An Anti-Inflammatory PPAR-γ Agonist from the Jellyfish-Derived

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An Anti-Inflammatory PPAR‑γ Agonist from the Jellyfish-Derived Fungus Penicillium chrysogenum J08NF‑4 Sen Liu,† Mingzhi Su,† Shao-Jiang Song,‡ Jongki Hong,§ Hae Young Chung,† and Jee H. Jung*,† †

College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 10016, People’s Republic of China § College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea ‡

S Supporting Information *

ABSTRACT: An investigation of the jellyfish-derived fungus Penicillium chrysogenum J08NF-4 led to the isolation of two new meroterpene derivatives, chrysogenester (1) and 5farnesyl-2-methyl-1-O-methylhydroquinone (2), and four known farnesyl meroterpenes. Docking analysis of 1 showed that it binds to PPAR-γ in the same manner as the natural PPAR-γ agonist amorfrutin B (7). Compound 1 activated PPAR-γ in murine Ac2F liver cells and increased nuclear PPAR-γ protein levels in murine RAW 264.7 macrophages. Because one of the main biological functions of PPAR-γ agonists is to suppress inflammatory response, an in vitro study was performed to explore the anti-inflammatory potency of 1 and the mechanism involved. In RAW 264.7 macrophages, 1 inhibited phosphorylation of the NF-κB p65 subunit and suppressed the expression of the pro-inflammatory mediators iNOS, NO, COX-2, TNF-α, IL-1β, and IL-6. We propose 1 suppresses inflammatory responses by activating PPAR-γ and subsequently downregulating the NF-κB signaling pathway, thus reducing the expressions of pro-inflammatory mediators. metabolism, inflammation, and metabolic homeostasis.10 PPAR-γ is the master regulator of adipocyte differentiation, fatty acid storage, and glucose metabolism and is recognized as an antidiabetic drug target. Thiazolidinediones (TZDs), such as rosiglitazone, are synthetic PPAR-γ agonists and have been used as a basis for the development of antidiabetic drugs. Recently, PPAR-γ agonists were found to demonstrate antiinflammatory effects through several mechanisms, which include direct inhibition of NF-κB expression,11 competing with NF-κB for coactivators,12 and promoting the nuclear export of p65 (RelA; a subunit of NF-κB).13 PPAR-γ agonists have also been reported to fundamentally affect the immune response through the inhibition of pro-inflammatory cytokines.14,15 Endogenous PPAR-γ agonists, such as 15-deoxyΔ12,14-prostaglandin J2 (15d-PGJ2), can protect kidneys from ischemic injury by inhibiting the activation of NF-κB and those of other pro-inflammatory mediators, such as AP-1, ICAM, and iNOS.16 During our investigation of the jellyfish-derived fungus Penicillium chrysogenum J08NF-4, two new meroterpenes, chrysogenester (1) and 5-farnesyl-2-methyl-1-O-methylhydroquinone (2), were isolated along with known farnesyl meroterpenes (3−6). Meroterpenes were previously reported from marine invertebrates such as sponges,17 ascidians,18 and soft corals19 and in microorganisms.20,21 These metabolites

Natural products have historically proven their value as a source of therapeutic drug candidates. In the past few decades, scientific interest in drug discovery was focused on synthetic pharmaceutical agents. However, due to the development and improvements made in biotechnology, a new era of bioprospecting for new natural products has begun. Revolutionary target screening techniques have further contributed to improve the efficiency of drug discovery from natural sources.1,2 In addition, leading edge genomics studies of biological symbiosis broaden a new frontier in the discovery of pharmaceutical agents, and symbiotic marine microorganisms are considered a promising source of new bioactive leads. In particular, endozoic fungi from diverse marine invertebrates offer prolific sources of structurally unique and biologically active metabolites.3−6 Metabolic disorders and inflammation caused by obesity now constitute a serious threat to global health.7 Some natural products and medicinal plants are traditionally used for the treatment of metabolic and inflammatory disease.8 However, only a few targets or pathways to mediate the beneficial effects of natural products in the context of metabolic disorders and inflammation have been defined. The peroxisome proliferatoractivated receptor (PPAR) family was reported as one of the most potential modulators for the prevention or therapy of metabolic and inflammatory diseases.9 PPARs are ligandactivated transcription factors and members of the nuclear receptor superfamily. The three PPAR isoforms (α, δ/β, and γ) play essential roles in the regulation of adipogenesis, lipid © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 5, 2017

A

DOI: 10.1021/acs.jnatprod.7b00846 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and 14.9 (C-12′) (E configuration, δCH3: 15−17 ppm; Z configuration, δCH3: 23−24 ppm).31 Compound 1 (chrysogenester) has a unique structure due to truncation of the 3carbon isopropenyl tail from the farnesyl chain. 5-Farnesyl-2-methyl-1-O-methylhydroquinone (2) was obtained as a yellow oil. Its molecular formula was assigned as C23H34O2 based its [M − H]− ion at m/z 341.2486 in the HRESIMS spectrum. Co-isolated compound 3 had the same molecular formula. The 1H and 13C NMR data of 2 and 3 closely resembled those of 4, except for an additional methoxy signal. Compounds 2 and 3 were considered isomers based on a comparison of their NMR data and were differentiated by HMBC correlations between methoxy protons and aromatic carbons (C-1 in 2 and C-4 in 3). The configurations of the two double bonds in the farnesyl moieties were assigned as E as described for 1 above (δC‑14′ and δC‑15: ∼16 ppm). Therefore, compound 2 was identified as 5-farnesyl-2-methyl-1-Omethylhydroquinone. In addition, compounds 4−6 were identified by comparison of 1H and 13C NMR and MS data with those in the literature. The configuration of 5 was determined by comparison of the specific rotation and ECD data with those reported.32 Amorfrutin B (7) was recently reported to be a partial PPARγ agonist with significant binding affinity for the PPAR-γ LBD (EC50 0.050 μM; rosiglitazone, EC50 0.004 μM).33 The crystal structure of the PPAR-γ LBD/amorfrutin B complex contains two amorfrutin molecules bound to chain A and chain B (inactive) of the PPAR-γ protein. In the active conformation of chain A, 7 stabilizes helix H3 and the β-sheet region of the binding pocket of PPAR-γ LBD by hydrogen bonding to Ser342. Multiple hydrophobic interactions around the geranyl side chain of 7 have been suggested to be essential for its high binding affinity to PPAR-γ. Because 1 and 7 possess a common structural motif, we performed an in silico study to investigate 1 for PPAR-γ binding. When 1 was docked to PPAR-γ LBD, it bound to the PPAR-γ LBD in a similar manner to 7 in terms of localization and orientation. The calculated binding affinity of 1 (−8.7 kcal/moL) was comparable to that of 7 (−9.4 kcal/ moL). Furthermore, additional hydrogen bonds to Cys285 and Arg288 of helix H3 and additional hydrophobic contacts to its helix H4/5 and β-sheet were suggested (Figure 1). Therefore, we speculate 1 may function as a PPAR-γ ligand and bind in a manner similar to 7. Prior to evaluating PPAR-γ agonist activities of 1−6 in murine Ac2F liver cells and RAW 264.7 macrophage cells, we assessed their cytotoxicities to gauge test concentrations. Only 1 was found to be noncytotoxic at concentrations up to 50 μM (Figure S15). PPAR-γ is a ligand-activated transcriptional factor. When a ligand binds to PPAR-γ, the conformation of its LBD is changed to facilitate coactivator binding. The ligand/PPAR-γ complex together with another nuclear receptor, retinoid X receptor (RXR), then form a heterodimer, which binds to the peroxisome proliferator response element (PPRE) region of DNA to initiate gene transcription.29 Initially, we investigated PPAR-γ activation activity using a luciferase assay. Ac2F cells (a murine liver cell line) transfected with the PPRE-X3-TK luc plasmid (PPRE-luc) and the PPAR-γ expression vector were treated with vehicle, 1, or rosiglitazone (positive control). Compound 1 was found to activate PPAR-γ in a concentrationdependent manner (Figure 2); at a concentration of 20 μM, 1 increased luciferase expression to a level comparable to that of

demonstrate a wide range of biological properties, which include cytotoxic,22 anti-inflammatory,23 antiviral,24 antimicrobial,25 and immunomodulatory activities.26,27 Meroterpenes 1−6 have structural motifs similar to those of amorfrutins. Amorfrutins A, B (7), and 2 are isoprenoidsubstituted benzoic acid derivatives and were initially isolated from the fruits of the legume Amorpha f ruticosa, an ingredient in some condiments. Amorfrutins act as partial PPAR-γ agonists and possess potent anti-inflammatory and antidiabetic properties.28,29 These agonists selectively activate PPAR-γ and repress side effects induced by TZDs, such as weight gain, osteoporosis, cardiovascular complications, and edema. In silico analysis of 1 showed that it may bind to the PPAR-γ ligand binding domain (LBD) in a manner similar to amorfrutin B (7). Our in vitro study revealed that 1 modulates inflammatory responses by activating PPAR-γ, which leads to suppression of the NF-κB signaling pathway and then suppression of proinflammatory mediators (iNOS, NO, COX-2, TNF-α, IL-1β, and IL-6). Herein, we describe the structure elucidation of the new compounds (1 and 2) and PPAR-γ/NF-κB-mediated antiinflammatory activity of 1.

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RESULTS AND DISCUSSION Chrysogenester (1) was obtained as a yellow oil. Its molecular formula was assigned as C20H28O4 based its [M + H]− ion at m/z 333.2056 and [M + Na] − ion at m/z 355.1874 in the HRESIMS spectrum. An IR absorption band observed at 1740 cm−1 suggested the presence of an ester group, and UV absorption maxima (λmax) at 224, 252, and 295 nm indicated the presence of a hydroquinone chromophore. Analysis of its 1 H and 13C NMR data (Table 1) revealed structural features similar to those of 5-farnesyl-2-methylhydroquinone (4).30 The aromatic moiety (δH‑3 6.48 and δH‑6 6.47) of 1 was presumed to be connected to the isoprenyl moiety, as it exhibited the same NMR pattern as for 4. However, the farnesyl chain of 1 was lacking the 3-carbon isopropenyl terminus of 4 and had been replaced by an ester group. In addition, two typical olefinic protons (δH‑2′ 5.28 and δH‑6′ 5.14) of the isoprenyl moieties and one additional carbonyl signal (δC‑10′ 174.4) of the ester moiety were observed. The connection between these two moieties was established by HMBC correlations from H-8′ (δH 2.24) to C-10′, C-6′, and C-11′ and a COSY correlation between H-8′ and H-9′ (δH 2.35). The configurations of the two double bonds in the isoprenyl moieties were assigned as E according to a ROESY correlation between H-2′ and H-4′ (δH 2.02) and H6′and H-8′, and the assignment was further confirmed by chemical shifts of the two methyl carbon signals at 14.6 (C-11′) B

DOI: 10.1021/acs.jnatprod.7b00846 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H (500 MHz) and 13C NMR (100 MHz) Data for Compounds 1−3 (CD3OD) 1 position

δC type

1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ OCH3

147.3, C 121.9, C 116.9, CH 147.7, C 125.8, C 115.6, CH 14.6, CH3 27.6, CH2 123.3, CH 134.8, C 39.5, CH2 26.2, CH2 124.9, CH 133.3, C 34.5, CH2 32.6, CH2 174.4, C 14.6, CH3 14.9, CH3

50.7

2 δH (J in Hz)

6.48, s

6.47, 2.08, 3.19, 5.28,

s s d (7.5) td (7.0, 1.5)

2.02, m 2.11, m 5.14, td (7.0, 1.5) 2.24, m 2.35, m 1.59, s 1.69, s

3.62, s

δC type

3 δH (J in Hz)

152.4, C 125.5, C 118.4, CH 149.2, C 126.8, C 113.2, CH 15.9, CH3 29.1, CH2 124.4, CH 136.5, C 40.9, CH2 27.6, CH2 125.4, CH 135.9, C 40.9, CH2 27.8, CH2 125.4, CH 132.0, C 25.9, CH3 17.7, CH3 16.1, CH3 16.2, CH3 56.5

6.54, s

6.58, 2.07, 3.25, 5.32,

s s d (7.5) t (7.0)

2.04, m 2.11, m 5.12, t (7.0) 1.93, m 2.03, m 5.06, t (7.0) 1.64, 1.57, 1.58, 1.70, 3.70,

s s s s s

δC type 149.8, C 123.1, C 114.7, CH 151.8, C 129.2, C 117.1, CH 16.1, CH3 28.9, CH2 124.4, CH 136.2, C 40.9, CH2 27.6, CH2 125.4, CH 135.9, C 40.8, CH2 27.8, CH2 125.4, CH 132.0, C 25.9, CH3 17.7, CH3 16.2, CH3 16.2, CH3 56.6

δH (J in Hz)

6.63, s

6.54, 2.14, 3.19, 5.25,

s s d (7.5) t (7.0)

2.01, m 2.10, m 5.10, t (7.0) 1.93, m 2.02, m 5.08, t (7.0) 1.65, 1.58, 1.58, 1.69, 3.73,

s s s s s

Figure 1. In silico analysis of the interactions between the PPAR-γ LBD and amorfrutin B (7) or 1. (A) Amorfrutin B (7; green) forms a direct hydrogen bond with Ser342 of the β-sheet of PPAR-γ LBD and interacts hydrophobically with Helix H3 (cyan: Ile281, Gly284, Cys285, Arg288 and Ile326), Helix H4/5 (red: Met329 and Leu330), Helix H2′ (orange: Leu255), and the β-sheet (yellow: Ile341) of PPAR-γ LBD (affinity value: −9.4 kcal/moL). (B) Compound 1 (green) forms direct hydrogen bonds with Ser342 of the β-sheet and with Cys285 and Arg288 of the helix H-3 and interacts hydrophobically with Helix H3 (cyan: Gly284 and Ile326), Helix H4/5 (red: Leu330), Helix H6 (Met364), and the β-sheet (yellow: Ile341) of PPAR-γ LBD (affinity value: −8.7 kcal/moL). The docking poses of amorfrutin B (7) and 1 were simulated using the reported crystal structure of amorfrutin B (7) and the human PPAR-γ protein (PDB: 4A4W).

5 μM rosiglitazone, which represented a 3-fold increase versus untreated controls. PPAR-γ is present in the cytoplasm and nuclei, but after ligand binding, the PPAR-γ/ligand complex is translocated to the nucleus. We measured 1-activated PPAR-γ protein levels in nuclear fractions by Western blot. When RAW 264.7 cells were treated with 1 or rosiglitazone for 24 h, 1 at 30 μM significantly increased nuclear PPAR-γ protein levels (∼1.8-fold increase), whereas rosiglitazone at 10 μM elicited a ∼1.9-fold increase and

30 μM elicited a ∼2.3-fold increase (Figure 3). In the nuclei, PPAR-γ regulates gene transcription by transactivation or transrepression.34 During transactivation, PPAR-γ forms a heterodimer complex with RXR and recognizes PPRE in the promotor regions of target genes, which results in transcription of PPAR-γ target genes ultimately involved in adipocyte differentiation and glucose and lipid metabolism. During transrepression, PPAR-γ suppresses gene transcription by inhibiting the NF-κB signaling pathway. The inducible C

DOI: 10.1021/acs.jnatprod.7b00846 J. Nat. Prod. XXXX, XXX, XXX−XXX

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levels induced by 1 in LPS-activated RAW 264.7 cells. Phosphorylation of p65 induces a conformational change, which impacts p65 ubiquitination and stability, as well as protein−protein interactions leading to activation of the NF-κB signal pathway.37 Cells were collected 30 min after LPS stimulation to detect the early state of NF-κB signaling. Levels of phosphorylated NF-κB p65 were significantly increased by LPS, but 1 repressed NF-κB p65 phosphorylation in a concentration-dependent manner. Furthermore, 1 at 30 μM more potently suppressed NF-κB p65 phosphorylation (by ∼2.8-fold) than dexamethasone at 10 μM (∼2.3-fold) (Figure 4). As expected, the specific PPAR-γ antagonist (GW9662)

Figure 2. Activation of PPAR-γ by 1 or by rosiglitazone (Rosi) in Ac2F cells. In vitro assay of PPAR-γ activation by 1 at concentrations of 5, 10, and 20 μM in murine Ac2F liver cells. Cells were transiently transfected with PPRE and pFlag-PPAR-γ1. Rosi (5 μM) was employed as a positive control to monitor the activation of the luciferase reporter. Blank cells were transfected with plasmid containing pcDNA3. Control cells were transfected with plasmid containing PPRE. Luciferase expressions (folds of controls) are presented as means ± SDs (n = 3). **p < 0.01 and ***p < 0.001 vs control cells.

Figure 4. Effect of 1 on the phosphorylation of NF-κB p65 protein in RAW 264.7 macrophages as determined by Western blot. Cells were pretreated with GW9662 (10 μM) for 1 h, then treated with 1 (10 or 30 μM) or dexamethasone (DEX, 10 μM) for 1 h, and then stimulated with lipopolysaccharide (LPS, 30 ng/mL) for 30 min. β-Actin was used as an internal control. The results shown are representative of three independent experiments. ###p < 0.001 vs untreated controls. **p < 0.01 and ***p < 0.001 vs LPS-treated cells.

dramatically reversed the effect of 1 (30 μM). Several studies have explored the interaction of a PPAR-γ agonist with NF-κB signaling by focusing on the early events of NF-κB signaling, such as IκBα expression, phosphorylation, and its subsequent degradation or p65 nuclear translocation, which may interfere with the p65 phosphorylation.38−41 Our results suggest that 1 may suppress the early stage of the NF-κB signaling pathway via activation of PPAR-γ. The transcription of pro-inflammatory mediators, such as TNF-α, IL-1β, IL-6, iNOS, and COX-2, can be regulated by NF-κB.42 To investigate the possible anti-inflammatory effects of NF-κB repression by 1, we measured the mRNA levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in LPS-induced RAW 264.7 macrophages by reverse transcriptase-PCR (RT-PCR). We found the mRNA levels of these pro-inflammatory mediators were dramatically and concentration-dependently diminished by pretreating with 1 (Figure 5). For example, at 50 μM, 1 significantly reduced the mRNA levels of iNOS, COX-2,

Figure 3. Endonuclear PPAR-γ protein levels in RAW 264.7 macrophages treated with 1 (10 or 30 μM) or rosiglitazone (Rosi, 10 or 30 μM) for 24 h, as determined by Western blot. Nuclear levels of transcriptional factor IIB (TF IIB) were used for reference purposes. The results shown are representative of three independent experiments. *p < 0.05, **p < 0.01 vs untreated controls.

transcription factor NF-κB consists of five subunits, RelA/p65, c-Rel, RelB, p50, and p52, and phosphorylation of the NF-κB subunit may enhance or reduce the transcription of target genes or modulate NF-κB activation during inflammatory processes, and thus, activated NF-κB signal upregulates the expression of multiple pro-inflammatory mediators.35 As a PPAR-γ agonist, rosiglitazone has also been reported to inhibit the expression of pro-inflammatory cytokines (TNF-α and IL-1β) and to suppress activation of p65 during lipopolysaccharide (LPS)induced acute kidney injury.36 In the present study, we resorted to Western blotting to investigate NF-κB p65 phosphorylation D

DOI: 10.1021/acs.jnatprod.7b00846 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 5. Effects of 1 on the cellular levels of pro-inflammatory mediators as determined by reverse transcriptase-PCR (RT-PCR). RAW 264.7 cells were pretreated with the indicated concentrations of compound 1 (10, 30, and 50 μM) or dexamethasone (DEX, 10 μM) for 1 h and then treated with LPS (1 μg/mL, 6 h). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The results shown are representative of three independent experiments. ##p < 0.01, ###p < 0.001 vs untreated controls. *p < 0.05, **p < 0.01, and ***p < 0.001 vs LPStreated cells.

TNF-α, IL-1β, and IL-6 by ∼5-, 2-, 2-, 2.5-, and 5-fold, respectively, which was better than that achieved by pretreating with 10 μM dexamethasone. Notably, 1 reduced iNOS and TNF-α levels as effectively as dexamethasone. We also confirmed these effects by investigating the effects of 1 on the protein levels of iNOS and COX-2 by Western blot (Figure 6). Compound 1 was found to consistently and concentrationdependently decrease iNOS levels with a potency comparable to that of dexamethasone, although its effect on iNOS protein was greater than that on COX-2. The effects of 1 on NO, TNFα, IL-1β, and IL-6 levels were also investigated using a Griess assay and an enzyme-linked immunosorbent assay (ELISA). As shown in Figure 7, the production of all four pro-inflammatory mediators was remarkably and concentration-dependently inhibited by 1. Moreover, 1 suppressed the production of NO and TNF-α with a potency comparable to those of dexamethasone, and these effects matched well with our gene expression results (Figures 5 and 7). These findings suggest 1 suppresses the expression of pro-inflammatory mediators via activating PPAR-γ and thus suppressing the NF-κB pathway. Summarizing, our chemical investigation of the jellyfishderived fungus P. chrysogenum J08NF-4 afforded two new meroterpenes and four known farnesyl meroterpenes. Molecular docking suggested that the new meroterpene 1 targets PPAR-γ LBD with high affinity by interacting with amino acid residues in helix H3 and the β-sheet region of PPAR-γ LBD in a

manner similar to the natural PPAR-γ agonist 7. In addition, 1 activated PPAR-γ in Ac2F cells (as gauged by a luciferase assay) and inhibited NF-κB p65 phosphorylation, and inhibition of the NF-κB pathway by 1 led to the concentration-dependent downregulation of the expression of iNOS, NO, COX-2, TNFα, IL-1β, and IL-6. Our findings suggest that 1 can be viewed as a starting point for the development of anti-inflammatory therapeutics.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were obtained on a Optizen UV/vis spectrophotometer, IR spectra were recorded on a Jasco 410 spectrometer, and 1D and 2D NMR spectra were recorded on Varian UNITY 400 and Varian INOVA 500 spectrometers, respectively. Chemical shifts are reported with reference to respective residual solvents or deuterated solvent peaks (δH 3.30 and δC 49.0 for CD3OD). HRESIMS data were obtained using an Agilent 1200 UHPLC accurate-mass Q-TOF MS spectrometer. HPLC was performed using a Gilson 307 pump, an ODS column (YMC-triart C18, 250 × 10 mm, i.d. 5 μm), and a Jasco UV-975 detector. Fungal Strain. The fungal strain P. chrysogenum J08NF-4 was isolated from the marine jellyfish Nemopilema nomurai, which was collected off the southern coast of South Korea in June 2007. The specimen was deposited at Marine Natural Product Laboratory, Pusan National University. After rinsing with filtered and sterilized seawater (National Institute of Fisheries Science), small pieces of the surface and inner tissues were homogenized and then inoculated on malt extract agar (MEA) in Petri dishes. The sterilized MEA medium used E

DOI: 10.1021/acs.jnatprod.7b00846 J. Nat. Prod. XXXX, XXX, XXX−XXX

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CD3OD) and 13C (100 MHz, CD3OD), Table 1; HRESIMS m/z 333.2056 [M + H]+ (calcd for C20H29O4, 333.2066) and [M + Na]+ m/z 355.1874 (calcd for C20H28O4Na, 355.1885). 5-Farnesyl-2-methyl-1-O-methylhydroquinone (2): yellow oil; UV (MeOH) λmax (log ε) 228 (3.40), 288 (3.18) nm; IR νmax 2926, 2864, 2340, 1723, 1653, 1510, 1455, 1395, 1197 cm−1; 1H (500 MHz, CD3OD) and 13C (100 MHz, CD3OD), Table 1; HRESIMS m/z 341.2486 [M − H]− (calcd for C23H33O2, 341.2481). Cell Culture and Cell Viability. RAW 264.7 murine macrophages were purchased from the Korean Cell Line Bank (KCLB), and rat liver Ac2F cells from the American Type Culture Collection (ATCC). Cells were cultured at 37 °C in a 5% CO2 humidified incubator and maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM/high glucose, HyClone) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100 units/mL penicillin (HyClone), and 100 μg/mL streptomycin (HyClone). Cells were seeded in 96well culture plates, cultured for 12 h, and then treated with various concentrations of samples for 24 h. Cell viabilities were evaluated using water-soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd.), which was added to each well (10 μL) and incubated at 37 °C for 1 h. Absorbances were read using an iMark microplate absorbance reader (Bio-Rad Laboratories) at a wavelength of 450 nm. Luciferase Assay. The TK-PPRE ×3-luciferase reporter plasmid containing three copies of the PPAR response element in an acyl CoA oxidase promoter was generously donated by Dr. Christopher K. Glass (University of California, San Diego). The pcDNA3 expression vector and full-length human PPAR-γ1 expression vector (pFlag-PPAR-γ1) were generously donated by Dr. Chatterjee (University of Cambridge, Addenbrooke’s Hospital). For luciferase assays, cells at 70−80% confluence were transfected in 48-well plates (5 × 104 cells/well) with effector plasmids and the PPRE-X3-TK-luciferase reporter plasmid (1 μg/well) plus pcDNA3 (0.1 μg/well) or pFlag-PPAR-γ1 (0.1 μg/well) using Lipofectamine 2000 (Invitrogen Co.), according to the manufacturer’s instructions. After transfection for 4 h, the conditioned medium was replaced with complete medium, and cells were incubated for an additional 10 h. The medium was then removed, and cells were treated with rosiglitazone or test compounds in serum-free media for 6 h. Cells were then lysed and assayed using the ONE-Glo luciferase assay system (Promega). Luciferase activities were measured using a GloMax-Multi microplate multimode reader (Promega Co.). Reverse Transcriptase-PCR (RT-PCR). To assess the expression of inflammatory markers in RAW 264.7 cells by RT-PCR, total RNA was isolated using Trizol reagent (Invitrogen), and first-strand cDNA was synthesized using total RNA and the M-MLV cDNA synthesis kit (Enzynomics). cDNA products and primers for each gene were used for PCR with AccuPower PCR PreMix (BIONEER) in a BIO-RAD T100 Thermal Cycler PCR unit (Hercules). Specific primers for iNOS (sense 5′-ACC TAC CAC ACC CGA GAT GGC CAG-3′, antisense 5′-AGG ATG TCC TGA ACA TAG ACC TTG GG-3′), COX-2 (sense 5′-CCG TGG GGA ATG TAT GAG CA-3′, antisense 5′-CCA GGT CCT CGC TTA TGA TCT G-3′), IL-1β (sense 5′-GGA GAA GCT GTG GCA GCT A-3′, antisense 5′-GCT GAT GTA CCA GTT GGG GA-3′), IL-6 (sense 5′-TGG GAA ATC GTG GAA ATG AG-3′, antisense 5′-GAA G GA CTC TGG CTT TGT CT-3′), and GAPDH (sense 5′-TTC ACC ACC ATG GAG AAG GC-3′, antisense 5′-GGC ATG GAC TGT GGT CAT GA-3′) were used to amplify gene fragments. PCR was performed over 27 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and elongation at 72 °C for 30 s. TNF-α (sense 5′-AGC ACA GAA AGC ATG ATC CG-3′, antisense 5′-GTT TGC TAC GAC GTG GGC TA-3′) was used to amplify gene fragments. PCR was performed over 27 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s. Aliquots (6 μL) were electrophoresed in 1.2% agarose gels and stained with ethidium bromide.44 Western Blot. Cells were harvested and suspended in lysis buffer containing protease and phosphatase inhibitor cocktails. Nuclear protein extraction was performed using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific). Concentrations of proteins were determined using a BCA protein assay (Thermo

Figure 6. Effects of 1 on inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expression in RAW 264.7 macrophages. Cells were pretreated with the indicated concentrations of 1 (10, 30, or 50 μM) or dexamethasone (DEX, 10 μM) for 1 h and then stimulated with lipopolysaccharide (LPS) (1 μg/mL, 24 h). iNOS and COX-2 levels were determined by Western blot. The results shown are representative of three independent experiments. ###p < 0.001 vs untreated controls. **p < 0.01 and ***p < 0.001 vs LPStreated cells. was prepared using seawater containing glucose (20 g/L), malt extract (20 g/L), agar (20 g/L), peptone (1 g/L), and antibiotics (final concentrations: 50 μg/mL penicillin and 50 μg/mL streptomycin). Emerging fungal colonies were transferred to the same medium in a Petri dish and incubated at 25 °C for 10−14 days to allow colony development.43 Twelve pure fungal strains (J08NF-1−J08NF-12) were isolated from the jellyfish. The fungal strain J08NF-4 was selected and further identified as P. chrysogenum according to its morphologic characteristics and ITS gene sequences (GenBank accession no. KR011759). P. chrysogenum J08NF-4 was then cultured in 44 L of MEA medium (prepared with 75% seawater) containing glucose (20 g/L), malt extract (20 g/L), and peptone (1 g/L) at 30 °C on a shaker platform at 130 rpm for 21 days. Extraction and Isolation. After a 21-day culture period, the culture medium and mycelia were extracted with EtOAc at room temperature. The EtOAc extract (18 g) was then partitioned between aqueous MeOH and n-hexane, and the aqueous MeOH layer was subjected to step-gradient medium-pressure liquid chromatography (MPLC) (ODS-A, 120 Å, S-30/50 mesh) and eluted with 30% to 100% MeOH/H2O to afford 30 fractions. Fraction 12 (85.2 mg) was subjected to RP-HPLC (YMC-triart C18, 250 × 10 mm, 5 μm) and eluted with 65% MeOH/H2O, which afforded 1 (12.8 mg). 2 (5.0 mg) and 3 (2.7 mg) were isolated from the n-hexane fraction by RP-HPLC (YMC-triart C18, 250 × 10 mm, 5 μm) with 65% MeCN/H2O elution and NP-HPLC (YMC-Pack SIL, 12 nm, 5 μm) with (1000:3.5) nhexane/2-propanol elution. Other fractions (8, 19, and 21) were also separated by RP-HPLC (YMC-triart C18, 250 × 10 mm, 5 μm) and yielded 4 (1.3 g), 5 (20.6 mg), and 6 (10.9 mg). Chrysogenester (1): yellow oil; UV (MeOH) λmax (log ε) 224 (2.72), 252 (2.73), 295 (2.18) nm; IR νmax 2925, 2880, 2348, 1740, 1738, 1660, 1517, 1507, 1480, 1460, 1198, 458 cm−1; 1H (500 MHz, F

DOI: 10.1021/acs.jnatprod.7b00846 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 7. Effects of 1 on the production of (A) nitric oxide (NO); (B) tumor necrosis factor α (TNF-α), (C) interleukin-1β (IL-1β), and (D) interleukin-6 (IL-6) in LPS-induced RAW 264.7 macrophages. Cells were pretreated with increasing concentrations of 1 (10, 30, or 50 μM) or dexamethasone (DEX, 10 μM) for 1 h and then treated with LPS. NO concentrations (LPS, 25 ng/mL, 24 h) in the medium were determined by the Griess method, and levels of IL-1β and IL-6 (LPS, 100 ng/mL, 24 h) and TNF-α (LPS, 100 ng/mL, 3 h) in the medium were determined by ELISA. The results shown are representative of three independent experiments. ###p < 0.001 vs untreated controls. p < 0.05, **p < 0.01, and ***p < 0.001 vs LPS-treated cells. Scientific). Equal amounts of proteins were resolved by 10% SDSpolyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skimmed milk for 1 h at room temperature, and then incubated with specific primary antibodies recognizing COX-2, iNOS, PPAR-γ, NF-κB p-p65, and β-actin (Cell Signaling Technology) overnight at 4 °C. Anti-rabbit horseradish-linked IgG was used as the secondary antibody. Signals were developed using the ChemiDoc Touch imaging system (Bio-Rad Laboratories). Concentrations of NO and Cytokines Released to the Medium. RAW 264.7 macrophages (1 × 104 cells/well) were seeded in a 96-well culture plate and cultured for 12 h, pretreated with various concentrations of samples or dexamethasone for 1 h, and then coincubated with 25 ng/mL of LPS for 24 h. NO concentrations in medium were determined using a Griess assay; Griess reagent (80 μL) was added to media supernatants (80 μL) and then incubated at 37 °C for 20 min in the dark. Absorbance was measured at 520 nm. NO concentrations were determined using 0−100 μM sodium nitrite standards. TNF-α, IL-1β, and IL-6 expression levels in culture media were quantified using sandwich-type ELISA kits (Biolegend). Molecular Docking Study. Docking calculations were performed using AutoDock Vina 1.1.2 software (The Scripps Research Institute). Default settings and the Vina scoring function were applied. For PPAR-γ ligand preparation, Chem3D Ultra 14.0 software (CambridgeSoft Corporation) was used to convert the 2D structures of

candidates into 3D structural data. Protein coordinates were downloaded from the Protein Data Bank (accession code: 4A4W). Chain A of the PPAR γ protein was prepared for docking within the molecular modeling software package Chimera 1.5.3 (National Institutes of Health), by removing chain B, all ligands, and water molecules. Polar hydrogens and grid box parameter settings were added using MGLTools 1.5.4 (The Scripps Research Institute). The analysis and visual investigation of ligand−protein interactions of docking poses were performed using Discovery Studio 4.3 (Accelrys Inc.). Docking success was evaluated using lowest affinity values. Statistics. The significances of intergroup differences were determined by ANOVA. Results are expressed as the means ± SDs of the indicated numbers of independent experiments. Statistical significance was accepted for p-values of