Two pure compounds were collected (A and B, figure 1). 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 the two pure compounds were identified by physical and spectral data (mp, EIMS, 1HNMR, UV, IR) compared with previous research values.

Cell cultures

The murine macrophage cell lines RAW 264.7 and J774A.1 were obtained from the American Type Culture Collection (Rockville, MD). RAW 264.7 macrophages stably transfected with the NF-B reporter gene (RAW-Blue™ cells) were purchased from InvivoGen (San Diego, CA). All cells were 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. In the case of RAW-Blue™ cells, 100 g/ml of zeocin was added to the medium. 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 μg/ml streptomycin, 2.5 μg/ml amphotericin B, and 10 % FBS.


AlamarBlue® assay for cell viability

RAW 264.7 macrophages were seeded in 96-well flat-bottom plates at a density of 5000 cells in 100 l of RPMI 1640 medium containing 10 % FBS per well and incubated for 24 h at 37 °C in a 5 % CO2 incubator. The cells were incubated for 24 h with or without PXT. The AlamarBlue® assay was used to determine the cytotoxicity of PXT. The procedure was conducted following the protocol described in the manufacturer’s instructions (AbD Serotec, Oxford).


Detection of pro-inflammatory mediators

RAW 264.7 macrophages were incubated for 30 min with or without PXT, then for 24 h with or without 1 g/ml of LPS. The levels of NO in the culture medium were measured by the Griess reaction. The levels of IL-6 and TNF-α in the culture medium were measured by ELISA. The levels of iNOS and COX-2 in cells were measured by Western blot (Chao et al., 2010).


Detection of IL-1β secretion and caspase-1 activation

For IL-1β secretion in Fig. 4A, and 5F, J774A.1 macrophages were incubated for 5.5 h with or without 1 g/ml of LPS, then with or without PXT for 30 min, then with or without 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), ATP (5 mM, 0.5 h), nigericin (10 µM, 0.5 h), FLA-ST (100 ng/ml, 24 h), or MDP (100 µg/ml, 24 h). The levels of IL-1 in the culture medium were measured by ELISA. For IL-1β secretion and caspase-1 activation in Fig. 5D, J774A.1 macrophages were incubated with or without PXT for 30 min, then for 5.5 h with or without 1 g/ml of LPS, then with or without 5 mM ATP and 10 µM nigericin for 0.5 h. The levels of IL-1 in the culture medium were measured by ELISA and the activated caspase-1 (p10) and pro-caspase-1 (p45) in the cells were measured by Western blot.


Detection of the phosphorylation levels of MAPK and PKC

RAW 264.7 macrophages were incubated for 30 min with or without PXT, then for 0-60 min with or without 1 g/ml of LPS. The phosphorylation levels of ERK1/2, JNK1/2, p38, and PKC-α/ were analyzed by Western blot (Chao et al., 2010).


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). For LPS-induced ROS generation, J774A.1 macrophages were incubated for 30 min with or without 40 µM PXT or 10 mM N-acetyl cysteine (NAC), then incubated for 30 min with 2 µM 2’,7’-dichlorofluorescein diacetate, and then stimulated for 10 min with 1 µg/ml of LPS. For ATP-, MSU-, and CPPD-induced ROS generation, J774A.1 macrophages were incubated for 5.5 h with 1 µg/ml of LPS, then incubated for 30 min with or without 40 µM PXT or 10 mM NAC, then incubated for 30 min with 2 µM 2’,7’-dichlorofluorescein diacetate. The cells were incubated for 10 min with 5 mM ATP, 500 µg/ml MSU, or 100 µg/ml CPPD crystals. 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.). For mitochondrial ROS generation, J774A.1 macrophages were incubated with 1 µg/ml of LPS for 5.5 h, then incubated for 30 min with or without 40 µM PXT, then incubated for 10 min with or without 500 µg/ml MSU or 100 µg/ml CPPD crystals, and then stained with 5 µM MitoSOX (Invitrogen, Carlsbad, CA) for 20 min. 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.


Detection of mitochondrial DNA release

The detection of mitochondrial DNA (mt-DNA) in the cytosol was performed as described (Nakahira et al., 2011). Briefly, J774A.1 macrophages were incubated for 5.5 h with or without 1 g/ml of LPS, then with or without PXT for 30 min, then with or without 500 µg/ml MSU or 100 µg/ml CPPD crystals for 30 min. Cells were homogenized in 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 700 x g at 4°C. The protein concentration and volume of the supernatant was normalized and was then centrifuged 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 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 ribosomal RNA) levels using a ratio of cytochrome c oxidase I DNA to nuclear DNA. The results were expressed as fold changes between the sample and the control group. The quality of the mitochondrial fraction and cytosolic fraction was monitored by detecting the protein expression level of voltage-dependent anion channel (VDAC, a marker protein for mitochondria). We found that VDAC can only be detected in the mitochondrial fraction and is not present in the cytosolic fraction (data not shown). These results indicate that the cytosolic fraction is free from mitochondria. The primers used were the following: mouse 18S, forward, 5’-TAGAGGGACAAGTGGCGTTC-3’, reverse, 5’-CGCTGAGCCAGTCAGTGT-3’; mouse cytochrome c oxidase I, forward, 5’-GCCCCAGATATAGCATTCCC-3’, reverse, 5’-GTTCATCCTGTTCCTGCTCC-3’.


NF-B reporter assay

RAW-Blue™ cells were seeded in 24-well plates at a density of 1 × 105 cells/ml and grown overnight in a 5% CO2 incubator at 37 °C. Cells were incubated for 30 min with or without PXT or 10 mM NAC, then for 24 h with or without 1 μg/ml of LPS. The medium was then harvested and 20 l aliquots were mixed with 200 l of QUANTI-Blue™ medium (Invitrogen, Carlsbad, CA) 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.


Phagocytosis assay

J774A.1 were seeded in 96-well flat-bottom plates at a density of 5 x 103 cells in 100 l of RPMI 1640 medium containing 10 % FBS per well and incubated with 1 g/ml of 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 g of fluorescence-conjugated E. coli. The fluorescence intensity was measured at an excitation wavelength of 509 nm and an emission wavelength of 533 nm. Net phagocytosis is calculated by subtracting the average fluorescence intensity of the no-cell negative-control wells from all experimental wells. The % of phagocytosis can then be calculated as a fraction of the net positive control phagocytosis as follows:




Bacterial infection

J774A.1 macrophages were infected with Klebsiella pneumoniae (NTUH K-2044) at 100 multiplicities of infection (MOI). 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 g/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. Data analysis involved one-way ANOVA with a subsequent Scheffé test.



Results
PXT reduces NO generation, inducible NO synthase expression, and IL-6 secretion in LPS-activated macrophages

PXT and auraptenol were isolated from the ethanol extract of fruits of C. monnieri along with osthole, and the chemical structure of these compounds is similar (Fig. 1A). The anti-inflammatory activity of PXT was investigated using LPS-activated RAW 264.7 macrophages. As shown in Fig. 1B, NO generation was inhibited by PXT in a dose-dependent manner. In the same system, we found that IL-6 secretion was inhibited by PXT in a dose-dependent manner (Fig. 1C), whereas TNF-α secretion was not reduced significantly (Fig. 1D). The protein expression levels of iNOS were also reduced by PXT in a dose-dependent manner, whereas the protein expression levels of COX-2 were not affected (Fig. 1E). To examine whether the effects on NO generation, iNOS expression, and IL-6 secretion were due to reduced cell viability, the toxicity of PXT for RAW 264.7 macrophages was examined. We found that PXT had no significant effect on cell survival at concentrations up to at least 40 M (Fig. 1F).


PXT reduces the phosphorylation levels of MAPK and PKC-/ in LPS-activated macrophages

Mitogen-activated protein kinase (MAPK), including ERK1/2, JNK1/2, and p38, is activated by LPS and plays important roles in the expression of inflammatory mediators in macrophages (Ho et al., 2013; Hua et al., 2013a; Liao et al., 2010; Su et al., 2006). In the present study, we investigated whether PXT is able to affect the activation of ERK1/2, JNK1/2, and p38 in LPS-stimulated RAW 264.7 macrophages. As shown in Fig. 2, LPS induced increase in the phosphorylation levels of ERK1/2, JNK1/2, and p38 and these effects were reduced by PXT except ERK1/2. In addition, PKC is one of the components of the TLR4 signaling pathway and plays important roles in macrophage activation in response to LPS (Ho et al., 2013; Hua et al., 2013a; Liao et al., 2010; Su et al., 2006). As shown in Fig. 3A-C, the LPS induced increase in phosphorylation levels of PKC- and PKC- and these effects were reduced by PXT.


PXT reduces the activation of NLRP3 inflammasome by reducing mitochondrial damage

We examined whether PXT is able to inhibit IL-1 secretion in LPS-primed J774A.1 macrophages induced by NLRP3 inflammasome activators, including monosodium urate (MSU), calcium pyrophosphate dihydrate (CPPD), SiO2 nanoparticles, alum crystals, nigericin, and ATP. We found that IL-1β secretion after the treatment of MSU, CPPD, SiO2 nanoparticles, and alum crystals were significantly reduced by PXT, but PXT was not able to inhibit IL-1β secretion induced by nigericin and ATP (Fig. 4A). Generation of ROS is one of the crucial elements for NLRP3 activation (Tschopp and Schroder, 2010; Bauernfeind et al., 2011). To determine whether the inhibition of IL-1β secretion by PXT occurred via inhibition of ROS generation, LPS-primed J774A.1 macrophages were incubated with PXT or the anti-oxidant N-acetyl-cysteine (NAC) for 30 min before the addition of CPPD, MSU and ATP. We found that PXT reduced CPPD- and MSU-induced increase in ROS generation; however, PXT was not able to reduce ATP-induced increase in ROS generation (Fig. 4B). Damaged and ROS-generating mitochondria activate the NLRP3 inflammasome (Kepp et al., 2011; Nakahira et al., 2011; Zhou et al., 2011). To determine whether PXT inhibited the NLRP3 inflammasome by preventing mitochondrial damage, we used fluorescent probe MitoSOX to detect the ROS released from damaged mitochondria. Mitochondrial ROS (mt-ROS) levels were increased when LPS-primed macrophages were treated with MSU and CPPD, and treatment with PXT reduced the mt-ROS levels in these cells (Fig. 4C). Over-generation 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 (Zhou et al., 2011). We examined whether the translocation of mt-DNA into the cytosol was decreased by PXT. We found that the levels of mt-DNA release into the cytosol were increased in MSU- and CPPD-activated macrophages, but they were significantly reduced by PXT (Fig. 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 (Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase 11. Nature 2011; 479, 117–121); however, PXT did not affect the expression levels of caspase-11 induced by LPS and interferon-γ (Fig. 4E).


PXT reduces NLRP3 inflammasome-derived IL-1β secretion by reducing proIL-1β expression

NLRP3 induction is a critical checkpoint for the priming step of NLRP3 activation and this is regulated by NF-κB (Bauernfeind et al., 2009). We investigated whether PXT inhibits the LPS-mediated priming signal for the NLRP3 inflammasome. We incubated J774A.1 macrophages with PXT for 30 min before treatment for 6 h with LPS and found that PXT did not inhibit the expression level of NLRP3, although it reduced the expression level of IL-1β precursor, proIL-1β (Fig. 5A). We then examined whether PXT is able to inhibit NF-B activation using NF-B-dependent alkaline phosphatase reporter cells (RAW-BlueTM cells) and, as shown in Fig. 5B, found that NF-B transcriptional activity in LPS-stimulated macrophages was not affected by PXT, although markedly inhibited by NAC used as the 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 LPS-induced expression of proIL-1β (Liao et al., 2013). We found that the LPS-induced increase in ROS generation was reduced by PXT and NAC (Fig. 5C). Although PXT was not able to reduce ATP-induced expression of IL-1β in LPS-primed macrophages (Fig. 4A), we assessed the secretion levels of IL-1β will be 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 (Fig. 5A). We found that ATP-induced IL-1β secretion was significantly inhibited by PXT when it was added before LPS priming (Fig. 5D, left); notably, the activation levels of caspase-1 were also inhibited by PXT under the same condition (Fig. 5D, right). In the same condition, PXT also inhibited IL-1β secretion in primary peritoneal macrophages (Fig. 5E). Furthermore, PXT is not selective for NLRP3-dependent inflammasome stimuli, as PXT reduced IL-1β secretion triggered by FLA-ST (flagellin from S. typhimurium) or muramyl dipeptide, which are dependent on the NLRP1 and NLRC4 inflammasomes (Moayeri et al., 2012; Franchi et al., 2012), respectively, and independent of NLRP3 (Fig. 5F).


PXT reduces the phagocytic efficiency of macrophages and decreases the expression levels of IL-1β in bacteria-infected macrophages

PXT reduced the expression levels of IL-1β induced by crystal NLRP3 inflammasome inducers, including alum crystals, CPPD, and SiO2 nanoparticles (Fig. 4A). These inducers induced the activation of NLRP3 inflammasome is depending on the uptake by phagocytosis. In view of this, we investigated the hypothesis that PXT inhibits crystal-mediated activation of 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 (microtubule polymerization inhibitor) for 30 min and then the phagocytosis activity was assessed by measuring uptake of fluorescence-conjugated E. coli. We found that both PXT and colchicine decreased the fluorescence-conjugated E. coli uptake by macrophages (Fig. 6A). In addition, we tested whether PXT affects the production of IL-1β in macrophages infected with live Klebsiella pneumoniae. We found that PXT reduced the levels of IL-1β in K. pneumoniae-infected macrophages (Fig. 6B). These results suggest that PXT may modulate macrophage function in K. pneumoniae infected sites by impairing phagocytosis and reducing the cytokine production.


Peroxide group of PXT is important for its anti-inflammatory activity

PXT contains a unique peroxide group (–OOH) on its carbon chain. We investigated the hypothesis that the unique peroxide group is important for the anti-inflammatory activity of PXT. We isolated a PXT analogue, auraptenol, from the ethanol extract of fruits of C. monnieri. Auraptenol contains a hydroxyl group (–OH) on the corresponding position of PXT, which is the peroxide group (Fig. 1A). We compared the anti-inflammatory activity of PXT and auraptenol in LPS-activated macrophages and we found that 100 μM auraptenol reduced LPS-induced IL-6 secretion (23.5 ± 1.5 ng/ml) to 17.5 ± 0.9 ng/ml, whereas 10 μM PXT reduced LPS-induced IL-6 secretion to 7.2 ± 1.0 ng/ml (Fig. 7A). In addition, 100 μM auraptenol reduced LPS-induced NO production (39 ± 5 μM) to 25 ± 3 μM, whereas 10 μM PXT reduced LPS-induced NO production to 5 ± 2 μM (Fig. 7B). Furthermore, auraptenol did not inhibit the expression levels of iNOS and proIL-1β in LPS-activated macrophages (Fig. 7C), whereas PXT significantly inhibited the expression levels of iNOS (Fig. 1E) and proIL-1β (Fig. 5A). These results indicated that the anti-inflammatory activity of auraptenol is lower than PXT, and suggested that the unique peroxide group is important for the anti-inflammatory activity of PXT.



Discussion

PXT was purified along with osthole from the ethanol extracts of the seeds of Cnidium monnieri. The structure of PXT is similar to osthole; however, their immune modulation properties in LPS-activated macrophages are quite different (Liao et al., 2010). The different biological outcomes between PXT and osthole are 1) PXT reduces the levels of IL-6, but osthole increases it. 2) PXT does not affect the levels of TNF-α and COX-2, but osthole reduces them. 3) PXT does not affect the phosphorylation levels of ERK1/2, but osthole increases it. 4) PXT does not affect the activation levels of NF-B, but osthole reduces it. 5) PXT reduces the phosphorylation levels of PKC- phosphorylation, but osthole doesn’t.

The priming step of the NLRP3 inflammasome is regulated by ROS, which are required for NLRP3 expression (Bauernfeind et al., 2011). PXT did not affect the priming step of the NLRP3 inflammasome, although PXT reduced the levels of ROS in LPS-activated macrophages (Fig. 5C). 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 macrophages resulted in the activation of PI3K, promoting caspase-1 activation and IL-1β secretion (Cruz et al., 2007). The inhibition of MSU- and CPPD-induced ROS generation may be responsible for the reduced NLRP3 inflammasome-derived IL-1β secretion by PXT. Furthermore, we found that PXT 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). PXT also reduced the cytosolic release of mitochondrial DNA, indicating that mitochondrial stabilization may be responsible for the reduced activation of NLRP3 inflammasome by PXT.

PXT inhibited neither ATP-induced IL-1β secretion (Fig. 4A) nor ROS generation (Fig. 4B) in LPS-primed macrophages. PXT also had no effect on LPS-induced NLRP3 expression (Fig. 5A). These results indicated that PXT did not affect the priming signal and the activation signal of NLRP3 inflammasome from LPS and ATP, respectively. Notably, however, PXT reduced ATP-induced caspase-1 activation when it was added before LPS and ATP (Fig. 5D). One of the possible mechanisms is that PXT might increase the cellular levels of cAMP, which has been demonstrated to inhibit inflammasome assembly by binding to NLRP3 directly (Lee et al., 2012). Osthole increased tissue cAMP content by inhibiting the activity of cAMP phosphodiesterase in guinea-pig trachea (Teng et al., 1994). The other possible mechanisms is that PXT might increase autophagy, which limits the 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). Furthermore, caspase-11 induction by LPS or interferon-γ synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation (Rathinam et al., 2012); however, PXT did not affect the expression levels of caspase-11 induced by LPS and interferon-γ.

K+ efflux, has a role in the regulation of NLRP3 inflammasome, is either achieved directly by nigericin or indirectly by the purinergic receptor for ATP (Pelegrin and Surprenant, 2006 and 2007). It has been reported that crystal-induced NALP3 inflammasome activation was inhibited by phagocytosis inhibitor (Hornung et al., 2008). These results indicated that crystal uptake by phagocytosis as prerequisites for activation of the NARP3 inflammasome. In this study we demonstrated that PXT reduced phagocytosis, which partially explain why PXT inhibited 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 nigericin and ATP.

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 liable to degrade into hydroxyl radical (•OH), a most destructive free radical which reacts easily with the metallic core atoms of intracellular enzymes and in turn modulates secretion of certain cytokines. However, under certain circumstances, when the concentrations of these free radicals are adequate, they may partake in important intracellular metabolism and become a line of defense in prevention or fighting diseases. In this study, we found that freshly extracted ethanolic PXT tended to decrease in concentration along with storage time. Storage at room temperature for more than 6 months, this became notable. For storage at -20℃, the situation was much improved. For another extracted compound, auraptenol, however, the ethanolic solution stored under room temperature for more than 6 months resulted in unchanged concentrations based on HPLC and NMR analyses, indicating the compound to have high stability.

Although PXT exhibited anti-inflammatory activities in LPS-activated macrophages, PXT did not cause general unresponsiveness to LPS, as expression of the COX-2 and TNF-α was not reduced by PXT. These indicate that administration of PXT might prevent excessive inflammatory response, but does not cause immunodeficiency. The C. monnieri fruits have been used for treating gout in traditional medicine; however, there is no scientific evidence supports this finding. Here we demonstrated that PXT reduced MSU-induced activation of NLRP3 inflammasome by inhibiting ROS generation and mitochondrial DNA release, explaining why Cnidium monnieri fruits are able to ameliorate gout (Martinon et al., 2006). In summary, we have demonstrated that PXT has anti-inflammatory activity by reducing the expression levels of NO and IL-6, as well as by reducing the activation of NLRP3 inflammasome. These results suggest that PXT may have potential for use as a natural anti-inflammation agent or nutraceutical for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases.
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Figure captions

Figure 1. Effect of PXT on the expression levels of inflammatory mediator. (A) Chemical structure of PXT, osthole and auraptenol. (B-D) RAW 264.7 macrophages were incubated for 30 min with or without PXT, then for 24 h with or without 1 µg/ml of LPS. The levels of NO (B), IL-6 (C), and TNF-α (D) in the culture medium were measured by Griess reaction and ELISA, respectively. (E) RAW 264.7 macrophages were incubated for 30 min with or without PXT, then for 24 h with or without 1 µg/ml of LPS. The levels of iNOS and COX-2 were assayed by Western blot. (F) RAW 264.7 macrophages were incubated for 24 h with or without PXT, and then the cell viability was assayed by AlamarBlue® assay. 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 actin compared to the control group. * and *** indicates a significant difference at the level of p < 0.05 and p < 0.001, respectively.
Figure 2. Effect of PXT on the phosphorylation levels of MAPK. (A) RAW 264.7 macrophages were incubated for 30 min with or without PXT, then for 0-60 with or without 1 µg/ml of LPS. The phosphorylation levels of ERK1/2, JNK1/2, and p38 were assayed by Western blot. (B) Quantitative for the phosphorylation levels of ERK1/2. (C) Quantitative for the phosphorylation levels of JNK1/2. (D) Quantitative for the phosphorylation levels of p38. 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 actin compared to the control group. * indicates a significant difference at the level of p < 0.05.
Figure 3. Effect of PXT on the phosphorylation levels of PKC. (A) RAW 264.7 macrophages were incubated for 30 min with or without PXT, then for 0-60 with or without 1 µg/ml of LPS. The phosphorylation levels of PKC-α and PKC-δ were assayed by Western blot. (B) Quantitative for the phosphorylation levels of PKC-α. (C) Quantitative for the phosphorylation levels of PKC-δ. 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 actin compared to the control group. * indicates a significant difference at the level of p < 0.05.
Figure 4. PXT 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 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 of 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 of 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 macrophages were incubated for 5.5 h with 1 µg/ml of 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 detection of cytochrome c oxidase I DNA in the cytosol. 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. Effect of PXT on the priming step of NLRP3 inflammasome. (A) J774A.1 macrophages were incubated for 30 min with or without PXT, then for 6 h with or without 1 µg/ml of LPS in the continued presence or absence of PXT, and then the protein expression levels of NLRP3 and proIL-1β in the cells were measured by Western blot. (B) RAW-BlueTM cells were incubated for 30 min with or without PXT or 10 mM NAC, then for 24 h with or without 1 µg/ml of LPS in the continued presence or absence of PXT or NAC, and then the activation levels of NF-κB were measured by NF-κB reporter assay. (C) J774A.1 macrophages were incubated for 30 min with or without 40 μM PXT or 10 mM NAC, then for 10 min with or without 1 µg/ml of 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 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. (E) Mice primary peritoneal macrophages were incubated for 30 min with or without PXT, 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 were measured by ELISA. (F) J774A.1 macrophages were incubated for 5.5 h with or without 1 µg/ml of LPS, then for 30 min with or without PXT in the continued presence or absence of LPS, and then with or without 100 g/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 blot 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. * and *** indicates a significant difference at the level of p < 0.05 and p < 0.001, respectively.
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 1 μg/ml LPS for 6 h, then for 30 min with or without 40 μM PXT or 10 μM colchicine. Cells were incubated with 100 μg 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, respectively.
Figure 7. Peroxide group of PXT is important for its anti-inflammatory activity. (A and B) RAW 264.7 macrophages were incubated for 30 min with or without PXT or auraptenol (aura), then for 24 h with or without 1 µg/ml of LPS. The levels of IL-6 (A) and NO (B) in the culture medium were measured by ELISA and Griess reaction, respectively. (C) RAW 264.7 macrophages were incubated for 30 min with or without PXT or aura, then for 6 h (for proIL-1β) and 24 h (for iNOS) with or without 1 µg/ml of LPS. The levels of proIL-1β and iNOS in the cells were measured by Western blot. 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 *** indicates a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively.


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