IKKβ-Targeted Anti-Inflammatory Activities of a Butanol Fraction of Artificially Cultivated Cordyceps pruinosa Fruit Bodies

The inhibitory activities of the Cordyceps pruinosa butanol fraction (Cp-BF) were investigated by determining inflammatory responses of lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells and by evaluating HCl/ethanol (EtOH)-triggered gastric ulcers in mice. The molecular mechanisms of the inhibitory effects of Cp-BF were investigated by identifying target enzymes using biochemical and molecular biological approaches. Cp-BF strongly inhibited the production of NO and TNF-α, release of reactive oxygen species (ROS), phagocytic uptake of FITC-dextran, and mRNA expression levels of interleukin (IL)-6, inducible NO synthase (iNOS), and tumour necrosis factor-alpha (TNF)-α in activated RAW264.7 cells. Cp-BF also strongly downregulated the NF-κB pathway by suppressing IKKβ according to luciferase reporter assays and immunoblot analysis. Furthermore, Cp-BF blocked both increased levels of NF-κB-mediated luciferase activities and phosphorylation of p65/p50 observed by IKKβ overexpression. Finally, orally administered Cp-BF was found to attenuate gastric ulcer and block the phosphorylation of IκBα induced by HCl/EtOH. Therefore, these results suggest that the anti-inflammatory activity of Cp-BF may be mediated by suppression of IKKα and its downstream NF-κB activation. Since our group has established the mass cultivation conditions by developing culture conditions for Cordyceps pruinosa, the information presented in this study may be useful for developing new anti-inflammatory agents.


Introduction
Inflammation is a complicated defensive response against various infecting pathogens and toxins and serves as one of the body's innate immunity barriers. As a major inflammatory cell population, macrophages respond to inflammatory events by phagocytosing infected materials and producing cytokines such as interleukin (IL)-1 and tumour necrosis factor (TNF)-, as well as inflammatory mediators such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ). For these responses, macrophages need to activate cell surface receptors (e.g., toll-like receptors) by interacting with their ligands, such as lipopolysaccharide (LPS) from Gram (−) bacteria. Extracellular signals received by plasma membrane receptors 2 Evidence-Based Complementary and Alternative Medicine will then generate intracellular signalling events through nonreceptor type protein tyrosine kinases, including Syk, Src, phosphoinositide-3-kinase (PI3K), and mitogen activated protein kinase (MAPK), upstream of extracellular signalrelated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Ultimately, all of these intracellular responses reach activation of transcription factors including CREB, nuclear factor-B (NF-B), and activator protein-1 (AP-1) [1,2]. The outcomes of such activation are linked to the expression of various proinflammatory genes and inflammatory mediators [3][4][5]. In addition, recent findings have raised the possibility that sustained inflammatory responses can cause serious diseases such as septic shock, cancer, diabetes, gastritis, and atherosclerosis [5][6][7]. Therefore, development of stronger and safer anti-inflammatory remedies may contribute to more promising strategies to effectively treat inflammation-derived diseases.
The genus Cordyceps, which includes Cordyceps sinensis, Cordyceps militaris, Cordyceps pruinosa, and Cordyceps bassiana, comprises ethnopharmacologically valuable mushrooms in Korea, China, and Japan [8]. The genus Cordyceps has been traditionally used for the treatment of various inflammatory and infectious diseases, such as eczema, skin diseases, chronic bronchitis, asthma, and tuberculosis, or used as a tonic for longevity, endurance, and vitality [8,9]. The mushroom Cordyceps was also described in ancient traditional Chinese medicine literatures as a medicinal ingredient prescribed for relieving chronic bronchitis, chronic obstructive pulmonary disease, and tuberculosis [10,11]. Additional studies have found that these mushrooms have numerous biological activities based on antioxidative, anticancer, antidiabetes, antibacterial, antifungal, and antifatigue properties [12,13]. From phytochemical approaches, it has been reported that cordycepin, ergosterol peroxide, and polysaccharides are the major components responsible for the various pharmacological activities of Cordyceps [14]. However, in spite of their excellent pharmaceutical and nutraceutical potential, it is difficult to mass produce these mushrooms. To overcome the limitations of mass production of Cordyceps species, our group has developed an artificial culturing condition with a grain rice-enriched medium that is able to achieve mass production of fruit bodies of Cordyceps militaris, Cordyceps pruinosa, and Cordyceps bassiana under defined culture conditions.
Among the mushrooms that we demonstrated to have grown in a mass production setting, we focused especially on Cordyceps pruinosa (Figure 1(a)), as only a few papers have reported its pharmacological activities towards cancer and inflammation, [15,16] and the cultivation rate of its fruit body is much higher than other species. Although the antiinflammatory activity of the methanol extract of Cordyceps pruinosa has been suggested previously by determination of IL-1 , TNF-, NO, and PGE 2 levels [15], the exact molecular mechanism for these effects has not been fully elucidated. Therefore, in this study,we aimed to explore antiinflammatory mechanism of Cordyceps pruinosa butanol fraction by using kinase assays, luciferase reporter assays, molecular binding assays, and overexpression strategies, as well as a mouse model of gastritis.

Preparation and Characteristics of Cp-BF.
After drying the fruiting bodies of Cordyceps pruinosa at 50 ∘ C, an ethanol extract was prepared and several solvent fractions were subsequently prepared with n-hexane, n-butanol, and ethyl acetate. After evaporation of the fractions under reduced pressure, each fraction was dried using a freeze-dryer to give solid subfractions. Among the various fractions, the butanol fraction (Cp-BF) gave a 7% yield. The phytochemical characteristics of Cp-BF were identified by high performance liquid chromatography (HPLC) analysis as reported previously [17]; the system was equipped with KNAUER (Wellchrom HPLCpump, K-1001, Wellchrom fast scanning spectrophotometer K-2600, and 4 channel degasser K-500). The elution solvents consisted of distilled water and acetonitrile. The gradient step  The elution solvents were distilled water and acetonitrile. The gradient step of the solvent was "water to acetonitrile 1%/min" performed using a Vydac C18 Column.

Determination of Reactive Oxygen Species Generation.
Levels of intracellular ROS were determined by analysing the change in fluorescence resulting from the oxidation of the fluorescent probe H 2 DCFDA. Briefly, 5 × 10 5 RAW264.7 cells were exposed to Cp-BF (0-100 g/mL) for 30 min. After incubation, cells were incubated with LPS (1 g/mL) as an inducer of ROS production at 37 ∘ C for 6 h. Cells were then incubated with 50 M H 2 DCFDA for 1 h at 37 ∘ C. The degree of fluorescence, corresponding to the level of intracellular ROS, was determined using a FACScan flow cytometer (Beckton-Dikinson, San Jose, CA, USA) as reported previously [16].

Flow
Cytometric Analysis. RAW264.7 cells (2 × 10 6 cells/mL) treated with Cp-BF (0-100 g/mL) and FITCdextran or H 2 DCFDA were washed with a staining buffer (containing 2% rabbit serum and 1% sodium azide in PBS) and incubated with directly labelled antibodies for a further 45 min on ice. After washing three times with staining buffer, cells were analysed on a FACScan flow cytometer (Becton-Dickinson).

Cell Viability Test.
After preincubation of RAW264.7 cells (1 × 10 6 cells/mL) for 18 h, the Cp-BF (0-200 g/mL) was added to the cells and incubated for an additional 24 h. The cytotoxic effect of the Cp-BF was then evaluated by a conventional MTT assay as described previously [22]. Briefly, 3 h prior to culture termination, 10 L of a MTT solution (10 mg/mL in phosphate buffered saline, pH 7.4; PBS) was added and cells were continuously cultured until termination of the experiment. Incubation was halted by the addition of 15% sodium dodecyl sulphate into each well to solubilise the produced formazan [23]. The absorbance at 570 nm (OD 570 ) was measured using a Spectramax 250 microplate reader.

mRNA analysis by Reverse Transcription (RT) Polymerase Chain Reaction (PCR).
To determine cytokine mRNA expression levels, total RNA was isolated from LPS-treated RAW264.7 cells with TRIzol Reagent (Gibco BRL) according to the manufacturer's instructions. Total RNA was stored at −70 ∘ C until use. Determination of mRNA levels was performed using RT-PCR as reported previously [24]. Results are expressed as the ratio of optimal density to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of the primers used in this analysis are shown in Table 1.

Transfection of DNA and Luciferase Reporter Gene
Activity Assay. HEK293 or RAW264.7 cells (5 × 10 6 cells/mL) were transfected with 1 g/mL of empty vectors or FLAG-IKK . Transfections were performed with lipofectamine 2000 (Invitrogen, Grand Island, NY) in 100 mm cell culture dishes in the presence or absence of MHNC. For luciferase assays, HEK293 cells (1 × 10 6 cells/mL) were transfected with 1 g of plasmids containing NF-B-Luc, CREB-Luc, or AP-1-Luc, as well as -galactosidase using the PEI method [25,26] in 12-well plates according to the manufacturer's protocol. Cells were used for experiments 48 h after transfection. Luciferase assays were performed using the Luciferase Assay System (Promega, Madison, WI) as reported previously [27].

Preparation of Total Lysates and Nuclear Fractions,
Immunoblotting, and Immunoprecipitation. Stomach tissues or cultured cells (HEK293 and RAW264.7) (5 × 10 6 cells/mL) were washed three times in cold PBS with 1 mM sodium orthovanadate and lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM ethylenediaminetetraacetic acid, 50 mM -glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1% Triton X-100, 10% glycerol, 10 g/mL aprotinin, 10 g/mL pepstatin, 1 mM benzamide, and 2 mM phenylmethanesulfonyl fluoride) for 30 min with rotation at 4 ∘ C. Next, lysates were clarified by centrifugation at 16,000 ×g for 10 min at 4 ∘ C and stored at −20 ∘ C until needed. Nuclear lysates were prepared in a three-step procedure [28]. After treatment, cells were collected with a rubber policeman, washed with 1 × PBS, and lysed in 500 L of lysis buffer on ice for 4 min. Cell lysates were then centrifuged at 19,326 ×g for 1 min in a microcentrifuge. In the second step, the pellet (the nuclear fraction) was washed once in washing buffer, which was the same as the lysis buffer with the omission of Nonidet P-40. In the final step, nuclei were treated with an extraction buffer containing 500 mM KCl, 10% glycerol, and the inhibitors noted in the lysis buffer. The nuclei/extraction buffer mixture was frozen at −80 ∘ C and then thawed on ice and centrifuged at 19,326 ×g for 5 min. Finally, the supernatant was collected as the nuclear extract.
For immunoprecipitation, cell lysates containing equal amounts of protein (500 g) from RAW264.7 cells (1 × 10 7 cells/mL) treated with or without LPS (1 g/mL) for 2.5 min were pre-cleared with 10 L of protein A-coupled Sepharose beads (50% v/v) (Amersham, Buckinghamshire, UK) for 1 h at 4 ∘ C. Precleared samples were then incubated with 5 L of anti-IKK antibody overnight at 4 ∘ C. Immune complexes were mixed with 10 L of protein A-coupled Sepharose beads (50% v/v) and rotated for 3 h at 4 ∘ C.
Evidence-Based Complementary and Alternative Medicine 5 2.13. IKK and IKK Kinase Assays. To evaluate the ability of extracts to inhibit IKK and IKK kinase activities using purified enzymes, a kinase profiler service from Millipore (Billerica, MA) was used. In a final reaction volume of 25 L, IKK or IKK (human; 1-5 mU) was incubated with the reaction buffer and the reaction was initiated by the addition of MgATP. After incubation for 40 min at room temperature, the reaction was stopped by the addition of 5 mL of 3% phosphoric acid solution. Next, 10 L of the reaction product was spotted onto a P30 filtermat and washed three times for 5 min each in 75 mM phosphoric acid. The filtermats were washed once in methanol, dried, and then subjected to scintillation counting.

HCl/Ethanol (EtOH)-Induced Gastritis.
Inflammation of the stomach was induced with HCl/EtOH, according to a published method [30]. Fasted ICR mice were orally treated with Cp-BF (200 mg/kg) or ranitidine (40 mg/kg) twice per day for 3 days. Thirty min after the final injection, 400 L of 60% EtOH in 150 mM HCl was administered orally. Each animal was anesthetized with an overdose of urethane 1 h after the administration of necrotizing agents. The stomach was then excised and gently rinsed under running tap water. After opening the stomach along the greater curvature and spreading it out on a board, the area (mm 2 ) of mucosal erosive lesions was measured using a pixel-counter under blind condition as reported previously [31].

Statistical Analyses.
Data are expressed as the mean ± standard deviation (SD) calculated from at least three independent experiments, each performed in triplicate, or representative of three different experiments with similar results. For statistical comparisons, results were analysed using analysis of variance/Scheffe's post hoc test and Kruskal-Wallis/Mann-Whitney test. Values of < 0.05 were taken to indicate statistically significant differences. All statistical tests were carried out using the SPSS computer program (SPSS, Chicago, IL).

Cp-BF Suppresses the Inflammatory Responses of
Macrophages. We first tested the effect of Cp-BFin macrophages. As shown in Figures 2(a) and 2(b), Cp-BF dosedependently suppressed the production of NO and TNFstimulated from LPS-treated RAW264.7 cells. In particular, 200 g/mL of Cp-ME greatly inhibited the production of NO up to 95% and the release of TNF-by 43%. In addition, this fraction significantly decreased the uptake of FITCdextran by up to 41% at 100 g/mL (Figure 2(c)). Moreover, Cp-BF almost completely suppressed the generation of ROS stimulated by LPS at 50 and 100 g/mL (Figure 2(d)). Importantly, Cp-BF at 200 g/mL did not affect the viability of either RAW264.7 or HEK293 cells (Figure 2(e)).

Cp-BF Suppresses the Inflammatory Responses at the Transcription Level.
To determine the mechanism by which Cp-BF exerts its anti-inflammatory effects, we next analysed the mRNA levels of genes involved in inflammation. As shown in Figure 3(a) Cp-BF strongly suppressed the expression of iNOS and IL-6 between 100 and 200 g/mL, while this fraction weakly inhibited the mRNA expression of TNFat these concentrations. The regulatory role of Cp-BF on the activation of inflammatory transcription factors was determined by luciferase assay. Thus, under the described conditions, the activity of luciferase was clearly enhanced by PMA treatment for NF-B and AP-1 activation, and by forskolin exposure for CREB (Figure 3(b)). Interestingly, Cp-BF remarkably diminished the NF-B-mediated luciferase activity in a dose-dependent manner, whereas others were marginally suppressed (Figure 3(b)). In agreement with this result, treatment with 200 g/mL Cp-BF decreased the nuclear translocation of p50 but not p65 triggered by LPS treatment in RAW264.7 cells (Figure 3(c)).

Cp-BF Inhibits NF-B Activation Pathway by Suppressing
IKK Activity. Immunoblot analysis revealed that Cp-BF was able to suppress the upstream signalling pathway for NF-B activation. Thus, Cp-BF clearly blocked the phosphorylation of I B at 5, 30, and 60 min (Figure 4(a) left panel). In contrast, this fraction did not block the phosphorylation of p38, JNK, and ERK from 5 to 60 min (Figure 4(a) left panel). In addition, there was no alteration of phosphorylation levels of Akt and IKK / between 2 to 5 min (Figure 4(a) right panel). Since there was no inhibition of IKK / phosphorylation but suppression of I B phosphorylation, Cp-BF was assumed to have dampened the enzymatic activities of IKK and IKK . In order to test our theory, we employed kinase assays to evaluate IKK / kinase activity. As shown in Figure 4(b), Cp-BF significantly suppressed the kinase activity of IKK -but not IKK -up to 44% at 200 g/mL. Similarly, overexpression of IKK -induced luciferase activity and phosphorylation of I B was completely diminished by Cp-BF (50 to 200 g/mL) (Figure 4(c)). In agreement with this result, the phosphorylation of NF-B subunits (p65 and p50) and I B induced by IKK overexpression in HEK293 cells was strongly suppressed by Cp-BF (Figure 4(d)). Finally, we found that Cp-BF was able to block the molecular interaction between IKK and I B (Figure 4(e)).
The importance of IKK in inflammatory responses was also demonstrated using the strong IKK inhibitor BAY11-7082. Specifically, BAY11-7082 inhibited the release of NO and suppressed the phagocytic uptake of FITC-dextran (Figure 4(f)).

Cp-BF Ameliorates HCl/EtOH-Induced Gastritis via Suppression of NF-B Pathway.
To demonstrate the antiinflammatory activity of Cp-BF in vivo, we analysed the effects of orally administered Cp-BF in a model of gastritis. As shown in Figures 5(a) and 5(b), orally administered Cp-BF ameliorated the inflammatory symptoms of HCl/EtOHinduced gastritis in vivo. Cp-BF (200 mg/kg) clearly suppressed the formation of inflammatory lesions in stomach during HCl/EtOH treatment and was more effective than ranitidine (40 mg/kg). Interestingly, Cp-BF also suppressed the phosphorylation of I B induced by gastric damage (Figure 5(c)).

Discussion
As part of the strategy to develop fruit bodies of artificially cultured Cordyceps species, Cordyceps pruinosa was selected and cultivation conditions were established with grain ricecontaining medium. Recently, our group succeeded in manufacturing fruit bodies of Cordyceps pruinosa (Figure 1(a)) at the mass production level, and as the next step we decided to explore its pharmacological activity. Thus, in the present study, we evaluated the anti-inflammatory mechanism of a butanol extract (Cp-BF) of the Cordyceps pruinosa fruit body.
Similar to a previous study of a methanol extract of the Cordyceps pruinosa fruit body, we found that Cp-BF could suppress the functional activation of macrophages in inflammatory responses. Thus, Cp-BF suppressed the essential outcomes of inflammatory process from macrophage activation such as production of NO and TNF- (Figures 2(a) and 2(b)), ROS generation (Figure 2(c)), and phagocytic uptake (Figure 2(d)), without altering cell viability (Figure 2(e)) [32,33]. Considering the importance of macrophages in inflammation, these data strongly implied that Cp-BF could negatively modulate macrophage-mediated inflammatory responses. These results are consistent with the previous reports that extracts of Cordyceps militaris can diminish inflammatory gene expression in LPS-treated RAW264.7 cells [34]. Furthermore, a chemical derivative of militarin from Cordyceps militaris exhibits strong antiinflammatory properties in LPS-treated macrophages [35]. The ethanol extract of Cordyceps bassiana also blocks the production of IL-12 in LPS-treated RAW264.7 cells [36]. Since the majority of inflammatory diseases such as hepatitis, gastritis, colitis, and nephritis are known to be caused by overactivated macrophages, our data strongly suggest that the inhibitory activity of macrophage function may contribute to the immunopharmacological action of Cordyceps. In fact, the curative efficacy of these mushrooms against macrophage-mediated diseases has been previously demonstrated in a number of different disease animal models [37,38].
Understanding the molecular mechanism of Cp-BF mediated suppression of macrophage inflammatory responses is a significant goal for immunopharmacological research of the genus Cordyceps. However, only a few reports to date have attempted to explore the molecular target of Cordyceps species with respect to their anti-inflammatory actions. Specifically, we previously suggested that the phosphorylation of p38 might target the inhibition of IL-12 expression in Cordyceps bassiana [36]. Cordycepin was found to suppress the phosphorylation of Akt for NF-B inhibition in LPS-treated conditions [39]. It was also reported that NF-B could be targeted by a methanol extract of Cordyceps pruinosa, although no further evaluation of its upstream signalling enzymes was performed [15]. Importantly, none of these reports clearly identified a target protein responsible for the activity of Cordyceps as they only tested the phosphorylation levels of relevant pathways. To improve such methodological limitations, we employed a direct kinase assay followed by an overexpression approach for target proteins to confirm their involvement. Through this approach, we identified IKK as a new potential target enzyme of Cp-BF.
We next analysed the mRNA levels of inflammatory genes (Figure 3(a)), luciferase promoter activity (Figure 3(b)), and nuclear levels of transcription factors (Figure 3(c)). Our results suggested that Cp-BF was able to suppress macrophage-mediated inflammatory responses at the transcriptional level by suppression of the NF-B pathway. Through determination of upstream phosphorylation patterns regulating the activation and translocation of NF-B, we confirmed the presence of a target event between IKK and I B based on clear suppression of the phosphorylation of I B by Cp-BF (Figure 4(a) left panel) and lack of inhibition of upstream phosphorylation events for the activation of IKK and Akt (Figure 4(a) right panel). Although the inhibitory activity was not strong, direct kinase assay with IKK indicated that approximately 40% of IKK enzyme activity was suppressed by Cp-BF (200 g/mL), implying that IKK may be a potential target enzyme. Indeed, additional experiment data strongly supported the idea. Cp-BF treatment remarkably blocked NF-B-mediated luciferase activity induced by overexpressed IKK (Figure 4(c)), upregulation of p65/p50 phosphorylation (Figure 4(d)), and molecular binding between IKK and I B (Figure 4(e)). However, the discrepancy between kinase assay (Figure 4(b)) and IKK overexpression experiments (Figures 4(c) and 4(d)) should be further explored. Presently, we speculate that there might be different pharmacological sensitivities between purified human IKK used for kinase assays and transfected IKK expressed in HEK293 cells.
The ethnopharmacological value of Cordyceps is now greatly accepted in Asian countries, since these mushrooms have been traditionally prescribed for the treatment of various inflammatory and metabolic diseases. Unlike ginseng, one of most famous herbal plants worldwide, for which cultivation methods have been traditionally established, the majority of Cordyceps products are still harvested in nature. Although natural fruit bodies of Cordyceps may be best in terms of pharmacological activities, large-scale production remains a key issue. Therefore, recent studies with Cordyceps species have focused on the development of cultural methods for mass production of Cordyceps. In fact, our group has established a suitable cultivation method and plant manufacturing system that achieves high productivity of Cordyceps fruit bodies [40]. Using the approach, we analysed the pharmacological efficacies of artificially cultivated fruit bodies of Cordyceps militaris, Cordyceps bassiana, and Cordyceps pruinosa maintained under identical conditions. To specifically test the activity of Cordyceps pruinosa, we employed a mouse in vivo disease model with HCl/EtOHinduced gastritis under oral administration of Cp-BF. Interestingly, this fraction strongly ameliorated gastritis symptoms (Figures 5(a) and 5(b)) and suppressed the phosphorylation of I B increased by HCl/EtOH treatment ( Figure 5(c)). These results strongly implied that the artificially cultivated fruit bodies exhibit similar pharmacological activity as the natural forms of this mushroom. Indeed, we previously observed that the artificially cultivated fruit body of Cordyceps bassiana can effectively treat symptoms of atopic dermatitis [41]. Moreover, the artificially cultivated fruit body of Cordyceps militaris possesses antihepatitis activity (data not shown). Taken together, these results strongly suggest that the artificially cultivated fruit bodies of Cordyceps pruinosa can be developed for the purpose of pharmaceutical and nutraceutical remedies.
To date little information on the phytochemical characteristics of Cordyceps pruinosa has been reported. A total of 5 alcohols, 21 amino acids, 15 organic acids, 4 purines, 3 pyrimidines, 7 sugars, 11 fatty acids, and 5 other metabolites in Cordyceps pruinosa mycelia cultivated with various media have been characterized by metabolic profile analysis [42]. This same study also showed that Cordyceps pruinosa mycelia contains a high phenolic content and exhibits good antioxidative properties, although the specific compounds responsible for this effect have not yet been identified [42]. According to the analysis of cordycepin by HPLC, this compound might not be included in this mushroom (Figure 1(c)). Therefore, future studies will need to include the understanding of active components with anti-inflammatory activity in this mushroom.
In summary, we have shown that Cp-BF can block macrophage-mediated inflammatory responses such as NO and TNF-production, ROS generation, and phagocytic uptake at the transcriptional level. Orally administered Cp-BF clearly ameliorates gastric ulcer formation induced by HCl/EtOH treatment. By analysing transcription factors and intracellular signalling enzymes, our results suggest that Cp-BF can directly inhibit IKK linked to the suppression of NF-B pathway as summarized in Figure 6. The data from the present study strongly support the pharmaceutical and nutraceutical values of artificially cultivated fruit bodies of Cordyceps pruinosa.