Mol Cell Biochem DOI 10.1007/s11010-015-2457-4

Anti-inflammatory effect of procyanidin B1 on LPS-treated THP1 cells via interaction with the TLR4–MD-2 heterodimer and p38 MAPK and NF-jB signaling Jing Xing1 • Rui Li1 • Nan Li1 • Jian Zhang1 • Yueqing Li2 • Ping Gong1 Dongna Gao1 • Hui Liu1 • Yu Zhang1

•

Received: 6 January 2015 / Accepted: 16 May 2015 Ó Springer Science+Business Media New York 2015

Abstract Anti-inflammatory effects of procyanidin B1 have been documented; however, the molecular mechanisms that are involved have not been fully elucidated. Molecular docking models were applied to evaluate the binding capacity of lipopolysaccharide (LPS) and procyanidin B1 with the toll-like receptor (TLR)4/myeloid differentiation factor (MD)-2 complex. LPS-induced production of the proinflammatory cytokine tumor necrosis factor (TNF)-a in a human monocyte cell line (THP1) was measured by ELISA. mRNA expression of MD-2, TLR4, TNF receptor-associated factor (TRAF)-6, and nuclear factor (NF)-jB was measured by real-time PCR with or without an 18-h co-treatment with procyanidin B1. In addition, protein expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) and NF-jB was determined by Western blotting. Structural modeling studies identified Tyr296 in TLR4 and Ser120 in MD-2 as critical sites for hydrogen bonding with procyanidin B1, similar to the sites occupied by LPS. The production of TNF-a was significantly decreased by procyanidin B1 in LPS-treated THP1 cells (p \ 0.05). Procyanidin B1 also significantly suppressed levels of phosphorylated p38 MAPK and NF-jB protein, as well as mRNA levels of MD-2, TRAF-6, and NF-jB (all p \ 0.05). Procyanidin B1 can compete with LPS for binding to the TLR4–MD-2 heterodimer and suppress downstream activation of p38 MAPK and NF-jB signaling pathways. & Yu Zhang [email protected] 1

Emergency Department, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China

2

College of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian 116024, China

Keywords Anti-inflammatory  LPS  Procyanidin B1  THP1 cells  TLR4–MD-2 heterodimer

Introduction Procyanidins are polyphenolic flavonoids that are abundant in commonly eaten fruits and fruit juices, for which procyanidins B1, B2, C1, and C2 possess a wide range of physiologic activities [1–4]. The consumption of these and other bioactive food compounds has been suggested to have beneficial effects on inflammation-related diseases [5, 6]. Indeed, procyanidins show anti-inflammatory and immune modulatory activities in vitro [7]. Procyanidins B1–5 differ by the position and configuration of monomeric linkages [8], which can lead to different pharmacologic effects. Recently, procyanidin B1 was shown to regulate innate and adaptive immunity by inhibiting inducible nitric oxide production and release of the proinflammatory cytokines interleukin-6 and tumor necrosis factor (TNF)-a by macrophages treated with lipopolysaccharide (LPS) [1]. Procyanidin B1 can impair inflammatory response signaling in human monocytes by suppressing the LPS-induced production of reactive oxygen species, thereby attenuating the activity of the inhibitor of nuclear factor (NF)-jB kinase subunit b and extracellular signalregulated kinase [9]. Furthermore, procyanidin B1 inhibits NF-jB, interferon-inducible protein-10, interleukin-8, and signal transducers and activators of transcription-1 [10]. The mechanism by which procyanidin B1 attenuates the effects of LPS is not clear, but may involve signaling through the toll-like receptors (TLRs), which are pathogenrecognition proteins that play important roles in the activation of the innate immune response [11–13]. TLR4 is one of the most widely studied of these receptors, and, in

123

Mol Cell Biochem

association with its co-receptor myeloid differentiation factor (MD)-2 (also known as lymphocyte antigen 96), is responsible for the physiologic recognition of LPS [14, 15]. The TLR4–MD-2 heterodimer has complex ligand specificity and can be activated by structurally diverse LPS molecules, including synthetic derivatives of LPS [16, 17]. Modulation of the TLR4/MD-2 signaling pathway is important to prevent hyperactivation of immune responses that could contribute to the pathogenesis of autoimmune, chronic inflammatory, and infectious diseases [18, 19]. The TLR4 signaling pathway can be suppressed via inhibitory alternative splice variants of essential signaling components [20–24] that are recruited to the TLR4 complex and lead to early-phase activation of TNF receptor-associated factor (TRAF)6 and NF-jB, and subsequent expression of proinflammatory cytokines (TNF-a and interleukin-6) and monocyte chemotactic protein-1 [1, 4]. To date, there are no data regarding a direct effect of procyanidin B1 on NF-jB or mitogen-activated protein kinase (MAPK) signaling pathways via the TLR4/MD-2 complex. Therefore, the aim of this study was to explore the possible underlying intracellular signaling mechanisms driving the anti-inflammatory effects of procyanidin B1 in human monocytes in order to establish its potential properties as a pharmacologic agent.

Cell culture The human acute monocytic cell line (THP1), derived from the blood of a one-year-old boy with acute monocytic leukemia, was obtained from the Beijing Union Cell Resource Center. THP1 cells were grown in RPMI 1640 medium (Gibco of Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10 % (v/v) heat-inactivated fetal bovine serum (HyClone of Thermo Fisher Scientific) and antibiotics (1 9 105 U/L penicillin and 100 mg/L streptomycin) at 37 °C in a humidified atmosphere of 5 % CO2. In all experiments, cells were plated at a density of 4 9 105 cells/well in a 96-well plate and were allowed to pre-incubate for 24 h prior to treatment. Cytotoxicity analysis To investigate the cytotoxic effect of procyanidin B1 (Dalian Meilun Biology Technology Co., Ltd., Dalian, China), viability of THP1 cells was assessed using the cell counting kit-8 (CCK8) assay. THP1 cells were treated with procyanidin B1 for 18 h, and 10 lL of CCK8 solution was then added to each well and the cultures were incubated for 4 h at 37 °C. The optical density (OD) at 450 nm was measured using an ELx808 Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA). The procyanidin B1 concentration tested ranged from 50 to 200 lg/mL. Each sample was tested in triplicate.

Methods Measurement of cytokine production Molecular docking study The X-ray crystallographic structure of the TLR4/MD-2 complex was obtained from the Protein Data Bank (http:// www.rcsb.org/pdb). The two-dimensional structures of procyanidin B1 and LPS were drawn by MDL ISIS Draw 2.5 standalone software and converted into three-dimensional structures using the Accelrys Discovery Studio 2.5 software (http://www.accelrys.com/). The docking procedures involved removing the ligand and crystallographic water molecules from the protein and adding hydrogens, and correcting the chemistry of the protein for missing atoms, bounds, contacts, etc. The Energy Grid Force Field parameter was set to ‘‘Dreiding’’ for computing the ligand– protein interaction energy. The Energy Grid parameters control the grid base docking used in the initial evaluation of the positions. In the Dreiding force field, the Gasteiger method was used to calculate the partial charges of ligands and proteins. The LigandFit module was used for molecular modeling on a Windows XP workstation (Microsoft Corp., Redmond, WA, USA). The analysis of resultant structures and visualization of complexes were performed with the Accelrys Discovery Studio 2.5.

123

THP1 cells were treated for 18 h with 1 lg/mL LPS (L2880; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of 100 lg/mL procyanidin B1. Culture medium was then collected to assay TNF-a levels using commercially available multiplex sandwich enzyme-linked immunosorbent assays (ELISA) (Shanghai Westang BioTech Inc., Shanghai, China) and measuring the OD at 450 nm. Each sample was tested in triplicate. Real-time PCR quantification of MD-2, TLR4, TRAF6, and NF-jB mRNAs Total RNA was extracted from THP1 cells using the RNAiso Plus kit (Takara Biotechnology Co., Ltd. Dalian, China). Isolated total RNA was reverse transcribed using an RT master mix kit (Takara Biotechnology Co., Ltd.) for 15 min at 37 °C and 5 s at 85 °C. cDNAs were amplified using a 7500 Real-Time PCR System (Applied Biosystems of Thermo Fisher Scientific) with sequence-specific primers for MD-2, TLR4, TRAF6, and NF-jB (Table 1) designed and synthesized by Takara Biotechnology. Relative mRNA expression was determined after normalizing

Mol Cell Biochem Table 1 Primer sequences for real-time RT-PCR analysis

Gene

Primers

Accession numbera

LY96/MD2

Forward: 50 -GAGCTCTGAAGGGAGAGACTGTGAA-30

NM015364

Reverse: 50 -GTGTAGGATGACAAACTCCAAGCAA-30 TRAF6

Forward: 50 -CCCAATTCCATGCACATTCAGTA-30

NM145803.2

Reverse: 50 -AACAGCCTGGGCCAACATTC-30 NFKB1

Forward: 50 -GAGGTGTATTTCACGGGACC-30 0

Reverse: 5 -GAAGTCCATGTCCGCAATGG-3 TLR4

NM003998.3

0

Forward: 50 -CCAAGAACCTGGACCTGAGCTTTA-30

NM138554.4

Reverse: 50 -CCATCTTCAATTGTCTGGATTTCAC-30 GAPDH

Forward: 50 -AGGCTAGCTGGCCCGATTTC-30

NM001256799.1

Reverse: 50 -TGGCAACAATATCCACTTTACCAGA-30 a

GenBank accession number, available at http://www.ncbi.nlm.nih.gov/

to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated using the 2DCt equation (Ct was defined as the PCR cycle where the florescence signal from the specific amplified product first increased above the background), setting the blank control group as 1. Each sample was tested in triplicate.

hoc test for multiple comparisons. Data are presented as mean ± SD, with p \ 0.05 considered as statistically significant.

Western blot analysis of phosphorylated p38 MAPK and NF-jB

Procyanidin B1 binds the TLR4/MD-2 complex similarly to LPS

Proteins were extracted using cell lysis buffer (0.1 mol/L NaCl, 0.01 mol/L TrisHCl [pH 7.6], 0.001 mol/L EDTA [pH 8.0], 1 lg/L aprotinin, and 100 lg/L benzyl sulfuryl fluoride). Protein samples (30 lg/lane) were separated on a 15 % polyacrylamide gel and electophoretically transferred to nitrocellulose membranes. After blocking with 5 % nonfat milk in PBS containing 0.2 % Tween-20, membranes were incubated at 4 °C overnight with primary antibody, including rabbit polyclonal anti-NF-jB or anti-pp38 MAPK and mouse polyclonal anti-GAPDH (all 1:1000; Abcam, Cambridge, UK). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:7500; Sigma-Aldrich) for 40 min at room temperature. ECL reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used for protein detection. ODs of the immunoreactive bands were measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA), and expression of NF-jB and phosphorylated p38 MAPK was normalized to GAPDH.

Molecular docking models of procyanidin B1 and TLR4/ MD-2 complex are shown in Fig. 1. Procyanidin B1 was fitted into the red and magenta partitions of the binding site (Fig. 1a). The molecular docking model of procyanidin B1 to the TLR4/MD-2 complex was similar to the TLR4/MD2 complex containing bound LPS ligand (Fig. 1b). The crystal structure shows two hydrogen bonds between procyanidin B1 and the binding pocket of the TLR4/MD-2 complex, one linking the hydroxyl groups of procyanidin B1 and Tyr296 in TLR4, and the other linking the hydroxyl groups of procyanidin B1 and Ser120 in MD-2 (Fig. 1c). The structure of procyanidin B1 aligned with the inner core carbohydrates of LPS (lipid A or endotoxin) (Fig. 1d). Consensus scores of the resultant structures were 6 , as shown in Fig. 1c, d. The ligScore1 was 6.31–6.37, ligScore2 was 7.32–7.40, and the DockScore was 85.124–85.145.

Statistical analysis

The viability of THP1 cells treated with various concentrations of procyanidin B1 for 18 h was assessed using the CCK8 assay. As shown in Fig. 2, treatment of THP1 cells with procyanidin B1 at concentrations [100 lg/mL induced cellular toxicity. Thus, procyanidin B1 was used at a concentration of 100 lg/mL for subsequent experiments.

Statistical analyses were performed with the software package SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Oneway analysis of variance was used to assess the overall differences among groups, followed by a Bonferroni post

Results

Maximum tolerated concentration of procyanidin B1

123

Mol Cell Biochem Fig. 1 Molecular docking of procyanidin B1 to the TLR4/ MD-2 complex. a The lipopolysaccharide (LPS) binding site in the crystal structure (PDB code 3FXI) is shown divided into different parts in different colors (hydrogen bonds, black dotted lines; carbon atoms, blue; carbon atoms of LPS, green). b The hydrogen bonds between the inner core carbohydrates of LPS and the residues around the binding site are shown. c Docking conformation of procyanidin B1 to the crystal structure of the TLR4/MD-2 complex identifying hydrogen bonds between procyanidin B1 and residues around the binding site. d Molecular modeling of procyanidin B1 superimposed onto the carbohydrates of LPS shows a high degree of similarity of their interactions with the receptor complex. (Color figure online)

Fig. 2 Optimization of procyanidin B1 dosage in THP1 cells. Cell viability was assessed after 18-h incubation with 50, 100, 150, and 200 lg/mL of procyanidin B1; *p \ 0.05 versus control

Production of TNF-a is suppressed by procyanidin B1 To examine the anti-inflammatory effect of procyanidin B1 on THP1 cells, we first measured the levels of the

123

Fig. 3 Procyanidin B1 co-treatment inhibits LPS-induced TNF-a production in THP1 cells. Cells were treated with LPS (1 lg/mL) alone or co-treated with 100 lg/mL procyanidin B1; *p \ 0.05 versus control; #p \ 0.05 versus LPS alone. LPS lipopolysaccharide

inflammatory cytokine TNF-a in the culture supernatants by ELISA. As shown in Fig. 3, the production of TNF-a increased dramatically upon treatment with LPS (1 lg/mL) (p \ 0.05) and was significantly inhibited by co-treatment with procyanidin B1 (p \ 0.05).

Mol Cell Biochem

Expression of MD-2, TRAF6, and NF-jB mRNA is reduced by procyanidin B1

Expression of phosphorylated p38 MAPK and NFjB is reduced by procyanidin B1

LPS (1 lg/mL) caused an increase in expression of MD-2 (70-fold), TLR4 (fivefold), TRAF6 (sevenfold), and NF-jB (eightfold) mRNA in THP1 cells (Fig. 4). Procyanidin B1 co-treatment significantly reduced the LPS-induced mRNA expression of three of these (MD-2, TRAF6, and NF-jB) by 60–75 % (p \ 0.05).

LPS (1 lg/mL) caused a sixfold increase in the level of phosphorylated p38 MAPK in THP1 cells, which was attenuated by *50 % with procyanidin B1 co-treatment (Fig. 5a, b). Similarly, LPS caused a threefold increase in the production of NF-jB, which was reduced by 25 % after co-treatment with procyanidin B1 (Fig. 5c, d).

Fig. 4 Procyanidin B1 cotreatment inhibits LPS-induced expression of MD-2, TRAF6, and NF-jB mRNA. Expression is relative to GAPDH; *p \ 0.05 versus control; #p \ 0.05 versus LPS alone. LPS lipopolysaccharide, MD-2 myeloid differentiation factor 2, NF-jB nuclear factor-jB, TRAF6 tumor necrosis factor receptor-associated factor 6

Fig. 5 Procyanidin B1 cotreatment inhibits LPS-induced expression of phosphorylated p38 MAPK and NF-jB protein. Representative Western blots are shown in the left panels and quantification of the band intensities in the right panels. GAPDH was used as a loading control; *p \ 0.05 versus control; #p \ 0.05 versus LPS alone. LPS lipopolysaccharide, MAPK mitogen-activated protein kinase, NF-jB nuclear factor-jB

123

Mol Cell Biochem

Discussion Procyanidin B1 is the major procyanidin found in most commonly eaten fruits and fruit juices [2, 25–27] and produces anti-inflammatory effects in human monocytes [1, 9, 10]. The data presented here indicate that procyanidin B1 may mediate these effects by acting as a competitive antagonist against TLR4/MD-2 activation of MAPK and NF-jB signaling and production of TNF-a. Inhibition of TLR4 signaling has become a focus of research on the innate immune system. Together, TLR4 and one of its co-receptors (MD-2) are responsible for the physiologic recognition of LPS [4, 14]. MD-2 has a b-cup fold structure composed of two anti-parallel b sheets that form a large hydrophobic pocket that serves as the ligandbinding site [28, 29] for interaction with the lipid side chains of LPS. The crystal structure of LPS shows the ester and amide groups connecting the lipids to the glucosamine backbone or to the other exposed lipid chains. These groups can interact with hydrophilic side chains located on the b strand of the MD-2 pocket and on the surface of TLR4 [30]. The two phosphate groups of lipid A dock LPS to the TLR4/MD-2 complex by interacting with positively charged residues on the receptors and forming a hydrogen bond to Ser118 of MD-2 [30]. Our analysis indicates that procyanidin B1 docks to the TLR4/MD-2 complex in a manner similar to LPS. Furthermore, we identified Tyr296 in TLR4 and Ser120 in MD-2 as critical sites for hydrogen bonding with procyanidin B1, similar to those sites occupied by LPS. Thus, the binding capacity of procyanidin B1 to the TLR4/MD-2 complex is similar to LPS. Therefore, we postulate that procyanidin B1 binds to the TLR4–MD-2 heterodimer through a competitive interaction with LPS. Binding of LPS to the TLR4/MD-2 complex triggers a signaling cascade that involves activation of NF-jB, which subsequently induces expression of various immune and inflammatory genes [33], including TNF-a. To determine the anti-inflammatory effects of procyanidin B1, we treated cells with a concentration (100 lg/mL) that was determined to be safe, similar to the previous reports [31, 32]. Co-treatment of cells with procyanidin B1 significantly attenuated the expression of TRAF6 and NF-jB. These data indicate that procyanidin B1 interferes with the activation of TLR4/MD-2 signaling pathways. These results are consistent with the previous studies showing reduced NF-jB mRNA expression with procyanidin B1 treatment [1, 34]. Furthermore, co-treatment reduced phosphorylation of p38 MAPK, which represents one of the primary signaling pathways involved in LPS-induced cytokine production [35]. The results of our study also show that procyanidin B1 reduced levels of MD-2, but not TLR4 mRNA, suggesting that procyanidin B1 may produce a

123

long-term anti-inflammatory effect via reducing levels of co-receptor MD-2 or the formation of a TLR4–MD-2 heterodimer, rather than through effects on a TLR4 homodimer. In conclusion, procyanidin B1 can bind to the TLR4– MD-2 heterodimer through a competitive interaction with LPS, which suppresses the activation of NF-jB and p38 MAPK pathways. These results provide a new insight into the role of procyanidin B1 as an antagonist of the immune response involved in the development and progression of many inflammatory diseases. Future clinical studies are needed to confirm the therapeutic efficacy of this compound. Acknowledgments This study was supported by the Young Starting Foundation of the First Affiliated Hospital, Dalian Medical University (QN2012008) and the 2013 Dalian Science and Technology Planning Project (guidance project). Conflict of interest to declare.

The authors do not have any conflicts of interest

References 1. Byun EB, Sung NY, Byun EH, Song DS, Kim JK, Park JH, Song BS, Park SH, Lee JW, Byun MW et al (2013) The procyanidin trimer C1 inhibits LPS-induced MAPK and NF-kB signaling through TLR4 in macrophages. Int Immunopharmacol 15:450–456. doi:10.1016/j.intimp.2012.11.021 2. Hwang SJ, Yoon WB, Lee OH, Cha SJ, Kim JD (2014) Radicalscavenging-linked antioxidant activities of extracts from black chokeberry and blueberry cultivated in Korea. Food Chem 146:71–77. doi:10.1016/j.foodchem.2013.09.035 3. Serra AT, Rocha J, Sepodes B, Matias AA, Feliciano RP, de Carvalho A, Bronze MR, Duarte CM, Figueira ME (2012) Evaluation of cardiovascular protective effect of different apple varieties—correlation of response with composition. Food Chem 135:2378–2386. doi:10.1016/j.foodchem.2012.07.067 4. Zhao J, Wang J, Chen Y, Agarwal R (1999) Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-30 -gallate as the most effective antioxidant constituent. Carcinogenesis 20:1737– 1745 5. Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Pruess HG (2000) Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology 148:187–197 6. Moini H, Rimbach G, Packer L (2000) Molecular aspects of procyanidin biological activity: disease preventative and therapeutic potentials. Drug Metab Drug Interact 17:237–259 7. Terra X, Valls J, Vitrac X, Merrillon JM, Arola L, Ardevol A, Blade C, Fernandez-Larrea J, Pujadas G, Salvado J et al (2007) Grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway. J Agric Food Chem 55:4357–4365. doi:10.1021/jf0633185 8. Prasain JK, Peng N, Dai Y, Moore R, Arabshahi A, Wilson L, Barnes S, Michael Wyss J, Kim H, Watts RL (2009) Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape

Mol Cell Biochem

9.

10.

11. 12.

13. 14.

15.

16. 17.

18.

19.

20.

21.

22.

23.

24.

seed extract. Phytomedicine 16:233–243. doi:10.1016/j.phymed. 2008.08.006 Terra X, Palozza P, Fernandez-Larrea J, Ardevol A, Blade C, Pujadas G, Salvado J, Arola L, Blay MT (2011) Procyanidin dimer B1 and trimer C1 impair inflammatory response signalling in human monocytes. Free Radic Res 45:611–619. doi:10.3109/ 10715762.2011 Jung M, Triebel S, Anke T, Richling E, Erkel G (2009) Influence of apple polyphenols on inflammatory gene expression. Mol Nutr Food Res 53:1263–1280. doi:10.1002/mnfr.200800575 Brikos C, O’Neill LA (2008) Signalling of toll-like receptors. Handb Exp Pharmacol 183:21–50 Coll RC, O’Neill LA (2010) New insights into the regulation of signalling by toll-like receptors and nod-like receptors. J Innate Immun 2:406–421. doi:10.1159/000315469 Takeda K, Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17:1–14. doi:10.1093/intimm/dxh186 Medzhitov R, Preston-Hurlburt P, Janeway CA Jr (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394–397 Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M (1999) MD-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4. J Exp Med 189:1777–1782 Erridge C, Bennett-Guerrero E, Poxton IR (2002) Structure and function of lipopolysaccharides. Microbes Infect 4:837–851 Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700. doi:10.1146/annurev.biochem. 71.110601.135414 Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K (2002) Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 196:1645–1651 Kolek MJ, Carlquist JF, Muhlestein JB, Whiting BM, Horne BD, Bair TL, Anderson JL (2004) Toll-like receptor 4 gene Asp299Gly polymorphism is associated with reductions in vascular inflammation, angiographic coronary artery disease, and clinical diabetes. Am Heart J 148:1034–1040 Guo J, Friedman SL (2010) Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis. Fibrogenesis Tissue Repair 3:21. doi:10.1186/1755-1536-3-21 Iwami KI, Matsuguchi T, Masuda A, Kikuchi T, Musikacharoen T, Yoshikai Y (2000) Cutting edge: naturally occurring soluble form of mouse toll-like receptor 4 inhibits lipopolysaccharide signaling. J Immunol 165:6682–6686 Janssens S, Burns K, Tschopp J, Beyaert R (2002) Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88. Curr Biol 12:467–471 Ohta S, Bahrun U, Tanaka M, Kimoto M (2004) Identification of a novel isoform of MD-2 that downregulates lipopolysaccharide signaling. Biochem Biophys Res Commun 323:1103–1108 Palsson-McDermott EM, Doyle SL, McGettrick AF, Hardy M, Husebye H, Banahan K, Gong M, Golenbock D, Espevik T, O’Neill LA (2009) TAG, a splice variant of the adaptor TRAM,

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

negatively regulates the adaptor MyD88-independent TLR4 pathway. Nat Immunol 10:579–586. doi:10.1038/ni.1727 Hosoi S, Shimizu E, Arimori K, Okumura M, Hidaka M, Yamada M, Sakushima A (2008) Analysis of CYP3A inhibitory components of star fruit (Averrhoa carambola L.) using liquid chromatographymass spectrometry. J Nat Med 62:345–348. doi:10.1007/s11418008-0239-y Lima Mdos S, Silani Ide S, Toaldo IM, Correa LC, Biasoto AC, Pereira GE, Bordignon-Luiz MT, Ninow JL (2014) Phenolic compounds, organic acids and antioxidant activity of grape juices produced from new Brazilian varieties planted in the Northeast Region of Brazil. Food Chem 161:94–103. doi:10.1016/j.food chem.2014.03.109 Shimada T, Tokuhara D, Tsubata M, Kamiya T, Kamiya-Sameshima M, Nagamine R, Takagaki K, Sai Y, Miyamoto K, Aburada M (2012) Flavangenol (pine bark extract) and its major component procyanidin B1 enhance fatty acid oxidation in fatloaded models. Eur J Pharmacol 677:147–153. doi:10.1016/j. ejphar.2011.12.034 Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO (2007) Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071–1082. doi:10.1016/j.cell.2007.09.008 Ohto U, Fukase K, Miyake K, Satow Y (2007) Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316:1632–1634 Park BS, Song DH, Kim HM, Choi BS, Lee H, Lee JO (2009) The structural basis of lipopolysaccharide recognition by the TLR4MD-2 complex. Nature 458:1191–1195. doi:10.1038/nature07830 Sung NY, Yang MS, Song DS, Kim JK, Park JH, Song BS, Park SH, Lee JW, Park HJ, Kim JH et al (2013) Procyanidin dimer B2mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages. Biochem Biophys Res Commun 438:122–128. doi:10.1016/j.bbrc.2013.07.038 Chacon MR, Ceperuelo-Mallafre V, Maymo-Masip E, MateoSanz JM, Arola L, Guitierrez C, Fernandez-Real JM, Ardevol A, Simon I, Vendrell J (2009) Grape-seed procyanidins modulate inflammation on human differentiated adipocytes in vitro. Cytokine 47:137–142. doi:10.1016/j.cyto.2009.06.001 Gray P, Michelsen KS, Sirois CM, Lowe E, Shimada K, Crother TR, Chen S, Brikos C, Bulut Y, Latz E et al (2010) Identification of a novel human MD-2 splice variant that negatively regulates lipopolysaccharide-induced TLR4 signaling. J Immunol 184:6359–6366. doi:10.4049/jimmunol.0903543 Sung NY, Yang MS, Song DS, Byun EB, Kim JK, Park JH, Song BS, Lee JW, Park SH, Park HJ et al (2013) The procyanidin trimer C1 induces macrophage activation via NF-kB and MAPK pathways, leading to Th1 polarization in murine splenocytes. Eur J Pharmacol 714:218–228. doi:10.1016/j.ejphar.2013.02.059 Diya Z, Lili C, Shenglai L, Zhiyuan G, Jie Y (2008) Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS. Innate Immun 14:99–107. doi:10.1177/1753425907088244

123