Chaetoglobosin Fex from the Marine-Derived Endophytic Fungus ...

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Dec 8, 2011 - 40) Lien E., Chow J. C., Hawkins L. D., McGuinness P. D., Miyake K., Es- pevik T., Gusovsky F., Golenbock D. T., J. Biol. Chem., 276, 1873—.
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Biol. Pharm. Bull. 34(12) 1864—1873 (2011)

Regular Article

Vol. 34, No. 12

Chaetoglobosin Fex from the Marine-Derived Endophytic Fungus Inhibits Induction of Inflammatory Mediators via Toll-Like Receptor 4 Signaling in Macrophages Huan DOU,a Yuxian SONG,a Xianqin LIU,a Wei GONG,a Erguang LI,a Renxiang TAN,*,b and Yayi HOU*, a a

Immunology and Reproductive Biology Lab & Jiangsu Key Laboratory of Molecular Medicine, School of Medicine, Nanjing University; and b Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, School of Lifesciences, Nanjing University; Nanjing 210093, P. R. China. Received August 11, 2011; accepted September 26, 2011; published online September 29, 2011 Chaetoglobosin Fex (Cha Fex), a cytochalasan-based alkaloid, was isolated from marine-derived endophytic fungus Chaetomium globosum QEN-14. The knowledge of its biological function is still limited. We investigated the effects and mechanism of Cha Fex on inflammatory mediators via Toll-like receptor 4 (TLR4) signaling in macrophages. Lipopolysaccharide (LPS), TLR4 ligand, was therefore designed to active TLR4 signaling pathway, and Cha Fex significantly inhibited the LPS-induced production of tumor necrosis factor-alpha (TNF-a ), interleukin 6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in peritoneal macrophages and murine macrophage cell line RAW264.7. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detection also found that Cha Fex down-regulated the mRNA expressions of these pro-inflammtory cytokines. Moreover, Cha Fex significantly attenuated the LPS-stimulated degradation of inhibitory kappa B-alpha and the subsequent translocation of the p65 subunit of nuclear factor-kappa B (NF-k B) to the nucleus. Cha Fex also reduced the phosphorylations of extracellular-signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2). Furthermore, we confirmed that Cha Fex didn’t affect LPS binding to the RAW264.7 cells and human monocytes, while Cha Fex was able to inhibit the increase of membrane-associated CD14 (mCD14) expression both on RAW cells and human monocytes induced by LPS to a certain degree. These results suggest that the anti-inflammatory property of Cha Fex may be attributed to NF-k B inhibition as well as the negative regulation of ERK1/2, p38, and JNK1/2 phosphorylations. On the other hand, these inhibitory effects may also be due to the blocking of mCD14 expression. Key words Chaetoglobosin Fex; Toll-like receptor 4; lipopolysaccharide; nuclear factor-kappa B; mitogen-activated protein kinases; membrane-associated CD14

Studies have demonstrated that inflammatory response represents the “common soil” of the multifactorial diseases.1) Toll-like receptors (TLRs) are pattern-recognition receptors playing a key role in human and murine innate immunity,2,3) and their signaling have been strongly implicated in the inflammatory response. One of the TLRs, TLR4, recognizes lipopolysaccharide (LPS) by forming a receptor complex with myeloid differentiation protein-2 (MD2) and cluster of differentiation antigen 14 (CD14), the latter of which also has an important role in the recognition of LPS.4) LPS, a cell wall component of Gram-negative bacteria, is one of potent stimulators of the immune response on certain monocytes and macrophages.5) TLR4 signals through the shared adaptor proteins, myeloid differentiation factor88 (MyD88) and Tollinterleukin (IL)-1R domain-containing adaptor-inducing interferon (IFN)-b (TRIF) leading to activation of NF-k B, mitogen-activated protein kinases (MAPKs) and IFN-regulatory factor 3 (IRF3) pathway, and production of inflammatory mediators, including cytokines (TNF-a , IL-6, interleukin-1 beta (IL-1b )) and chemokines (MCP-1/CCL2, interferoninducible protein-10 (IP-10)/CXCL10).6) When in excess, LPS/TLR4 signaling leads to serious systemic disorders with a high mortality rate, for example, during sepsis.7) Thus, suppression and/or inhibition of the above-mentioned signaling molecules may have great potential for preventing and treating of inflammation-associated diseases. Cytochalasans are a large family of fungal polyketideamino acid hybrid metabolites with a wide range of biologi∗ To whom correspondence should be addressed.

cal functions. Some of them possess phytotoxic, cytotoxic, antimicrobial activities, and exhibit inhibition of cholesterol synthesis, or interference with glucose transport and hormone release.8) Chaetoglobosins are cytochalasans with the phenyl group in the molecules being replaced by an indolyl group and to date around 40 of which have been isolated and identified from the fungal genus Chaetomium.9—14) Thus the biological effects of Chaetoglobosins have been exploited,15—17) but the knowledge of their biological function are still scarce. Cha Fex, isolated from Chaetomium globosum QEN-14, an endophytic fungus derived from the marine green alga UlVa pertusa, was investigated for the anti-inflammatory effects and mechanisms for its potential therapeutic exploitation. Thus in the present study, to explore the molecular mechanism underlying the repression effect of Cha Fex, we used LPS to stimulate macrophages and found for the first time that Cha Fex inhibited significantly LPS-induced inflammatory mediators production both in RAW264.7 cell line and mouse peritoneal macrophages. And its anti-inflammatory property was exibited by inhibiting NF-k B and MAPKs activation in LPS-stimulated macrophages, and blocking membrane-associated CD14 (mCD14) expession. MATERIALS AND METHODS Reagents LPS (Escherichia coli 0111: B4), fluorescein isothiocyanate-conjugated LPS (FITC-LPS) (from E. coli

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© 2011 Pharmaceutical Society of Japan

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0111:B4), dexamethasone (Dex), paraformaldehyde (PFA), dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich (St. Louis, MO, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), penicillin G, streptomycin and fetal bovine serum (FBS) were purchased from Gibco Inc. (Grand Island, NY, U.S.A.). Enzyme-linked immunosorbent assay (ELISA) MaxTM Sets were obtained from BioLegend (San Diego, CA, U.S.A.). Anti-mouse CD14-PE, anti-human CD14-APC antibodies were ordered from eBioscience (San Diego, CA, U.S.A.). Anti-phospho-c-Jun N-terminal kinase (JNK), anti-phospho-p38, anti-phospho-extracellular-signalregulated kinase (ERK)1/2, anti-inhibitory (I)k Ba , anti-NFk B p65, and anti-b -tubulin were all purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). All electrophoresis chemicals were purchased from Bio-Rad Labs (Hercules, CA, U.S.A.). Cell counting kit-8 (CCK-8) was purchased from DojinDo Laboratories (Kyushu, Japan). Preparation of Cha Fex The culture broth of a marine derived fungus named Chaetomium globosum QEN-14 was extracted three times with EtOAc to give an extract, while mycelia were homogenized and extracted three times with MeOH to give another extract. Then the two extracts were combined together, and they were subjected to column chromatography over silica gel eluting with different solvents of increasing polarity (from petroleum ether to MeOH) to yield 14 fractions on the basis of TLC analysis. The CHCl3– MeOH solvent partition fraction was further separated by CC on Sephadex LH-20 (CHCl3–MeOH, 1 : 1) and on reversedphase silica gel C18 (MeOH–H2O, 4 : 1) to afford a compound identified as Cha Fex, whose stereostructure (Fig. 1) was identified on the basis of high resolution-electrospray ionization (HR-ESI)-MS and NMR data by comparison with literature values.18) The purity of Cha Fex was 98% by HPLC analysis. Cells and Cell Culture Conditions The murine macrophage cell line RAW264.7 was purchased from the China Cell Bank of SIBS (Shanghai, China). RAW264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS. The cell culture medium all contained penicillin G (100 U/ml) and streptomycin (100 m g/ml). Cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C and subcultured every 2 d. In all experiments, cells were grown to 80—90% confluence and subjected to no more than 20 cell passages. For stimulation experiments, different cells including RAW264.7 and thioglycolate-elicited peritoneal macrophages (1106 cells/ml)19) were treated with indicated concentration of Cha Fex for 1—2 h and LPS for 1—20 h as indicated in the figure legends. All cell culture media and all other reagents were entirely free of endotoxin, as checked by Tachypleus amebocyte

Fig. 1. Stereostructure of Chaetoglobosin Fex (Cha Fex)

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lysate assay (Xiamen Houshiji, Ltd., Fujian, China). Cell Viability The cytotoxicity of Cha Fex was evaluated using the CCK-8. In brief, 5103 cells per well were plated in a 96-well plate and incubated at 37 °C for 18 h. Cells were then treated with the indicated concentrations of Cha Fex and incubated at 37 °C for an additional 24 h. Ten ml of the CCK-8 solution (contains WST-8 [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt]) was added to each well, and cells were incubated for 3 h. The absorbance was measured using microplate reader (Synergy HT, Bio-Tek) at 450/650 nm as measure reference wavelength respectively. The average optical density formed in control cells was taken as 100% viability, and the results of treatments were expressed as a percentage of the control. Determination of Cytokine Concentrations Release of cytokines (tumor necrosis factor (TNF)-a , IL-6 and MCP-1) was measured in the culture supernatants by ELISA using Mouse TNF-a /IL-6/MCP-1 ELISA MAXTM Standard Sets, according to the manufacturer’s instructions. Briefly, 96-microwell plates were coated with anti-mouse cytokine antibody in coating buffer (pH 9.5) and were incubated overnight at 4 °C. Plates were then washed by 0.05% Tween-20/phosphate-buffered saline (PBS) and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. After additional washes, samples were added and incubated at 4 °C overnight. Wells were then washed and the detection antibody (biotinylated mouse cytokine monoclonal antibody) and the enzyme reagent (avidin-horseradish peroxidase conjugate) diluted with assay diluent (PBS supplemented with 1% BSA) were added and incubated at room temperature for 30 min with shaking. After intensive washing, TMB substrate buffer was added to start the color-developing reaction. The reaction was terminated by reaction stop solution and the absorbance was measured at 450 nm using an automated microplate reader. A standard curve was run on each assay plate using cytokine in serial dilutions. The lower detection limits using standard curves were 7.8 pg/ml for TNF-a and IL-6, and 62.5 pg/ml for MCP-1, respectively. RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Total RNA was isolated from 1106 cells treated with LPS, LPSCha Fex at indicated concentrations, LPSDex and untreated RAW264.7 cells by Trizol method. The RNA quantitation was done spectrophotometrically at 260 nm. The integrity and purity of the RNA samples were checked by agarose gel electrophoresis. mRNA levels were measured by real-time quantitative PCR after reverse transcription of RNA. The first-strand cDNA (cDNA) was synthesized from 1 m g total RNA by using oligo dT primers and M-MLV reverse transcriptase in a total reaction volume of 20 m l. Quantitative reverse transcription (qRT-PCR) was performed in triplicate in 96-well plates, using the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.). The sequences of the primers are shown in Table 1. PCR reactions were carried out in a 20 m l reaction volume containing 10 m l of FastStart Universal SYBR Green Master (Rox) (Roche, Mannheim, Germany), 1 m l of 500 nM forward and reverse primer, and 1 m l of cDNA template. The following cycle parameters were used: 55 °C for 2 min, 95 °C for 10 min, and then 40 cycles of 95 °C for 30 s, 60 °C for 30 s. The relative expression levels of the tar-

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Table 1. Primers Used for Real-Time RT-PCR Studies in RAW264.7 Cells Primer TNF-a F TNF-a R IL-6 F IL-6 R MCP-1 F MCP-1 R GAPDH F GAPDH R

Sequence 5-CCCTCACACTCAGATCATCTTCT-3 5-GCTACGACGTGGGCTACAG-3 5-TAGTCCTTCCTACCCCAATTTCC-3 5-TTGGTCCTTAGCCACTCCTTC-3 5-TTAAAAACCTGGATCGGAACCAA-3 5-GCATTAGCTTCAGATTTACGGGT-3 5-GGTGAAGGTCGGTGTGAACG-3 5-CTCGCTCCTGGAAGATGGTG-3

get genes (TNF-a , IL-6 and MCP-1) against that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was defined as DCT(CTTargetCTGAPDH). The target mRNA/ GAPDH mRNA ratio was calculated as 2DCT according to the manufacture’s specification. Western Blotting Cells were rinsed twice with ice-cold PBS, and solubilized in lysis buffer containing 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% (m/v) NP-40, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM NaF and 1.0 mM dithiothreitol (DTT) for 30 min on ice. Lysates were centrifuged (15000 rpm) at 4 °C for 10 min. Protein concentrations were measured using a Bradford assay kit. Equal amounts of the soluble protein were denatured in sodium dodecyl sulfate (SDS), electrophoresed on a 10% SDS-polyacrylamide gel, and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then blocked for non-specific binding in blocking buffer (5% bovine serum albumin (BSA) in TBST) for 1 h and then washed with TBST 5 times. Subsequently, the membranes were incubated with antibodies at 1 : 1000 dilutions in antibody dilution buffer (5% BSA in TBST) with gentle shaking at 4 °C overnight and then washed with TBST. After washing, the membranes were exposed to HRP-conjugated secondary antibody at 1 : 3000 dilutions in antibody dilution buffer for 1 h at the room temperature and then washed again. After six washes with TBST, protein bands were visualized with enhanced chemiluminescence (ECL) Western blotting detection reagents. The ECL image was recorded using the FluorChem Xplor (Alpha Innotech, U.S.A.), and the optical density of an equal surface area for each band was determined using Image J software. All blots were stripped and reprobed with polyclonal anti-b -Tubulin antibody to as certain equal loading of proteins. Immuno-staining for Translocation of p65 RAW 264.7 cells (2105 cells/well) were cultured in 6-well plates containing a cover glass for 24 h at 37 °C. Cells were washed with PBS, replaced with fresh media, and then treated with the Cha Fex (2 m g/ml) for 1 h. Following stimulated with LPS (100 ng/ml) for 1 h, cells were fixed with 4% PFA for 30 min and permeabilized with 0.1% Triton X-100 for 15 min. After three washes with PBS, 3% PBS-BSA was then added to block non-specific binding for 1 h. After washing, p65 monoclonal antibody was diluted in PBS-BSA solution at 1 : 200 dilutions with gentle shaking at 4 °C overnight, and then cells were washed with PBS for 5 min three times. Subsequent antibody, Texas red conjugated anti-rabbit immunoglobulin G (IgG) was used against p65 antibody diluted 1 : 200 in PBSBSA, and the cells were incubated in the dark for 1 h at the

room temperature. Finally, cell nuclei were labeled with 4,6diamidino-2-phenylindole (DAPI; Nakalai Tesque, Kyoto, Japan) for 10 min. Cells were then washed and mounted with antifade mounting medium. Mounted slides were viewed under a confocal laser scanning microscope (LSM510 Axiovert; Zeiss) using a 60 oil immersion objective. The excitation wavelengths for DAPI and p65 were 350 and 596 nm, respectively. Measurement of Cha Fex Binding of FITC-LPS to Human Monocytes or Murine Macrophage Cell Line The binding of LPS and human monocyte or murine macrophage cell line (RAW264.7) assay were determined by flow cytometry (FCM), respectively. 1) Blood samples were obtained from healthy volunteers. After discarding the first 2 ml, the subsequent whole blood was collected in heparinized tubes. Immediately after sampling, 100 m l whole blood was divided into each group. Then the LPS group was treated with 4 m g/ml FITC-LPS (final concentration) and Cha Fex groups were done with the FITC-LPS which had been pre-incubated with 0.5—2 m g/ml Cha Fex (final concentration) for 15 min. These groups were incubated at room temperature in the dark for 30 min. In addition, the blood without adding anything as autofluorescence control was run in parallel. After lysis of erythrocytes, samples were measured with FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, U.S.A.). A set of 10000 gated cells was analyzed for their mean fluorescence intensity (MFI). 2) Five hundred microliters of RAW264.7 cell suspension was incubated with FITC-LPS (2—10 m g/ml) and Cha Fex (0.5—2 m g/ml) or PBS as control. The cells were held at 0 °C for 1 h, washed twice with ice-cold PBS, and then fixed with 1% PFA until flow cytometry was performed within 24 h. The number of cells stained with FITCLPS was determined by FCM. Specific LPS binding was estimated by subtracting the MFI of LPS-binding cells in the absence of LPS from that in the presence of LPS. The average MFI formed in LPS group was taken as 100% bingding rate, and the results of treatments were expressed as a percentage of the LPS group. CD14 Expressing Assay 1) The expression of mCD14 in RAW264.7 cells was determined in the same treated and untreated cultures. After 100 ng/ml LPS treated for 24 h, the cells were washed with PBS, scraped off the bottom of the wells, and collected by centrifugation. Then, cells from triplicate cultures were washed in PBS, and centrifuged for 5 min at 1300 rpm. Next, the cells were resuspended in PBS and stained with anti-mouse CD14-PE, following the manufacturer’s instructions. Cell-associated fluorescence was analyzed by a FACSCalibur flow cytometer supplied with CellQuest software. For each sample 10000 cells were acquired. Antibody binding was shown as MFI. 2) CD14 expressing assay on human monocytes was conducted according to previous protocol.20) Immediately after sampling, blood was treated with Cha Fex 0.5—2 m g/ml and/or LPS (final LPS concentration 50 ng/ml) for 60 min at 37 °C. Then samples were washed 3 times in PBS. For analysis of CD14 expression, all above blood samples were incubated with APC-labeled anti-human CD14 at 4 °C in the dark for 30 min. At the same time, autofluorescence and isotype controls were run in parallel. For lysis of erythrocytes, 1 ml of lysis buffer was applied for about 10 min at room temperature in the dark. The

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samples were then centrifuged at 1500 rpm for 5 min at 4 °C in PBS, and prepared for flow cytometric analysis. Monocytes were gated based on their forward- and side-scatter properties. The expression of each surface CD14 of all cells were shown as MFI. Statistical Analysis All results were were analyzed with Prism 4 (GraphPad Software, Inc., San Diego, CA, U.S.A.) and expressed as meansS.D. Student’s t-test was used to compare between two groups (e.g., treated and control). Oneway analysis of variance (ANOVA) was used to compare among 3 or more groups. Differences with p 0.05 were considered significant. RESULTS Cytotoxicity Studies of Cha Fex on Macrophages Macrophage is one of the key players in the early innate immune response, and releases inflammatory cytokines when activated. Thus we studied the effect of Cha Fex on the viability of peritoneal macrophages by using the CCK-8 assay. After exposure to 0.25, 0.5, 1, 2, 4 or 8 m g/ml Cha Fex for 24 h, the viabilities of cells were 96.232.40, 91.445.00, 97.638.50, 96.882.01, 92.391.24 and 91.791.55%, respectively, compared with the mean of 0.05% DMSO group (control 1003.38%). These results confirmed that up to a concentration of 8 m g/ml did not affect the cell viability at 24 h of incubation (Fig. 2). Concentrations of Cha Fex below the threshold were selected for further studies. Cha Fex Supresses Cytokine and Chemokine Production from LPS-Stimulated Macrophages While TLR4 is expressed on a variety of hematopoietic and non-hematopoietic cells, macrophages have been frequently studied as physiologically relevant LPS-sensitive cells.21,22) The hallmark of macrophage activation through TLR4 induced by LPS is the production of TNF-a , IL-6, MCP-1 and other cytokines and chemokines. To determine the activity of Cha Fex, we first exposed the peritoneal macrophages from three mice with Cha Fex for 2 h, and then stimulated with LPS (100 ng/ml). Dexamethasone (Dex) was used in this experiment as a positive control. Figure 3A shows that stimulation of peritoneal macrophages with LPS (100 ng/ml) for 20 h induced the production of TNF-a . Preincubation of these cells with Cha Fex ( 0.5 m g/ml) significantly inhibited LPS-induced intracellu-

Fig. 2. Effect of the Cha Fex on Cell Viability Cells were treated with different concentrations of Cha Fex for 24 h, and their viability were determined with the CCK-8 assay. Results of independent experiments were averaged as percentage cell viability, compared with control. Data are presented as meansS.E.M. of triple determinations.

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lar TNF-a production (15.2% inhibition by 0.5 m g/ml, 21.3% inhibition by 1 m g/ml and 56.7% inhibition by 2 m g/ml). And the treatment with different concentration of Cha Fex also blocked IL-6 secretion induced by LPS for 20 h. Figure 3A also illustrates the same inhibitory effect of Cha Fex on MCP-1 production. We also confirmed in the murine macrophage cell line, RAW264.7, to various concentrations of this compound and measured TNF-a , IL-6 and MCP-1 secretion by ELISA. Analysis (Fig. 3B) showed that RAW264.7 cells unstimulated by LPS synthetised basal level of TNF-a , while the stimulation of LPS without Cha Fex resulted in an increase in TNFa production. Differrent concentrations of Cha Fex all significantly inhibited the production of LPS-induced TNF-a . At the same time, we found Cha Fex inhibitory effcect on IL-6 production. As shown in Fig. 3B, when stimulated with LPS (100 ng/ml) alone for 20 h, macrophages produced IL-6 by 907.3 pg/ml in the culture medium. However, Cha Fex significantly inhibited the production of LPS-induced IL-6 by 30.9% at 0.5 m g/ml, 37.1% at 1 m g/ml and 50.1% at 2 m g/ml. And MCP-1 Elisa detection showed the preincubation of these cells with Cha Fex (0.5 m g/ml) had the same inhibitory effect. Cha Fex Inhibits TNF-a , IL-6 and MCP-1 mRNA Expressions in Murine Macrophages Because Cha Fex inhibited TNF-a production in RAW264.7 cells, as well as IL6 and MCP-1 secretion in RAW264.7 cells, quantitative realtime PCR was used to assess the effects of Cha Fex on LPSinduced TNF-a , IL-6 and MCP-1 gene expression in RAW264.7 cells. Upon LPS treatment, the mRNA expressions of these three genes were markedly augmented. The concentration response for inhibition of TNF-a , IL-6 and MCP-1 mRNA expressions is shown in Fig. 4. Cha Fex down-regulated TNF-a , IL-6 and MCP-1 mRNA significantly, indicating that Cha Fex could attenuate LPS-induced TNF-a , IL-6 and MCP-1 expression at the transcriptional level. Cha Fex Inhibits the Degradation of Ik Ba and the Nuclear Translocation of p65 Subunit of NF-k B In order to further investigate the inhibitory effect of Cha Fex upon LPS-mediated signaling related to pro-infammatory cytokines gene expression, we examined whether Cha Fex pretreatment of RAW264.7 cells alters the LPS-mediated downstream activation of NF-k B. Ik B degradation unmasks the nuclear localization signal motif of NF-k B, which allows the transcription factor to move into the nucleus. Here, we demonstrated that 100 ng/ml LPS rapidly induced the degradation of Ik Ba within 15 min compared to b -Tubulin (Fig. 5A), and Ik Ba returned to the basal level at around 60 min subsequent to LPS stimulation (Fig. 5A). By contrast, pre-exposure of RAW264.7 cells to Cha Fex resulted in the rapid inhibitory effect of the LPS-mediated degradation of Ik Ba at 30 min (Fig. 5B). Furthermore, we also examined whether Cha Fex inhibits nuclear translocation of p65 into the nucleus by immuno-staining, since p65 is the major component of NF-k B activated by LPS in macrophages. p65 translocated into the nucleus in response to LPS was markedly increased upon exposure to LPS alone, but pretreatment of Cha Fex reduced LPS-mediated changes of p65 into the nucleus (Fig. 5C). These results suggested that Cha Fex blocks LPS-induced NF-k B activation by suppression of Ik Ba degradation

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Fig. 3. Inhibitory Effect of Cha Fex on Cytokines and Chemokines Production from Macrophages Stimulated with LPS (A) Thioglycolate-elicited peritoneal macrophages and (B) RAW264.7 cells were stimulated with LPS (100 ng/ml) in the presence or absence of Cha Fex (0.5,1,2 m g/ml), the levels of TNF-a , IL-6 and MCP-1 in culture supernatants were detected by ELISA. Data are presented as meanS.E.M. of three independent experiments. # p 0.05, LPS group significantly different from control, ∗ p 0.05, significantly different from LPS group. Dex: dexamethasone, positive control.

and NF-k B subunit nucleus shift. Cha Fex Pretreatment Down-Regulates LPS-Induced the Activation of MAPKs in RAW264.7 Cells The three families of MAPKs, ERK, JNK, and p38, have all been shown to be activated in response to LPS in RAW264.7 cells. To determine the intracellular mechanism of Cha Fex, we investigated the activation of MAPKs involved in the TLR4signaling pathway using the RAW264.7 cell line. The JNK, ERK1/2 and p38 activation states were determined by immunoblot analysis using antibodies specifically directed against phosphorylated forms of the enzymes, compared to

data obtained with antibodies directed against total forms. LPS (100 ng/ml) markedly induced the activation of all three MAPKs in RAW264.7 cells (Fig. 6A). In the first instance, the phosphorylation level of ERK1/2 reached a peak at around 15 min subsequent to LPS stimulation, and returned to the basal level at around 60 min subsequent to LPS stimulation. The presence of phosphorylated JNK at the maximal level above baseline was detected at 15 min subsequent to LPS stimulation, and the level reduced significantly 60 min subsequent to LPS stimulation. The phosphorylation level of p38 quickly reached a significant level at around 15 min sub-

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Fig. 4. Inhibitory Effect of Cha Fex on TNF-a , IL-6 and MCP-1 mRNA Expressions RAW264.7 cells were stimulated with LPS (100 ng/ml) for 6 h in the presence or absence of Cha Fex (0.5, 1, 2 m g/ml). Expression levels of TNF-a , IL-6 and MCP-1 mRNA were determined by real-time quantitative PCR and expressed as relative fold expression to control. Data are shown as meanS.E.M. of three independent experiments. # p 0.05, LPS group significantly different from control, ∗ p 0.05, significantly different from LPS group.

sequent to LPS stimulation, and maintained the level till 120 min subsequent to LPS stimulation. However, pre-exposure of RAW264.7 cells to Cha Fex (0.5 or 2 m g/ml) resulted in the rapid suppress of the LPS-mediated activation of ERK1/2, JNK, and p38 (Fig. 6B). Cha Fex Does Not Influence LPS Binding Ability The results described above suggest that Cha Fex might target an upstream event in LPS signaling or inhibit LPS binding to the cells. It is known that LPS binds to CD14/TLR4/MD-2 complex on host cells such as monocytes and macrophages.23—25) We examined whether Cha Fex inhibit the interaction between LPS and RAW264.7 cells using flow cytometry technology. Polymyxin B (PMB) was used in this experiment to use as a positive control. The cells were incubated with fluorescein-conjugated LPS, and the LPS binding was analyzed by flow cytometry. Incubation of RAW cells with PMB resulted in complete inhibition of the binding of LPS to cells; in contrast to PMB, Cha Fex did not block the binding of LPS to RAW264.7 even at the highest concentration (Fig. 7). However, Cha Fex at a concentration of 0.5 m g/ml inhibited the production of TNF-a and IL-6 from RAW264.7 stimulated under conditions similar to those of the LPS binding assay. These results suggest that Cha Fex inhibits cytokine production without antagonizing the binding of LPS to CD14/TLR4/MD-2 complex. Next we confirmed this result on human monocytes. The MFI of FITC-LPS group was 160.672.52, higher than that of Contorol group (139.677.51), p 0.05 (Table 2). When blood samples were treated with different concentrations of Cha Fex (0.5, 2 m g/ml) and FITC-LPS, MFI of these groups showed no significant difference from FITC-LPS group. At the same time, addition of the LPS inhibitor PMB could efficiently blocked LPS binding response. All these results showed that LPS could bind RAW cells and the binding abil-

Table 2. Effect of Cha Fex on Binding of LPS to Human Monocytes (n6, xS.D.) Group

MFI

Control FITC-LPS FITC-LPSCha Fex-0.5 m g/ml FITC-LPSCha Fex-2 m g/ml FITC-LPSPMB

139.677.51 160.672.52# 156.670.58# 163.338.14# 137.334.72*

# p 0.05 vs. Control group; ∗ p 0.05 vs. FITC-LPS group. MFI: mean fluorescence intensity, LPS: inhibitor.

ity of LPS pretreated with Cha Fex was not significantly different, suggesting that Cha Fex could not inhibit the bioactivity of LPS and not affect the binding of LPS with cells. Cha Fex Decreases the Expression of mCD14 Induced by LPS mCD14 is important for transducing LPS-mediated signals via coordination between TLR4. We used flow cytometry to determine whether Cha Fex attenuates pro-inflammatory mediators production by blocking the mCD14 expression induced by LPS. First, the surface expression of CD14 in RAW cells was significantly modified by LPS alone (Fig. 8A). Compared to untreated controls, the expression of CD14 was up-regulated by LPS about 3-fold after a 24 h exposure period, and the increasing expression level was downregulated by the pretreatment with Cha Fax ( 0.5 m g/ml) (Fig. 8A). Similar effects were detected on human monocytes. The MFI of CD14 on human monocytes stimulated with 50 ng/ml LPS was 116.2513.79, significantly higher than Control group, showed that LPS could upregulate the expression of CD14 on the surface of monocytes. The MFI of LPSCha Fex-0.5/2 groups were 73.334.04 and 715.20, significantly lower than that of LPS group, and showed no significant difference between that of Control

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Fig. 5. Cha Fex Pretreatment Blocks LPS-Induced Ik Ba Degradation and Inhibits the Nuclear Translocation of NF-k B in LPS-Stimulated RAW264.7 Cells (A) RAW264.7 cells were treated with LPS (100 ng/ml) alone for the indicated time points. Levels of Ik Ba were analyzed by Western blotting. One of three experiments is presented. (B) RAW264.7 cells were pretreated for 1 h with Cha Fex (0.5 or 2 m g/ml) or medium and stimulated with LPS (100 ng/ml) for the 30 min. Levels of Ik Ba were analyzed by Western blotting. One of three experiments is presented. (C) Cells were treated with the Cha Fex for 1 h and then stimulated with LPS for 1 h. The cellular localization of NF-k B was determined using immunofluorescence analysis. After fixation, cells were stained with anti-p65 antibody, followed by Texas-red conjugated antibody. Nuclei of the corresponding cells were visualized with DAPI, and observed at 600 magnification. Control: untreated cells, LPS: LPS only (100 ng/ml), LPSCha Fex: Cha Fex (2 m g/ml) and LPS.

group (61.6710.50) and Cha Fex group (60.331.15), all with p 0.05. Therefore, Cha Fex could inhibit the increase of CD14 expression on the surface of monocytes induced by LPS completely (Fig. 8B). DISCUSSION Inflammation is a host response to external/internal challenge that leads to the release of a large amount of inflammatory mediators. Excess production of these inflammatory mediators is involved in many diseases including rheumatoid arthritis, atherosclerosis, asthma, pulmonary fibrosis and

cancer.26—30) The macrophage-mediated inflammatory response is a key etiologic component of the inflammatory diseases. TLR4, one of the gateways to inflammatory signaling in the macrophage, can start the inflammation response, thus has sparked great interest in therapeutic manipulation.31) In an effort to search for novel TLR4 signaling regulators from endophytic metabolites, more than 100 natural products were tested for their ability to reduce inflammatory mediators’ production from LPS-stimulated RAW264.7 macrophages. Although Cha Fex was isolated previously, to the best of our knowledge this is the first time this compound was shown to reduce inflammatory mediators production.

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Fig. 6. Cha Fex Pretreatment Down-Regulates Activation of MAPKs (A) RAW264.7 cells were treated with LPS (100 ng/ml) alone for the indicated time points. Phosphorylation and total level of ERK, JNK, and p38 were analyzed by Western blotting. One of three experiments is presented. (B) RAW264.7 cells were pretreated for 1 h with Cha Fex (0.5 or 2 m g/ml) or medium and stimulated with LPS (100 ng/ml) for the 30 min. Phosphorylation and total level of ERK, JNK, and p38 were analyzed by Western blotting. One of three experiments is presented.

Fig. 7. Effect of Cha Fex on Binding of LPS to RAW264.7 RAW264.7 cells were pretreated with 0.5 or 2 m g/ml Cha Fex (Cha Fex-0.5: Cha Fex 0.5 m g/ml, Cha Fex-2: Cha Fex 2 m g/ml) for 1 h and further incubated with 4 m g/ml FITC-conjugated LPS for 1 h at 37 °C. Samples were measured by flow cytometry. The results were expressed as meanS.E.M. of three independent experiments. ∗ p 0.05, significantly different from LPS group. PMB: LPS inhibitor as a positive control.

In this study, we selected Cha Fex to evaluate its pharmacological effects on the production of inflammatory mediators in LPS-induced macrophages. Cytokines, such as TNFa , IL-1b , IL-6, IL-12, and chemokines, such as MCP-1, MIP-1, were all shown to be up-regulated by LPS treatment.32,33) Amongst the cytokines notably produced by LPS stimulation in this study, Cha Fex significantly reduced the production of TNF-a , IL-6 and MCP-1 in primary mouse peritoneal macrophages, without any cytotoxic effect on these cells. We then obtained similar results in macrophage cell line RAW264.7. Furthermore, Cha Fex suppressed TNFa , IL-6 and MCP-1 at transcription level. These data suggested that Cha Fex could show suppressive effects on the

production of various types of inflammatory mediators, including those examined in this study. To further elucidate the molecular mechanism of this activity, the activations of MAPKs and the transcriptional factor NF-k B were investigated. Based on LPS/TLR4 signaling, TNF-a and IL-6 are thought to be MyD88-dependent cytokines, while MCP-1 are TRIF-dependent mediators.32) In the last, expression of these inflammatory mediators is all NF-k B and MAPKs dependent. NF-k B is a dimeric transcription factor formed by the hetero or homodimerization of proteins in the Rel family, including p50 and p65.34) The activation of NF-k B converges at the inhibitor of kappa B kinase (IKK) complex35); IKK activation initiates Ik Ba phosphorylation at specific amino-terminal serine residues, and then phosphorylation Ik Ba ubiquitinated; this ubiquitination targets it for degradation, thereby releasing NF-k B dimers from the cytoplasmic NF-k B-Ik Ba complex and allowing them to translocate to the nucleus, binding DNA recognition sites in regulatory regions of target genes.36) Phosphorylation of p38 MAPK, ERK1/2, and JNK induced by LPS leads to the regulation of TNF-a and IL-6.37,38) Our study indicated that Cha Fex inhibited the nuclear translocation of p65 protein via suppressing Ik Ba degradation, which provides strong evidence that Cha Fex inhibited the activation of NF-k B. Meanwhile, Cha Fex had the ability to block MAPKs activation. These might also explain that mRNA levels of TNF-a and MCP-1 were strongly inhibited by Cha Fex. After we examined the inflammatory mediator expression of extracellular and intracellular signaling molecules involved in the TLR4-mediated signaling pathways, we detected the TLR4 signal triggering on the membrane. Several compounds that targeting the upstream of TLR4 signal have been previously reported. The synthetic analogue of bacterial

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Fig. 8. Effect of Cha Fex on the Expression of CD14 Induced by LPS (A) RAW264.7 cells and (B) human mooncytes were divided to six groups: Control group, Cha Fex group, LPS group, LPSCha Fex-0.5 group (Cha Fex-0.5: Cha Fex 0.5 m g/ml), LPSCha Fex-2 group (Cha Fex-2: Cha Fex 2 m g/ml), LPSPMB group. Samples were measured by flow cytometry. The results were expressed as MFIS.E.M. of one of three representative experiments. # p 0.05, LPS group significantly different from control. ∗ p 0.05, significantly different from LPS group.

lipid A E5531 and a cyclic cationic polypeptide antibiotic, PMB inhibit TLR signaling via interactions with LPS.39,40) Using flow cytometric technique, we confirmed that Cha Fex didn’t affect LPS binding to the RAW264.7 cells. Then we confirmed this effect on human monocytes ex vivo. In addition to TLR4, MD-2 and serum lipopolysaccharide binding protein (LBP), mCD14, a glycerophosphatidylinositol-anchored macrophage/monocyte surface protein, is important in LPS-induced innate immunity.41,42) Emerging evidences indicate that upstream inhibition of bacterial LPS/TLR4/ CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation.43,44) Cha Fex was able to inhibit the increase of CD14 expression both on RAW264.7 cells and human monocytes induced by LPS to a certain degree. This effect may lead to the inhibition of TNF-a , IL-6 and MCP-1 production from LPS-stimulated macrophages. In summary, the findings of the present study suggest that Cha Fex is a potent inhibitor of LPS-induced TNF-a , IL-6 and MCP-1 production in macrophage cells, and that it produces these effects by acting at the level of transcription. Moreover, the inhibitory effects of Cha Fex were found to be associated with an inactivation of NF-k B and MAPKs that resulted from the degration of Ik Ba , and a blockade JNK, ERK, and p38 phosphorylation. At the same time, these inhibitory effects were due to the blocking of mCD14 expression. Since TLR4 singnalling is associated with inflammation, its inhibition by Cha Fex offers a possible approach to the treatment of severe inflammatory diseases. Acknowledgments This work was supported by the National Natural Science Foundation of China (90813036, 81072410), the Scientific Rearch Foundation of Graduate School of Jiangsu Province (CXZZ11_0036), and the Scientific Rearch Foundation of Graduate School of Nanjing University (2010CL04). REFERENCES 1) Scrivo R., Vasile M., Bartosiewicz I., Valesini G., Autoimmun. Rev., 10, 369—374 (2011).

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