Article pubs.acs.org/JAFC
Methanol Extract of Antrodia camphorata Protects against Lipopolysaccharide-Induced Acute Lung Injury by Suppressing NFκB and MAPK Pathways in Mice Guan-Jhong Huang,†,‡,∥ Jeng-Shyan Deng,§,∥ Chin-Chu Chen,⊥ Ching-Jang Huang,⊗ Ping-Jyun Sung,□,○ Shyh-Shyun Huang,*,# and Yueh-Hsiung Kuo*,‡,△ †
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan ‡ Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 404, Taiwan § Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan ⊥ Biotechnology Center of Grape King Inc., Chung-Li City 320, Taiwan ⊗ Department of Biochemical Science and Technology, National Taiwan University, Taipei 106, Taiwan ○ Graduate Institute of Marine Biotechnology and Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan □ National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan # School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan △ Department of Biotechnology, Asia University, Taichung 413, Taiwan ABSTRACT: Antrodia camphorata (AC) has been used as a herbal medicine for drug intoxication for the treatment of inflammation syndromes and liver-related diseases in Taiwan. This study demonstrates the protective effect of the methanol extract of AC (MAC) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Mice were treated with MAC 1 h before the intratracheal (I.T.) instillation of LPS challenge model. Lung injury was evaluated 6 h after LPS induction. Pretreatment with MAC markedly improved LPS-induced histological alterations and edema in lung tissues. Moreover, MAC also inhibited the release of pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 at 6 h in the bronchoalveolar lavage fluid (BALF) during LPS-induced lung injury. Furthermore, MAC reduced total cell number and protein concentrations in the BALF the pulmonary wet/dry weight (W/D) ratio, and myeloperoxidase activity and enhanced superoxide dismutase (SOD) activity in lung tissues. MAC also efficiently blocked protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibited the degradation of nuclear factor-kappa B (NF-κB) and IκBα. This is the first investigation in which MAC inhibited acute lung edema effectively, which may provide a potential target for treating ALI. MAC may utilize the NF-κB and MAPKs pathways and the regulation of SOD activity to attenuate LPS-induced nonspecific pulmonary inflammation. KEYWORDS: Chinese herb, Antrodia camphorata, acute lung injury, anti-inflammation, TNF-α
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overproduction of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) are involved in the pathogenesis of ALI in animals. Additionally, the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) has been an important step in the development of ALI, and it may serve as a novel therapeutic target.5 Antrodia camphorata (AC; Polyporaceae, Aphyllophorales) camphoratus) is a rare mushroom of the Polyporaceae family. It has been used to treat liver dysfunction, drug and alcohol intoxication, hypertension, pruritis, abdominal pain, cancer, and inflammatory disorders.6 A number of functional compounds of
INTRODUCTION Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are diseases induced by many extreme conditions. They are defined by a series of histologic changes such as pulmonary edema and neutrophil accumulation, which are characterized by diffuse injury to the alveolar-capillary membrane in the lung and associated with shock, sepsis, acidosis, and ischemia reperfusion.1 Excess production of inflammatory mediators of ALI/ARDS include cytokines, chemokines, and adhesion molecules.2 Lipopolysaccharide (LPS) has been well recognized for studying the pathogenesis of ALI/ARDS.3 Intratracheal administration of LPS has been used to induce macrophages and neutrophils and develop lung inflammation in this model ALI.4 LPS exposure to the lung tissues induced the nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions, and subsequently the © XXXX American Chemical Society
Received: March 10, 2014 Revised: May 6, 2014 Accepted: May 21, 2014
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infiltration assessment. The BALF samples were centrifuged (700g for 10 min at 4 °C) and their supernatants were frozen at −80 °C for protein and cytokine ELISA. The cell pellets were resuspended in PBS (1 mL) and the total cell counts were determined using a hemocytometer in the BALF. Lung tissues from mice were collected but not subjected to BALF collection and then stored at −80 °C for further experiments. Histopathological Analysis. At 6 h after treatment with LPS, mice were euthanized and lung tissues were harvested and fixed in 4% paraformaldehyde. For light microscopic examination, the lung tissue was dehydrated, then paraffin embedded, and sliced. After hematoxylin and eosin (H&E) staining of lung tissue slices, pathological changes were observed. Measurement of Myeloperoxidase (MPO) Activity. The MPO activity was determined to assess neutrophil accumulation, and it was measured as described previously.14 The lung tissues were homogenized in 80 mM phosphate buffer, pH 5.4, and then homogenate was centrifuged (12000g for 15 min, at 4 °C). Ten microliters of sample was added to 290 μL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine chloride and 0.0005% H2O2 as a substrate. Oxidized o-dianisidine was determined by spectrophotometry (OD460 nm) (Molecular Devices, Sunnyvale, CA, USA). MPO content was expressed as relative MPO activity (OD460 nm/mg protein of lung tissue). Lung Wet-to-Dry Weight (W/D) Ratio. The lung tissues were excised, immediately weighed, and then placed in an oven at 80 °C for 48 h to obtain the “dry” weight. The W/D ratio was then calculated. Measurement of BALF TNF-α, IL-1β, and IL-6 by an EnzymeLinked Immunosorbent Assay (ELISA). The concentrations of cytokine levels of TNF-α, IL-1β, and IL-6 in the BALF were measured by ELISA using commercially available reagents according to the manufacturer’s instruction (Biosource International Inc.). TNF-α, IL1β, and IL-6 were determined from a standard curve. Measurement of Nitric Oxide/Nitrite. Nitrite and nitrate could have a role by producing NO. The nitrite level was determined by the Griess reagent in BALF.15 One hundred microliters was applied to 96well plates, followed by 100 μL of Griess reagent. The Griess reagent was prepared by mixing equal amounts of 0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide. Absorbance at 540 nm was measured after 10 min of incubation at room temperature using a microplate reader (Molecular Devices). A standard curve was plotted with the concentration of sodium nitrite. Total SOD activity Measurements. Total SOD activity was determined by an indirect method involving the reduction of cytochrome c.16 For the cytochrome c reaction, the SOD mediated inhibition of ferricytochrome c reduction in the presence of a superoxide anion generating system (xanthine/xanthine oxidase) was determined by monitoring the change in absorbance at 550 nm. One SOD enzyme unit was defined as the amount of enzyme that causes a 50% inhibition. Western Blot Analysis. Lung tissues were homogenized in the protein extract solution and protease inhibitors and centrifuged at 12000g for 20 min. The supernatants were frozen at −20 °C until use. The amount of protein (50 μg) from the supernatant was resolved on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membrane was incubated with an appropriate dilution of specific primary antibodies (anti-iNOS, anti-COX-2, anti-COX-2, anti-IκBα, and anti-NF-κB) followed by appropriate horseradish peroxidaseconjugated (Sigma), goat anti-mouse, or anti-rabbit IgG. The bands were detected by enhanced chemiluminescence (ECL) by using hyperfilm and ECL reagent (Thermo). Band intensity on scanned films was quantified using Kodak Molecular Imaging software (version 4.0.5, Eastman Kodak Co., Rochester, NY, USA) and expressed as relative intensity compared with the control group. Statistical Analysis. Data are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed by oneway analysis of variance (ANOVA followed by Scheffe’s multiple-range tests). The levels of statistical significance are expressed as (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001.
AC were found, including polysaccharide, maleic/succinic acid derivatives, triterpenoids, benzenoids, and benzoquinone derivatives.7,8 Recent studies have various biological activities of AC, including antioxidant, anti-inflammation, liver fibrosis, antimetastasis, vasorelaxation, immunomodulation, and anticancer activities.9−11 In addition, different fermentation processes have been conducted in either liquid or solid states of A. camphorata mycelium. Ergostatrien-3β-ol (EK100), a triterpenoid isolated from the liquid fermentation of A. camphorata mycelia, was demonstrated to exhibit significant anti-inflammatory and hepatoprotective bioactivity in vitro.9,12 However, this study investigated whether oral treatment of MAC could attenuate ALI induced by LPS in vivo and explored the molecular mechanism of this regulatory pathway. The results demonstrated that MAC may protect against LPSinduced lung injury in mice and possibly exert its activity through the regulation of antioxidant enzyme activity, NF-κB, and MAPKs signaling pathways.
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MATERIALS AND METHODS
Chemicals. LPS (Escherichia coli 055:B5) and dexamethasone (Dex) were obtained from Sigma (St. Louis, MO, USA). TNF-α, IL1β, and IL-6 were obtained from Biosource International Inc. (Camarillo, CA, USA). The polyclonal antibodies against iNOS, COX-2, NF-κB, IκB-α, ERK, JNK, p38, p-ERK, p-JNK, p-p38, and βactin were purchased from Abcam (Cambridge, UK). Polyvinylidene fluoride transfer membranes (Immobilon P) were from Millipore Corp. (Bedford, MA, USA). Plant Material. The powder of AC of the submerged whole broth (batch MZ-247) was obtained from Biotechnology Center of Grape King Inc. (Chung-Li City, Taiwan). The preparative processes were all of commercial security. Fingerprint Chromatogram of MAC Extracts by HPLC. The chromatographic system consisted of a quaternary gradient pump (SFD 2100), a C-18 column (4.6 i.d. × 250 mm; TSK gel column, ODS-80TM; 5 μm particles, Merck), and an S-3210 photodiode array detector (PDA) (Schambeck SFD GmbH, Bad Honnef, Germany). The samples were detected with H2O (solvent A) and 100% methanol (solvent B) at 280 nm. The solvent gradient started with solvent A (10%) for 0 min and then linearly increased to solvent B (100%) for another 120 min. This linear gradient was followed by a solvent B elution until 180 min. The flow rate was 0.6 mL/min, and the injection volumes of standards and samples were 10 μL. Identification of EK100 was based on retention time and UV spectra by comparison with standards. Animals. All animal procedures adhered to the NIH Guide to the Care and Use of Laboratory Animals. All tests were conducted under the guidelines of the International Association for the Study of Pain (IASP). Male imprinting control region (ICR) mice (6 weeks old) were obtained from the BioLASCO Taiwan Co., Ltd. The animals were kept in isolation for at least 1 week before the experiment with the temperature maintained within 22−23 °C. Mice were given ad libitum access to food and water. LPS-Induced Acute Lung Injury in Mice. ALI was induced by intratracheal administration of LPS as previously described.13 Fiftyfour mice were randomly divided into six groups, the control group and five treatment groups. Normal mice were given 50 μL of normal saline without LPS. LPS (1.0 g/kg; 50 μL/per mouse) was administered intravenously to induce lung injury. MAC [25, 50, and 100 mg/kg; distilled water dissolved with 0.5% carboxymethyl cellulose (CMC)], dexamethasone (DEX; 10 mg/kg), or vehicle (PBS) was administered orally 1 h before 50 μL of LPS intratracheal (I.T.) instillation. Lung tissue samples were collected for Western blot assay, MPO assays, and antioxidant enzyme (SOD) activity assays. In these studies, bronchoalveolar lavage fluid (BALF) was collected for protein concentration assay (Bio-Rad, Hercules, CA, USA) and leukocyte B
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Figure 1. HPLC analysis of the compound identification of EK100 (retention time = 145.75 min) (A) and MAC (methanol extract of Antrodia camphorata) (B). Total volume of 20 μL was loaded into the HPLC column to measure the relative content of ST1 in MAC extract according to the concentration of standard compounds.
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activities (0.41 ± 0.04 OD 460 nm/mg protein) in mouse lung tissues, significantly (p < 0.001). Pretreatment with MAC (25, 50, 100 mg/kg) decreased MPO activity to 0.23 ± 0.03, 0.27 ± 0.04, and 0.32 ± 0.02 OD 460 nm/mg protein, respectively, and DEX decreased it to 0.26 ± 0.02 OD460 nm/mg protein. These data indicated that MAC prevented excessive infiltration of neutrophils into the lung tissues in LPS-induced ALI mice. MAC Attenuates the Lung Wet-to-Dry Ratio and Protein Concentration Induced in LPS-Induced ALI Mice. To assess lung edema, we determined the lung water content in ALI mice 6 h after LPS treatment. LPS challenge increased the lung vascular permeability, as shown by the lung W/D ratio as compared to the control group, significantly. Therefore, we further investigated the W/D ratios and protein concentrations of different groups, and as shown in Figure 4, the W/D ratio and protein concentration expanded in the LPS group compared with the control group and the pretreatment with MAC (100 mg/kg) and DEX ameliorated the W/D ratio and protein concentration compared with the LPS group. These results indicated that MAC could suppress lung edema in LPSinduced ALI mice. Effect of MAC on Anti-inflammatory Cytokines in Lung Tissues of LPS-Induced ALI mice. LPS-induced ALI is related to the various pro-inflammatory cytokines. In this study, the levels of pro-inflammatory cytokines (NO, TNF-α, IL-1β, and IL-6) in BALF were measured 6 h after LPS instillation. As shown in Figure 5, the pro-inflammatory cytokine NO, TNF-α, IL-1β, and IL-6 levels of LPS-induced ALI mice in the BALF were improved compared to those in the control group, significantly (p < 0.001). MAC (100 mg/kg) and DEX significantly reduced NO, TNF-α, IL-1β, and IL-6 production compared to the LPS-induced ALI group.
RESULTS Fingerprint Chromatogram of MAC by HPLC. The fingerprint chromatogram of MAC was established for the quality control of the liquid fermentation of A. camphorata mycelia. The structure of ergostatrien-3β-ol (EK100) was elucidated by its retention time, UV absorbance of purified standards, and comparison with the literature.17 Figure 1 shows the predominant constituents identified as EK100. MAC Reduces LPS-Induced Lung Injury in Vivo. To evaluate the LPS-induced lung injury after MAC treatment, we observed the histological changes of lungs in the different groups. In the control group, no obvious histological alteration was found in lung tissues (Figure 2). In the LPS-induced lung injury group H&E staining evidenced a large number of infiltrations of inflammatory cells, lung tissue damage, and marked alveolar wall edema. These pathological changes were improved by treatment with MAC (25, 50, and 100 mg/kg) and DEX (10 mg/kg), suggesting that MAC could protect against histological changes in the LPS-induced ALI model. Effects of MAC on Inflammatory Cell Count and MPO Activity on LPS-Induced ALI in Mice. Six hours after LPSinduced ALI, the total cell numbers in the BALF increased compared with the control group, significantly (p < 0.001). MAC (100 mg/kg) and DEX (10 mg/kg) significantly also decreased the number of total cells (p < 0.001) compared to LPS-induced ALI mice (Figure 3A). Polymorphonuclear leukocyte (PMN) infiltration plays a central role in inflammation, and it is also a major cause of tissue damage. MPO activity can be considered a good indicator of lipid peroxidation in lung tissues. As Figure 3B shows, the intratracheal instillation of LPS increased MPO C
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Figure 2. Effects of MAC on lung histopathological changes in LPS-induced ALI mice. Mice were killed at 6 h after LPS stimulation. The left lungs were excised and embedded in 10% formalin and sectioned, followed by H&E staining; magnification ×100. Images are representative of three experiments. Br, bronchi; DEX, dexamethasone.
Effects of MAC Protect against Oxidative Stress in LPS-Induced ALI Mice. Oxidative stress may lead to apoptosis and lung injury. SODs are a class of enzymes that can acting as the chief ROS scavenging enzyme, and the activity of SODs has decreased in some reports of LPS-induced ALI mice. The data presented demonstrated that the pretreatment with MAC (100 mg/kg) and DEX increased SOD activity in mice as compared to the LPS-induced ALI group (Figure 6). Therefore, in this study, we demonstrated that MAC might be attributed to its antioxidant enzyme activities in the inflammatory response og LPS-induced ALI mice. Effects of MAC on iNOS and COX-2 Protein Expressions in LPS-Induced ALI Mice. To investigate possible mechanisms of whether the level of NO was due to regulated iNOS and COX-2 protein expressions, the effects were studied by Western blot in the LPS-induced ALI. The results showed that the pretreatment with MAC (100 mg/kg) for 6 h inhibited iNOS and COX-2 protein expression in lung tissues of LPS-induced ALI mice (Figure 7A).
Effect of MAC on NF-κB and IκBα Inactivation in LPSInduced ALI Mice. NF-κB is activated by a variety of stimuli including LPS, TNFα, and IL-1β. The effect of MAC on NF-κB expression in lung tissue of LPS-induced ALI mice was assessed by Western blot. In this study, Figure 7B shows that LPSinduced IκBα and NF-κB degradation was inhibited by pretreatment with MAC in LPS-induced ALI mice. The signal of NF-κB inhibition is regulated by interaction with inhibitory IκBα proteins. Overall, our findings provide support that the NF-κB inhibitor IκBα contains a functional nuclear export signal. Effect of MAC on MAPK Signaling Pathway in LPSInduced ALI Mice. To examine the mechanisms of the LPSinduced ALI mice, we investigated the effect of MAC on the MAPKs signal pathway. As shown in Figure 8, MAPKs were activated in LPS-induced ALI. Furthermore, MAC (100 mg/ kg) and DEX dramatically inhibited the phosphorylation of ERK, JNK, and p38 protein expressions. These results D
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Figure 3. Effects of MAC on the number of total cells in the BALF of LPS-induced ALI mice: (A) number of total cells; (B) myeloperoxidase activity (MPO) performed at 6 h after LPS challenge. Its activity reflects the neutrophil infiltration in the lungs. Cell counts were performed with a hemocytometer. Each value represents the mean ± SEM. (###) p < 0.001 as compared with the control group; (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 as compared with the LPS group (one-way ANOVA followed by Scheffe’s multiple-range test).
Figure 4. Effects of MAC on the lung W/D ratio and total protein level in the BALF of LPS-induced ALI mice. Mice were given MAC (25, 50, or 100 mg/kg) 1 h prior to an intratracheal (I.T.) instillation administration of LPS. The lung W/D ratio (A) and total protein concentration in the BALF (B) were determined 6 h after the LPS challenge. Each value represents the mean ± SEM. (###) p < 0.001 as compared with the control group; (∗∗) p < 0.01 and (∗∗∗) p < 0.001 as compared with the LPS group (one-way ANOVA followed by Scheffe’s multiple-range test).
demonstrated that MAC inhibited the activity of MAPK pathways in LPS-induced ALI.
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added to the understanding of the epidemiology and pathogenesis, but the reason for the morbidity and mortality in critically ill patients is unclear.20 Administration of LPSinduced ALI in mice has been used to study the pathophysiological and biological pathways.21 Intratracheal or intranasal bacterial products such as LPS administration trigger the optimal ALI, which is characterized by increased capillary permeability, hemorrhage, intra-alveolar and interstitial edema, and an influx of inflammatory cells into the lung. Currently, DEX, a strong synthetic glucocorticoid, is the most used antiinflammatory and antioxidative drug in the clinical treatment of ALI because it regulates an efficient decrease of inflammation.22 Therefore, in this study, the anti-inflammatory effect of DEX was a positive control in LPS-induced mice ALI. Lung edema is one of the important features of ALI.23 Furthermore, we investigated the lung W/D ratio and protein concentration in the BALF to quantify the magnitude of lung edema. Here, we found that MAC decreased the lung W/D
DISCUSSION In this study, these data are the first to demonstrate the antiinflammatory activity of MAC in LPS-induced mice ALI. In our experiment, we used this model to determine whether MAC could prevent ALI induced by LPS intranasal instillation. The MAC has been reported to exhibit anti-inflammatory properties in vitro and in vivo.18 Thus, this study is the first to investigate the effects of MAC inhibition on NF-κB and MAPK activation occurring together with IκBα degradation in a mouse model of LPS-induced ALI. ALI/ARDS are diseases of multifactorial etiology by acute respiratory failure that cause acute lung tissue edema, lung injury, and inflammatory responses. Multiple clinical studies have reported the development of ALI associated with sepsis, pulmonary injury from pneumonia and aspiration, aspiration of gastric contents, and pulmonary injury from trauma.19 ALI has E
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Figure 5. Effect of MAC on NO, TNF-α, IL-1β, and IL-6 expression from mice with ALI. Mice were given MAC (25, 50, or 100 mg/kg) by intraperitoneal injection 1 h before challenge with LPS. BALF was collected at 6 h following LPS challenge to analyze the inflammatory cytokines NO (A), TNF-α (B), IL-1β (C), and IL-6 (D). Each value represents the mean ± SEM. (###) p < 0.001 as compared with the control group; (∗∗) p < 0.01 and (∗∗∗) p < 0.001 as compared with the LPS group (one-way ANOVA followed by Scheffe’s multiple-range test).
with corticosteroid resistance and increased the severe inflammatory response, including ALI/ARDS. LPS-induced mouse ALI is characterized by neutrophil infiltration in lung tissues that reduced SOD activity and increased MPO activity.26 It was supposed that LPS-induced mouse ALI stimulates the neutrophil to migrate into the BALF, and LPS induced proinflammatory mediator expressions.4 This paper suggests that MAC potently inhibited the neutrophil infiltration, MPO activity, and edema formation and increased the SOD activity. LPS-induced mouse ALI displays some characteristics such as neutrophil accumulation, pro-inflammatory cytokine production, and lung injury. The levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 can be used to evaluate lung injury and inflammatory level.27 Our previous studies also have reported that MAC can decrease iNOS and COX-2 protein expression in vitro and in vivo.18 In this experiment, we found that pretreatment with MAC reduced the TNF-α, IL-1β, and IL-6 expressions in BALF. These results indicate that MAC could effectively improve the LPS-induced ALI in mice. NO acts as a second messenger with multiple functions, including neural activity, blood flow, and pain modulation.28 Excessive levels of NO produced from iNOS lead to a reaction
ratio after pretreatment 1 h before mice were subjected to LPSinduced ALI. In addition, we also evaluated that lung sections were stained by H&E staining and analyzed by light microscopy after pretreatment with MAC in LPS-induced mice. This result identified the inhibition of MAC or DEX in LPS-induced pulmonary edema and inflammation by lung morphological examination. In vivo the I.T. administration of LPS is a well-known model for the induction of lung inflammation.23 Leukocytes move within 1 h to the lung tissue and are replaced by monocytes and macrophages cells and influence the level of inflammatory reaction. Neutrophil infiltration associated with a LPS-induced inflammatory reaction and the free radical productions from neutrophils have potent antimicrobial activity in acute infections consisting of oxidants, proteinases, and cationic peptides that lead to lung injury.24 In this study, we found that total numbers of cells and the expressed of MPO activity in neutrophils were decreased after pretreatment with MAC in LPS-induced mice and were relative to the oxidative stress, and free radicals may intensify inflammatory reaction.25 In this regard, it has been proposed that oxidative stress may be related F
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cellular constituents, and induction of cell apoptosis and necrosis.29 Transcription factor NF-κB plays a critical role that controls multiple key cellular processes. Members of the NF-κB family include p50 and p65 heterodimers. The translocation of NF-κB p65 is related to cancer, inflammation, and acute immune response.26 Inhibition of the NF-κB pathway has been shown to recover septic dysfunction and inhibit the expression of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6.30 The results from the present study show that pretreatment with MAC inhibits the degradation of IκBα and NF-κB in the LPSinduced mouse ALI. MAPK families (ERK, JNK, and p38) play an important role in regulating cytokine release and signal transduction pathways. JNK and p38 MAP kinase have been reported to identify regulatory pro-inflammatory cytokines (such as IL-1 and TNFα).29 MAPK pathways (ERK, p38, and JNK) have been demonstrated as participating in LPS-induced mouse ALI.31 In addition, p38 regulated the LPS-induced NF-κB activation by blocking neutrophil infiltration into the lung in LPS-induced mouse ALI.2 In this study, pretreatment of MAC inhibited LPSinduced phosphorylation of MAPKs (ERK, JNK, and p38). Thus, it is possible that the inhibition of pro-inflammatory (TNF-α, IL-1β, and IL-6) expressions by pretreatment with MAC occurs through pathways that inhibit the phosphorylation of MAPKs and IκB activation in LPS-induced acute lung injury. The fruiting body and mycelium of AC are well-known as a traditional medicine for many diseases. The anti-inflammatory
Figure 6. Effects of MAC on SOD activity in lungs from mice with ALI. Lung homogenates were prepared for measuring SOD activity. Each value represents the mean ± SEM. (###) p < 0.001 as compared with the control group; (∗∗) p < 0.01 and (∗∗∗) p < 0.001 as compared with the LPS group (one-way ANOVA followed by Scheffe’s multiple-range test).
of NO with superoxide to create ONOO−. ONOO− formation represents one of the major mechanisms of acute lung injury, including substantial oxidative/nitrative stress, destruction of
Figure 7. Inhibition of iNOS, COX-2 (A), IκBα, and NF-κB (B) protein expression by MAC in LPS-induced ALI mice. Tissues suspended were then prepared and subjected to Western blotting by using an antibody specific for iNOS, COX-2, IκBα, and NF-κB. β-Actin was used as an internal control. A representative Western blot from two separate experiments is shown. (###) Compared with sample of control group. Data represent the mean ± SD for the three different experiments performed in triplicate. (∗∗∗) p < 0.001 compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple-range test). G
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Figure 8. Effects of MAC on LPS-induced phosphorylation of MAPK in LPS-induced ALI mice. Mice were pretreated with different concentrations of MAC for 1 h and stimulated with LPS. Cellular proteins from the lungs were used for the detection of phosphorylated or total forms of three MAPK molecules, ERK, p38, and JNK. A representative Western blot from two separate experiments is shown. (###) Compared with sample of control group. Data represent the mean ± SD for the three different experiments performed in triplicate. (∗∗∗) p < 0.001 compared with the LPSalone group (one-way ANOVA followed by Scheffe’s multiple-range test).
activity of A. camphorata mycelium contributed to prevent inflammatory disease through repression of iNOS and COX-2 protein expression.18,28 Eburicoic acid and dehydroeburicoic acid, an active ingredient from solid-state culture of AC, were evaluated for anti-inflammatory and hepatoprotective effects.10,11 In our previous paper, we showed that ergostatrien3β-ol (EK100) had analgesic, anti-inflammatory,9 and hepatoprotective12 activities. Thus, we used EK100 to evaluate the quality of A. camphorata mycelium using high-performance liquid chromatography in this study. Antrocamphin A, antcin A, antcin B, and eburicol from AC have anti-inflammatory activity by inhibition of N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide anion generation.32 In summary, our study shows protective effects of MAC on LPS-induced mouse ALI. MAC pretreatment reduced lung histological changes, lung edema, and leukocyte infiltration in lungs, changed the wet/dry ratio in the lung tissue, and inhibited the release of inflammatory mediators in the BALF. These data suggested that MAC was proposed as a novel therapeutic strategy for inflammatory diseases through inhibition of the NF-κB pathway by directly blocking phosphorylation and degradation of inhibitory IκBα via inhibition of MAPK-responsive signaling pathways. Thus, MAC exerts an anti-inflammatory effect in rodents, and more research needs to be done before a clinical trial of this drug can be realized.
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China Medical University (CMU) (CMU100-ASIA-15 and CMU101-AWARD-08), Asia University (100-ASIA-15), and CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan, and the Department of Health Clinical Trial and Research Center of Excellent (DOH102-TD-C-111004), Taiwan. Notes
The authors declare no competing financial interest.
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AUTHOR INFORMATION
Corresponding Authors
*(S.-S.H.) Phone: +886 4 22053366, ext 5127. Fax: +886 4 22083362. E-mail:
[email protected]. *(Y.-H.K.) Phone: +886 4 22071693. Fax: +886 4 22071693. E-mail:
[email protected]. Author Contributions ∥
REFERENCES
(1) Tunceroglu, H.; Shah, A.; Porhomayon, J.; Nader, N. D. Biomarkers of lung injury in critical care medicine: past, present, and future. Immunol, Invest. 2013, 42, 247−261. (2) Chu, X.; Ci, X.; Wei, M.; Yang, X.; Cao, Q.; Guan, M.; Li, H.; Deng, Y.; Feng, H.; Deng, X. Licochalcone a inhibits lipopolysaccharide-induced inflammatory response in vitro and in vivo. J. Agric. Food Chem. 2012, 60, 3947−3954. (3) Liu, L.; Xiong, H.; Ping, J.; Ju, Y.; Zhang, X. Taraxacum of f icinale protects against lipopolysaccharide-induced acute lung injury in mice. J. Ethnopharmacol. 2010, 130, 392−397. (4) Wei, M.; Chu, X.; Jiang, L.; Yang, X.; Cai, Q.; Zheng, C.; Ci, X.; Guan, M.; Liu, J.; Deng, X. Protocatechuic acid attenuates lipolysaccharide-induced acute lung injury. Inflammation 2012, 35, 1169−1178. (5) Liu, Y.; Wu, H.; Nie, Y. C.; Chen, J. L.; Su, W. W.; Li, P. B. Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-κB pathway. Int. Immunopharmacol. 2011, 11, 1606−1612. (6) Liaw, C. C.; Chen, Y. C.; Huang, G. J.; Tsai, Y. C.; Chien, S. C.; Wu, J. H.; Wang, S. Y.; Chao, L. K.; Sung, P. J.; Huang, H. C.; Kuo, Y. H. Anti-inflammatory lanostanoids and lactone derivatives from Antrodia camphorata. J. Nat. Prod. 2013, 76, 489−494. (7) Chen, Y. C.; Chiu, H. L.; Chao, C. Y.; Lin, W. H.; Chao, L. K.; Huang, G. J.; Kuo, Y. H. New anti-inflammatory aromatic components from Antrodia camphorata. Int. J. Mol. Sci. 2013, 14, 4629−4639. (8) Hsieh, Y. H.; Chu, F. H.; Wang, Y. S.; Chien, S. C.; Chang, S. T.; Shaw, J. F.; Chen, C. Y.; Hsiao, W. W.; Kuo, Y. H.; Wang, S. Y. Antrocamphin A, an anti-inflammatory principal from the fruiting body of Taiwanof ungus camphoratus, and its mechanisms. J. Agric. Food Chem. 2010, 58, 3153−3158. (9) Huang, G. J.; Huang, S. S.; Lin, S. S.; Shao, Y. Y.; Chen, C. C.; Hou, W. C.; Kuo, Y. H. Analgesic effects and the mechanisms of antiinflammation of ergostatrien-3β-ol from Antrodia camphorata sub-
G.-J.H. and J.-S.D. contributed equally to this work.
Funding
The authors want to thank the financial supports from the National Science Council (NSC101-2313-B-039-002-MY3, NSC 102-2320-B-031-022-, and NSC 102-2320-B-468-004-), H
dx.doi.org/10.1021/jf405113g | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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merged whole broth in mice. J. Agric. Food Chem. 2010, 58, 7445− 7452. (10) Deng, J. S.; Huang, S. S.; Lin, T. H.; Lee, M. M.; Kuo, C. C.; Sung, P. J.; Hou, W. C.; Huang, G. J.; Kuo, Y. H. Analgesic and antiinflammatory bioactivities of eburicoic acid and dehydroeburicoic acid isolated from Antrodia camphorata on the inflammatory mediator expression in mice. J. Agric. Food Chem. 2013, 61, 5064−5071. (11) Huang, G. J.; Deng, J. S.; Huang, S. S.; Lee, C. Y.; Hou, W. C.; Wang, S. Y.; Sung, P. J.; Kuo, Y. H. Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia camphorata in a mouse model of acute hepatic injury. Food Chem. 2013, 141, 3020− 3027. (12) Huang, G. J.; Deng, J. S.; Huang, S. S.; Lin, S. S.; Shao, Y. Y.; Chen, C. C.; Hou, W. C.; Kuo, Y. H. Protective effects of antrosterol against carbon tetrachloride-induced acute liver injury in mice. Food Chem. 2012, 132, 709−716. (13) Sun, J.; Chi, G.; Soromou, L. W.; Chen, N.; Guan, M.; Wu, Q.; Wang, D.; Li, H. Preventive effect of imperatorin on acute lung injury induced by lipopolysaccharide in mice. Int. Immunopharmacol. 2012, 14, 369−374. (14) Bani, D.; Masini, E.; Bello, M. G.; Bigazzi, M.; Sacchi, T. B. Relaxin protects against myocardial injury caused by ischemia and reperfusion in rat heart. Am. J. Pathol. 1998, 152, 1367−1376. (15) Sheu, M. J.; Deng, J. S.; Huang, M. H.; Wu, C. H.; Huang, S. S.; Huang, G. J. Antioxidant and anti-inflammatory properties of Dichondra repens Forst. and its reference compounds. Food Chem. 2012, 132, 1010−1018. (16) Flohe, L.; Otting, F. Superoxide dismutase assays. Methods Enzymol. 1984, 105, 93−104. (17) Shao, Y. Y.; Chen, C. C.; Wang, H. Y.; Chiu, H. L.; Hseu, T. H.; Kuo, Y. H. Chemical constituents of Antrodia camphorata submerged whole broth. Nat. Prod. Res. 2008, 22, 1151−1157. (18) Wen, C. L.; Chang, C. C.; Huang, S. S.; Kuo, C. L.; Hsu, S. L.; Deng, J. S.; Huang, G. J. Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo. J. Ethnopharmacol. 2011, 137, 575−584. (19) Matthay, M. A.; Zimmerman, G. A. Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management. Am. J. Respir. Cell Mol. Biol. 2005, 33, 319−327. (20) Johnson, E. R.; Matthay, M. A. Acute lung injury: epidemiology, pathogenesis, and treatment. J. Aerosol Med. Pulm. Drug Delivery 2010, 23, 243−252. (21) Philippakis, G. E.; Lazaris, A. C.; Papathomas, T. G.; Zissis, C.; Agrogiannis, G.; Thomopoulou, G.; Nonni, A.; Xiromeritis, K.; Nikolopoulou-Stamati, P.; Bramis, J.; Patsouris, E.; Perrea, D.; Bellenis, I. Adrenaline attenuates the acute lung injury after intratracheal lipopolysaccharide instillation: an experimental study. Inhal. Toxicol. 2008, 20, 445−453. (22) Azoulay, É.; Canet, E.; Raffoux, E.; Lengliné, E.; Lemiale, V.; Vincent, F.; de Labarthe, A.; Seguin, A.; Boissel, N.; Dombret, H.; Schlemmer, B. Dexamethasone in patients with acute lung injury from acute monocytic leukaemia. Eur. Respir. J. 2012, 39, 648−653. (23) Zhang, S.; Rahman, M.; Zhang, S.; Song, L.; Herwald, H.; Thorlacius, H. Targeting rac1 signaling inhibits streptococcal m1 protein-induced CXC chemokine formation, neutrophil infiltration and lung injury. PLoS One 2013, 8, No. e71080. (24) Lin, Y. C.; Lai, Y. S.; Chou, T. C. The protective effect of αlipoic acid in lipopolysaccharide-induced acute lung injury is mediated by heme oxygenase-1. Evidence-Based Complement. Alternat. Med. 2013, 2013, No. 590363. (25) El-Sayed, N. S.; Mahran, L. G.; Khattab, M. M. Tempol, a membrane-permeable radical scavenger, ameliorates lipopolysaccharide-induced acute lung injury in mice: a key role for superoxide anion. Eur. J. Pharmacol. 2011, 663, 68−73. (26) Yang, R.; Yang, L.; Shen, X.; Cheng, W.; Zhao, B.; Ali, K. H.; Qian, Z.; Ji, H. Suppression of NF-κB pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice. Eur. J. Pharmacol. 2012, 674, 391−396.
(27) Ni, Y. F.; Kuai, J. K.; Lu, Z. F.; Yang, G. D.; Fu, H. Y.; Wang, J.; Tian, F.; Yan, X. L.; Zhao, Y. C.; Wang, Y. J.; Jiang, T. Glycyrrhizin treatment is associated with attenuation of lipopolysaccharide-induced acute lung injury by inhibiting cyclooxygenase-2 and inducible nitric oxide synthase expression. J. Surg. Res. 2011, 165, e29−e35. (28) Hseu, Y. C.; Wu, F. Y.; Wu, J. J.; Chen, J. Y.; Chang, W. H.; Lu, F. J.; Lai, Y. C.; Yang, H. L. Antiinflammatory potential of Antrodia camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-κB pathway. Int. Immunopharmacol. 2005, 5, 1914−1925. (29) Kosaka, J.; Morimatsu, H.; Takahashi, T.; Shimizu, H.; Kawanishi, S.; Omori, E.; Endo, Y.; Tamaki, N.; Morita, M.; Morita, K. Effects of biliverdin administration on acute lung injury induced by hemorrhagic shock and resuscitation in rats. PLoS One 2013, 8, No. e63606. (30) Kang, P.; Kim, K. Y.; Lee, H. S.; Min, S. S.; Seol, G. H. Antiinflammatory effects of anethole in lipopolysaccharide-induced acute lung injury in mice. Life Sci. 2013, 93, 955−961. (31) Chi, G.; Wei, M.; Xie, X.; Soromou, L. W.; Liu, F.; Zhao, S. Suppression of MAPK and NF-κB pathways by limonene contributes to attenuation of lipopolysaccharide-induced inflammatory responses in acute lung injury. Inflammation 2013, 36, 501−511. (32) Chen, J. J.; Lin, W. J.; Liao, C. H.; Shieh, P. C. Antiinflammatory benzenoids from Antrodia camphorata. J. Nat. Prod. 2007, 70, 989−992.
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