Seco-type β-Apocarotenoid Generated by β-Carotene Oxidation Exerts Anti-inflammatory Effects against Activated Macrophages.

β-Apocarotenoids are the cleavage products of β-carotene. They are found in plants, carotenoidcontaining foods, and animal tissues. However, limited information is available regarding the health benefits of β-apocarotenoids. Here, we prepared seco-type β-apocarotenoids through the chemical oxidation of β-carotene and investigated their anti-inflammatory effects against activated macrophages. Oxidation of β-carotene with potassium permanganate produced seco-β-apo-8'-carotenal, in which one end-group formed an "open" β-ring and the other was cleaved at the C-7',8' position. In lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells, seco-β-apo-8'-carotenal inhibited the secretion and mRNA expression of inflammatory mediators such as nitric oxide, interleukin (IL)-6 and IL-1β, and monocyte chemoattractant protein-1. Furthermore, seco-β-apo-8'-carotenal suppressed phosphorylation of c-Jun N-terminal kinase and the inhibitor of nuclear factor (NF)-κB as well as the nuclear accumulation of NF-κB p65. Notably, since seco-β-apo-8'-carotenal exhibited remarkable anti-inflammatory activity compared with β-apo-8'-carotenal, its anti-inflammatory action could depend on the opened β-ring structure. These results suggest that seco-β-apo-8'-carotenal has high potential for the prevention of inflammation-related diseases.


Introduction
Carotenoids are lipophilic pigments that are widely distributed in microbes, plants, and animals. These natural pigments comprising eight isoprene units C40 , are characterized by a polyene backbone with long conjugated double bonds and various end-group structures. In microbes and plants, carotenoids are ubiquitous and play essential roles in photosynthesis and photoprotection. Dietary carotenoids are incorporated and accumulated in the bodies of animals, including humans, owing their inability to biosynthesize these compounds innately. Among natural carotenoids, β-carotene, which comprises 11 conjugated double bonds and two cyclized end-groups β-rings , is one of the most abundant carotenoids in nature. Numerous studies have indicated that β-carotene has various nutritional functions such as pro-vitamin A activity, antioxidant and anti-cancer effects, and the prevention of receptor PPAR 8 10 . These findings suggest that asymmetric β-apocarotenoids possibly exert biological functions to the same extent as that of retinoids. Furthermore, β-apo-8 -carotenal is used commercially used and is routinely utilized as a food colorant 11 . β-Apocarotenoids, including opened β-ring seco-type carotenoids, are also easily generated via the oxidation of β-carotene 12,13 . In particular, seco-type β-apocarotenoids have unique structures such as opened β-ring with α,β-unsaturated carbonyl group in the molecule. Thus, it is worthwhile to investigate the potential health benefits of various β-apocarotenoids.
Inflammation is an adaptive response that is triggered by noxious stimuli and conditions such as infection and tissue injury 14 . As important inflammatory cells, macrophages are involved in initiating inflammatory responses. Upon activation by lipopolysaccharides LPS , which constitute a component of the outer membrane of gram-negative bacteria, the production of numerous inflammatory cytokines such as tumor necrosis factor α TNF-α , interleukin IL -6, and IL-1β is induced through mitogen-activated protein kinases MAPK and nuclear factor NF -κ B pathways 15 . Furthermore, nitric oxide NO , a major inflammatory mediator, is produced following nitric oxide synthase NOS gene expression, which is involved in innate immunity as a toxic agent against infectious organisms 16 . Monocyte chemoattractant protein-1 MCP-1 is also produced through the expression of C-C motif chemokine ligand 2 Ccl2 , which regulates immune cell migration and infiltration into inflammation foci 17 . Although these inflammatory factors contribute to host defenses against infectious organisms and to tissue homeostasis, the dysregulation of proinflammatory factor production can lead to many inflammatory disorders 18 . Hence, to prevent inflammation-related diseases, it is crucial to regulate the overexpression of inflammatory factors by activated macrophages.
Full-length β-carotene has been reported to attenuate excessive inflammatory responses in murine macrophagelike RAW264.7 cells stimulated by LPS via inhibition of the NF-κ B, JAK2/STAT3, and MAPK signaling pathways 19 . However, the anti-inflammatory mechanisms of β-carotenederived β-apocarotenoids against activated macrophages have not been established. I n t h e p r e s e n t s t u d y, w e s o u g h t t o p r e p a r e apo-β-carotene from β-carotene through chemical oxidation using potassium permanganate KMnO 4 and characterized the structure of seco-β-apo-8 -carotenal by proton nuclear magnetic resonance 1 H-NMR analysis for the first time. Furthermore, the inhibitory effects of this seco-type β-apocarotenoid against the overexpression of inflammatory mediators were investigated in LPS-activated macrophage-like RAW264.7 cells.

Chemicals
Murine macrophage-like RAW264.7 cells were obtained from the European Collection of Authenticated Cell Cultures Salisbury, UK . Fetal bovine serum FBS was purchased from Gibco Grand Island, NY, USA . RPMI 1640 medium, penicillin/streptomycin, and β-carotene were purchased from FUJIFILM Wako Pure Chemical Co., Ltd.

2.2
In vitro oxidation of β-carotene and chromatographic analysis First, 5 mg of β-carotene and 0.8 mg of CTAB were dissolved in a solution of 40 mL chloroform and 10 mL KMnO 4 solution 180 mg/10 mL distilled water . After 3 h of incubation at room temperature, 20 mL chloroform, 30 mL methanol, and 8 mL distilled water were added to the oxidation mixture, which separated into two phases. The chloroform phase lower was collected and dried using a rotary evaporator. Thin-layer chromatography TLC was performed using methanol on an RP-18 F 254S plate Merck Millipore, Burlington, MA, USA to detect β-carotene oxidation products. The major β-carotene oxidation product 30 µg was isolated by high-performance liquid chromatography HPLC under the following conditions, column: Develosil C30-UG-5 column 250 4.6 mm, Nomura Chemical Co., Aichi, Japan , mobile phase: methanol, flow rate: 1.0 mL/min, detection: 450 nm. Liquid chromatography-mass spectrometry LC-MS was performed to estimate the molecular ion mass of the purified oxidation product using LCMS8040 Shimadzu, Kyoto, Japan with an ODS-UG-3 column 150 2.0 mm, Nomura Chemical Co., Inc., Aichi, Japan and methanol as the mobile phase at a flow rate of 0.2 mL/min. The column temperature was maintained at 30 . A triple quadrupole mass spectrometer with electrospray ionization, in positive ion mode, was used for analyzing the total ion scanning range at m/z 50-700 under a nebulizer nitrogen gas flow rate of 2.0 L/min, drying nitrogen gas flow rate of 15.0 L/min, DL temperature of 250 , and heat block temperature of 400 .

Cell culture
RAW264.7 cells were cultured in RPMI 1640 with 10 FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere with 5 CO 2 at 37 . After pre-incubating RAW264.7 cells for 24 h, the culture medium was treated with carotenoids dissolved in dimethyl sulfoxide DMSO solution and incubated for an additional 2 h. Control cells were treated with DMSO alone. The final DMSO concentration was adjusted to 0.1 in the culture medium without cytotoxicity. Then, LPS 100 ng/mL was added to the medium in the presence of carotenoids, and the cells were stimulated for an additional 30 min and 2 h for western blotting , 6 h for gene expression analysis , or 24 h for NO, IL-6, IL-1β, and MCP-1 secretion analysis .

Cell viability
Cell viability was measured using WST-1 reagent. Briefly, RAW264.7 cells were treated with various concentrations of seco-β-apo-8 -carotenal for 24 h. After incubation, 10 µL of WST-1 was added to each well, and this was followed by incubation for 2 h. The absorbance of each well was determined at 450 nm using a microplate reader Molecular Devices, CA, USA . Media samples with each seco-βapo-8 -carotenal concentration without cells were prepared as blanks.
2.6 Determination of NO, IL-1β, and MCP-1 concentrations in culture media. NO production was determined by the Griess method 20 . Culture media collected after LPS stimulation for 24 h were mixed equally with the Griess reagent 2.5 phosphoric acid, 1 sulfanilamide, and 0.1 N-1-naphtyl ethylenediamine dihydrochloride in distilled water . The optical absorbance at 550 nm was measured, and the NO concentration was determined from a standard curve with NaNO 2 . Media containing carotenoids without cells were prepared as sample blanks. The levels of IL-6, IL-1β, and MCP-1 production in the culture medium were measured using a commercial enzyme-linked immunosorbent assay ELISA kit Thermo Scientific, Frederick, MD, USA ac-cording to the manufacturer s protocol.

Reverse transcription quantitative polymerase chain
reaction RT-qPCR Total RNA was extracted from RAW264.7 cells using QIAzol lysis reagent Qiagen, Hilden, Germany . Then, cDNA was synthesized from total RNA using ReverTra Ace TOYOBO, Osaka, Japan according to the manufacturer s instructions. RT-qPCR analysis was performed with the StepOnePlus real-time PCR system Applied Biosystems Japan Ltd, Tokyo, Japan . GeneAce Probe qPCR Mix II Nippon gene, Tokyo, Japan was used according to the manufacturer s protocol with the following PCR cycling conditions: 50 for 2 min, 95 for 10 min, and 40 cycles of 95 for 30 s and 60 for 1 min. mRNA expression levels were measured using TaqMan Gene Expression Assays Thermo Fisher Scientific, Frederick, MD, USA for Ccl2 Mm00441242_m1 , Il6 Mm00446190_m1 , Il1b Mm00434228_m1 , Rps18 Mm02601777_g1 , and Gapdh Mm99999915_g1 .

Western blotting
RAW264.7 cells were washed twice with ice-cold PBS and lysed with 50 µL ice-cold RIPA buffer 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 NP-40, 0.1 sodium deoxycholate, 0.1 sodium dodecyl sulfate SDS , 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 1 protease inhibitor cocktail, and 1 phosphatase inhibitor . Cell lysates were centrifuged at 12,000 g for 10 min at 4 . Nuclear proteins were fractionated using the LysoPure Nuclear and Cytoplasmic Extractor Kit FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan according to the manufacturer s protocol. Following centrifugation, the protein concentration in the supernatant was measured using a DC protein assay kit Bio-Rad Laboratories, Hercules, CA, USA . Then, proteins in the supernatant 10 µg of protein per lane were separated by 10 sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with Tris-HCl buffer saline containing 1 Tween 20 TBST and 1 BSA for 1 h and then incubated with primary antibodies JNK: 1:2500, phospho-JNK: 1:2000, NF-κ B p65: 1:2500, Iκ Bα: 1:5000, phospho-Iκ Bα: 1:3000, β-actin: 1:3000, and lamin B1: 1:3000 at room temperature for 1 h. After washing three times with TBST, the membranes were incubated with secondary antibodies 1:20000 for 1 h at room temperature. Proteins were detected using Clarity Western ECL Substrate Bio-Rad Laboratories, Hercules, CA, USA according to the manufacturer s instructions.

Statistics
Data are expressed as mean standard error of mean SEM . Statistical analysis was performed using one-way analysis of variance followed by the Tukey-Kramer test. Statistical significance was determined at p 0.05.

Results
3.1 Identification of seco-type β-apocarotenoid in β-carotene oxidation products Chemical oxidation of β-carotene Fig. 2B by KMnO 4 generated new TLC spots, although β-carotene itself no longer remained Fig. 1A . We focused on the characteristic Peak 1 detected at 6.3 min by HPLC analysis Fig. 1B , which showed a longer maximum absorption at 462 nm  Fig. 1C. Subsequently, we analyzed molecular structures using NMR. The 1 H-NMR peaks were assigned based on the 1 H-1 H COSY and NOESY spectra and the comparison with published 1 H-NMR data of carotenoid end-groups Table 1 and Fig. S2-S4 Fig. 2A .

S u p p re s s i o n o f i n fl a m m a to r y m e d i a to rs by
seco-β-apo-8 -carotenal in LPS-activated RAW264.7 cells We investigated the suppressive effects of seco-β-apo-8carotenal on inflammatory mediator production induced in LPS-activated RAW264.7 cells. Seco-β-apo-8 -carotenal significantly inhibited the secretion of inflammatory mediators and cytokines such as NO and MCP-1 Table 2 . Compared to the original β-carotene Fig. 2B , seco-β-apo-8 -carotenal significantly decreased MCP-1 levels Table 2 , indicating that β-carotene oxidation augmented anti-inflammatory action against activated macrophages. To clarify the mechanism of anti-inflammatory action against LPS-activated RAW264.7 cells, the activity of seco-β-apo-8 -carotenal was compared with that of β-apo-8 -carotenal, which contains a β-ionone ring Figs    Statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test.

Seco-β-apo-8 -carotenal inhibits protein phosphorylation in MAPK and NF-κ B signaling pathways
To elucidate the anti-inflammatory mechanism of seco-β-apo-8 -carotenal, phosphorylated protein levels in the MAPK and NF-κ B pathways associated with inflammatory signaling were investigated. As shown in Fig. 5A, the LPS-induced phosphorylation of JNK and the inhibitor of NF-κ B α Iκ Bα was diminished by seco-β-apo-8 -carotenal treatment. Furthermore, seco-β-apo-8 -carotenal signifi-cantly inhibited the nuclear accumulation of the transcription factor NF-κ B p65 Fig. 5B . These results indicate that seco-β-apo-8 -carotenal suppresses the mRNA expression of inflammatory factors by attenuating the MAPK and NF-κ B signaling pathways. The cells were pretreated with each carotenoid (4 µM) for 2 h and then stimulated with LPS (100 ng/mL) in the presence or absence of carotenoids for 24 h. Inflammatory factor secretion in the culture supernatant was quantified by the Griess method (NO) and commercialized ELISA kits (MCP-1). Data are presented as mean±SEM (n=3). Different letters indicate significant differences (p < 0.05). Statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test. Seco: seco-β-apo-8 -carotenal, N.D.: not detected, LPS: lipopolysaccharide, NO: nitric oxide, MCP-1: monocyte chemoattractant protein-1.

Discussion
Apocarotenoids are widely found in plants, foods, and mammalian tissues 22 . Previously, β-apocarotenoids have been reported to antagonize the activation of nuclear receptors, such as RAR, RXR, and PPAR, thereby inhibiting the agonist-induced expression of retinoid-responsive g e n e s 8 1 0 . H o w e v e r, t h e s u p p r e s s i v e e f f e c t o f β-apocarotenoids on inflammation remains unclear. Since chronic inflammation is associate with various diseases, we prepared a characteristic seco-type β-apocarotenoid via β-carotene oxidation and investigated its anti-inflammatory effects against activated macrophages.
Several studies have shown that β-carotene oxidation using KMnO 4 generates a series of β-apocarotenoids: β-apo-14 -carotenal, β-apo-12 -carotenal, β-apo-10 -carotenal, and β-apo-8 -carotenal 13,23 . The reaction mechanism for the oxidative cleavage of β-carotene is thought to involve isomerization of an E-to Z-double bond and synaddition of the permanganate ion, thus forming a cyclic permanganate ester, which is well established as an intermediate in this type of a reaction. These reactions give rise to the expected series of β-apo-carotenals due to oxidative cleavage of the double bonds of the polyene backbone. Furthermore, β-carotene oxidation products generated by KMnO 4 also include certain seco-type carotenoids that result from the cleavage of the permanent Z-configured double bonds C-5,6 and/or C-5 ,6 positions in the β-ring 13,23 . Here, the protonated ion at 449.4 M H of Peak 1 Fig. 1C of the β-carotene oxidation products corresponded to that of seco-type β-apocarotenoid, as described by Gurak et al. 23 Therefore, we identified for the first time the structure of Peak 1 as seco-β-apo-8 -carotenal by 1 H-NMR analysis, which was incompletely determined by Gurak et al. 23 through NMR. At present, seco-β-apo-8 -carotenal is neither found in foods nor in mammalian tissues, although several β-apocarotenoids are detected through intake of carotenoid-containing foods and oxidative metabolism of β-carotene in vivo. On the other hand, there are several seco-type carotenoids including apo-bodies in edible bivalves such as freshwater clams and oysters 24,25 : hence, these seco-carotenoids may be absorbed in the body. However, to the best of our knowledge, there is no information concerning the accumulation and distribution of secocarotenoids in mammalian tissues. Thus, future studies are greatly needed for the investigation of seco-carotenoid metabolism.
To verify the anti-inflammatory effect of seco-β-apo-8carotenal, we examined the production of inflammatory mediators in LPS-activated RAW264.7 cells. Seco-β-apo-8carotenal significantly inhibited the overproduction of NO The cells were pretreated with seco-β-apo-8 -carotenal 4 µM for 2 h and then stimulated with LPS 100 ng/mL in the presence or absence of seco-β-apo-8 -carotenal. Phosphorylation A and nuclear accumulation B levels of each protein were analyzed by western blotting after LPS stimulation for 30 min and 2 h, respectively. β-Actin and lamin B1 were used as the loading controls. Data are mean SEM n 3 . Bars with different letters are significantly different p 0.05 . Statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test. Seco: seco-β-apo-8 -carotenal. and MCP-1 Table 2 . Notably, seco-β-apo-8 -carotenal, but not the original β-carotene Fig. 2B , markedly decreased MCP-1 secretion Table 2 , indicating that the oxidative cleavage of β-carotene augments its anti-inflammatory action. Seco-β-apo-8 -carotenal significantly downregulated the mRNA expression of inflammatory factors such as Il6, Il1b, and Ccl2 Fig. 4 . In addition, seco-β-apo-8 -carotenal inhibited the phosphorylation of JNK and Iκ Bα as well as the nuclear accumulation of NF-κ B p65 Fig. 5 . Upon activation, JNK phosphorylates transcription factors such as c-Jun, c-Fos, and ATF. In turn, these factors constitute the activator protein-1 AP-1 transcription factor, which regulates the expression of several stress-responsive genes, including inflammatory mediators and cytokines 26,27 . Moreover, NF-κ B activation converges on activating the Iκ B kinase IKK complex, leading to Iκ B phosphorylation and subsequent degradation. NF-κ B released from Iκ B translocates to the nucleus to bind specific DNA sequences, thus activating the transcription of multiple genes, including inflammatory cytokines and chemokines 28 . Thus, seco-βapo-8 -carotenal suppresses the secretion and gene expression of inflammatory factors by regulating the activation of MAPK and NF-κ B signal cascades, thereby preventing excessive inflammation.
In total lipid extracts of RAW264.7 cells treated with seco-β-apo-8 -carotenal for 26 h, several new peaks, as well as seco-β-apo-8 -carotenal were detected, although the molecular structures are unknown. In contrast, those peaks, except for seco-β-apo-8 -carotenal, were not detected in the culture medium after 26 h of incubation without cells. Thus, future work for the identification of seco-β-apo-8 -carotenal metabolites in cells is desired to reveal their anti-inflammatory mechanisms.

Conclusion
We successfully determined the molecular structure of seco-β-apo-8 -carotenal as a β-carotene-derived apocarotenoid. Seco-β-apo-8 -carotenal exhibited a characteristic structure in which one end-group formed an opened β-ring and the other was cleaved at the C-7 ,8 position of β-carotene. In LPS-activated murine macrophage-like RAW264.7 cells, seco-β-apo-8 -carotenal, but not β-apo-8carotenal, suppressed the overexpression of inflammatory factors by inhibiting the activation of the MAPK and NF-κ B signaling pathways. Notably, the present results demonstrate that the anti-inflammatory action of seco-β-apo-8carotenal depends on the opened β-ring structure.

Conflicts of Interest
The authors declare that there are no conflicts of interest.