The immune-enhancing activity of Cervus nippon mantchuricus extract (NGE) in RAW264.7 macrophage cells and immunosuppressed mice

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The immune-enhancing activity of Cervus nippon mantchuricus extract (NGE) in RAW264.7 macrophage cells and immunosuppressed mice Se Hyang Honga, Jin Mo Kua, Hyo In Kima, Chang-Won Ahnb, Soo-Hyun Parkb, Hye Sook Seoc, Yong Cheol Shinc, Seong-Gyu Koc,⁎ a b c

Department of Science in Korean Medicine, Graduate School, Kyung Hee University, Seoul 02447, Democratic People's Republic of Korea Research and Development Center, Nong Shim Co., Ltd., Seoul 07057, Democratic People's Republic of Korea Department of Preventive Medicine, College of Korean Medicine, Kyung Hee University, Seoul 02447, Democratic People's Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Chemical compounds studied in this article: Lipopolysaccharide (PubChem CID: 11970143) Cyclophosphamide (PubChem CID: 2907)

Chemotherapeutics are often used to inhibit the proliferation of cancer cells. However, they can also harm healthy cells and cause side effects such as immunosuppression. Especially traditional oriental medicines long used in Asia, may be beneficial candidates for the alleviation of immune diseases. Cervus nippon mantchuricus extract (NGE) is currently sold in the market as coffee and health drinks. However, NGE was not widely investigated and efficacy remain unclear and essentially nothing is known about their potential immune-regulatory properties. As a result, NGE induced the differentiation of RAW264.7 macrophage cells. NGE-stimulated RAW264.7 macrophage cells elevated cytokines levels and NO production. NGE-stimulated RAW264.7 macrophage cells activated MAPKs and NF-κB signaling pathways. NGE encouraged the immuno-enhancing effects in immunosuppressed short-term treated with NGE mice model. NGE or Red ginseng encouraged the immunoenhancing effects in immunosuppressed long-term treated with NGE mice model. Our data clearly show that NGE contains immune-enhancing activity and can be used to treat immunodeficiency.

Keywords: Cervus nippon mantchuricus Extract Deer bone Immunosuppressed mice Macrophages Immune-enhancing activity MAPK NF-κB

1. Introduction The regulation of immune response plays a pivotal role in preventing and treating diseases, and increasing attention has been poured to the investigation of immunomodulation and immunostimulation induced by active substance. Previous reports indicated that immunomodulators could help the host defense responses, which was an efficient way to enhance resistance to disease (Yu, Kong, Zhang, Sun, & Chen, 2016). Many investigators have shown that the increase of physical immunity could efficiently defense against disease. Immunotherapy has been proposed for more than a century and has made spectacular progress in recent years. Lipopolysaccharide (LPS) interacts with the cell-surface protein Toll-like receptor 4 (TLR4) and intracellular connector protein MyD88 (Takeda, Kaisho, & Akira, 2003). The TLR4 is a member of the TLR superfamily, involved in the innate immune system and triggers LPS response (Aderem & Ulevitch, 2000). LPS is known to have ability to activate macrophages (Poltorak et al., 1998). Macrophages play essential role in immunity and inflammatory processes, being induced by TLR ligands, and they secrete various cytokines including tumor

necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β, IL-6, IL12, granulocyte macrophage colony stimulating factor (GM-CSF), nitric oxide (NO), inducible NO synthase (iNOS) and prostaglandin E2 (PGE2) (Agarwal, Piesco, Johns, & Riccelli, 1995). Various transduction pathways are related with differentiation, cell activation and cytokine secretion of macrophages. The mitogen activated protein kinase (MAPK) and Nuclear factor (NF)-κB signaling pathways are the most important intracellular transduction cascades for macrophages (Varin & Gordon, 2009). Extract from Cervus nippon mantchuricus, deer bone extract, called nok-gol (NGE) and Red ginseng (RG), is the most commonly used traditional remedies in oriental medicine. They have been widely used to invigorate Qi. Previous works have indicated that the RG is well known immune modulator (Lee, Hwang, et al., 2014; M. J. Lee et al., 2016; Lee, Han, et al., 2014). RG could be heat steamed and dried. As a consequence of this process, RG undergoes certain biochemical changes and acquires certain pharmacological properties such as anti-aging, anti-viral, anti-memory loss, anti-obesity and anti-cancer activities (T. H. Kang et al., 2009; Kim, Hahm, Yang, Lee, & Shim, 2005; M. H. Lee, Seo, Kang, Oh, & Choi, 2014; Y. Lee & Oh, 2015; C. Z. Wang, Anderson,

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Corresponding author. E-mail addresses: [email protected] (S.H. Hong), [email protected] (J.M. Ku), [email protected] (H. In Kim), [email protected] (C.-W. Ahn), [email protected] (S.-H. Park), [email protected] (H.S. Seo), [email protected] (Y.C. Shin), [email protected] (S.-G. Ko). http://dx.doi.org/10.1016/j.foodres.2017.06.053 Received 23 March 2017; Received in revised form 20 June 2017; Accepted 21 June 2017 0963-9969/ © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

Please cite this article as: Hong, S.H., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.06.053

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incubated for 30 min at room temperature. OD was determined at 570 nm using a ELISA reader (Versa Max, Molecular Devices, Sunnyvale, CA, USA).

Du, He, & Yuan, 2016). NGE, supplied by Nongshim Corporation is currently sold in the market as coffee and health drinks. In recent study, it was found that NGE oral administration is useful for the treatment of various inflammatory diseases, memory loss, bone resorption and neutropenia (Choi et al., 2013; Du et al., 2015; S. K. Kang et al., 2006; H. Lee, Choi, et al., 2014; Lee, Park, et al., 2014). However, NGE was not widely investigated and efficacy remains unclear and essentially nothing is known about their potential immune-regulatory properties. In this study, we evaluated the immune-enhancing activity of NGE in vitro measuring leukocytes levels, NO levels, and cytokines levels in RAW264.7 macrophage cells. The intervention of MAPK pathway and NF-κB pathway is also investigated. Moreover, we evaluated the immune-enhancing activity of NGE in vivo using immunosuppressed mice. Our data clearly show that NGE contains immune-enhancing activity and can be used to treat immunodeficiency.

2.6. Western blot analysis Harvested cells were lysed with buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 1 mM Na3VO4, 1 mM DTT, 1 mM NaF, 1 mM PMSF, and PI cocktail on ice for 30 min. The lysates were cleared by centrifugation at 13,000 rpm for 20 min at 4 °C. The supernatant was stored at −70 °C until use. The protein concentration was quantified using a Bio-Rad Bradford protein assay (Bio-Rad, Hercules, CA, USA). Next, total proteins (15–20 μg) were electrophoresed using 6–15% reducing SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 0.1% Tween-20 in PBS (PBST) containing 1% skim milk and 1% BSA for 1 h, the membranes were incubated overnight at 4 °C with the indicated primary antibodies. After washing in 1× PBST for 15 min (3 times × 5 min), the membranes were incubated with diluted enzyme-linked secondary antibodies. After washing in 1× PBST for 1 h (4 times × 15 min), the protein bands were detected using the EZ-western chemiluminescent detection kit and visualized by exposing the membranes to X-ray films. In a parallel experiment, cytoplasmic and nuclear proteins were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions. Each protein was blotted by appropriate antibodies as follows: anti-NF-κB, p-EKR1/2, p-P38, p-JNK and Lamin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-anti-iNOS, COX-2, p-NF-κB and GAPDH antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-α-tubulin antibody was purchased from Sigma (St. Louis, MO, USA).

2. Materials and methods 2.1. Cervus nippon mantchuricus extract (NGE) and Red ginseng (RG) preparation Cervus nippon mantchuricus extract (NGE) was provided by Nongshim Corporation (Seoul, Korea). NGE contained 88.8% of crude protein, 1.9% of crude fat, 2.2% of crude ash, and 3.0% of moisture. Total ganglioside content of the extract as sialic acid was 0.09%. Total amino acid and free amino acid amount of extract were 922.5 and 8.21 mg/g, respectively. NGE is white powder extracted by water and dissolved in water for experiments. Red ginseng (RG) was purchased from Korea Ginseng Corporation (KGC, Daejeon, Korea). RG product is made of concentrated six-years-grown Korean Red ginseng extract. More information on NGE (http://nongshim.co.kr) and RG (http:// www.kgc.co.kr) production processes could be requested to the company.

2.7. Animals 2.2. Cell culture

The RAW 264.7 cells (5 × 105 cells/mL) were cultured in the presence of LPS and NGE (25, 100, 500 μg/mL) for 24 h in 60 mm culture dish. Morphological changes were analyzed by taking images using a camera connected to a light microscopy (Olympus, Tokyo, Japan).

Six-week-old male BALB/c mice (20 ± 2 g) were purchased from Orient (Sungnam, Korea). The mice were randomized into eight (no treatment, cyclophosphamide, G-CSF, NGE 25 mg/kg, NGE 50 mg/kg, NGE 100 mg/kg, NGE 200 mg/kg, NGE 400 mg/kg) and seven groups (no treatment, cyclophosphamide, G-CSF, NGE pre-treatment, RG pretreatment, NGE post-treatment, RG post-treatment), each comprising six mice. All mice were kept under pathogen-free environment and allowed free access to the diet and water. All methods were carried out in accordance with relevant guidelines and regulations and procedures involving mice were approved by the animal care center of Kyung-Hee University (Approval Number KHUASP (SE)-14-014). At the end of the experiment, mice were sacrificed by CO2 inhalation, and cardiac blood was collected.

2.4. WST assay

2.8. Sensitization and treatment

Cell viability was determined using WST assay (Dogen, Seoul, Korea). RAW264.7 cells (1 × 104 cells/well) were seeded into 96-well plates and incubated overnight. Cells were then treated with different concentrations of NGE and incubated for another 24 h. 10 μL of WST solution was added to 100 μL cell culture medium, and plates were incubated for 2 h. Optical density (OD) was determined at 450 nm using a ELISA reader (Versa Max, Molecular Devices, Sunnyvale, CA, USA).

To induce immunosuppression, mice were intraperitoneally injected with 100 mg/kg of cyclophosphamide twice in one week and then treated with cyclophosphamide or G-CSF (1 μg/kg) two times in next week. Mice were rested for one week with no treatment when treatments were switched. NGE and RG (200 mg/kg) pre-treatment group was orally administered every day before the induction with cyclophosphamide or G-CSF. NGE and RG (200 mg/kg) post-treatment group was orally administered every day for the last procedure. At the end of the experiment, mice were sacrificed by CO2 inhalation, and cardiac blood was collected.

The RAW264.7 murine macrophage cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Welgene, Daegu, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Welgene, Daegu, Korea) and 1% antibiotics (Ab, Welgene, Daegu, Korea) at 37 °C in a 5% CO2 humidified incubator. 2.3. Cell morphology

2.5. Griess assay NO production was measured in RAW 264.7 culture supernatant using Griess reagent kit (Promega, Madison, WI, USA). In detail, 150 μL of supernatant from each well was transferred to 96-well plate and then mixed with 150 μL of Griess reagent solution. Mixtures were then

2.9. Spleen weight The Spleens from all mice were removed and weighed immediately after sacrifice. 2

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Student's t-test was used for single variable comparisons, and a P value < 0.05 was considered statistically significant. Statistical analyses were performed using PRISM software (GraphPad Software Inc., La Jolla, CA, USA,).

2.10. Blood analyses Mouse whole blood samples were collected by cardiac puncture. The blood was placed in Vacutainer TM tubes containing EDTA (BD science, San Jose, NJ, USA). Anti-coagulated blood was processed to determine hematological parameters (WBC, lymphocytes, monocytes, eosinophils, basophils and neutrophils) in a HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Dallas, TX, USA) in accordance to manufacturer's recommendation.

3. Results 3.1. Cervus nippon mantchuricus extract (NGE) induces RAW264.7 macrophage cells differentiation

2.11. ELISA assay

To examine the effect of NGE on cell viability, RAW264.7 cells were treated with NGE or LPS (1 μg/mL) for 24 h (Fig. 1A). NGE treatment did not affect the cell viability. Besides, both NGE and LPS increased WST activities (Fig. 1A) in RAW264.7 cells, indicating that NGE induced activities of dehydrogenases. Thus, for further experiments, the macrophages were treated with NGE in the concentration range 25, 100, and 500 μg/mL. We next investigated the morphology of the macrophage RAW264.7 cells under NGE or LPS treatment (Fig. 1B). LPS has been known to induce a differentiation of monocytic RAW264.7 cells to macrophages (Agarwal et al., 1995). Consistently, LPS induced Raw264.7 differentiation in our experiment. Likewise, NGE treatment also resulted in Raw264.7 cell differentiation in a dose-dependent manner. Taken together, these results demonstrate that NGE induces RAW264.7 cell differentiation to macrophages.

The levels of cytokines in the serum of mice and in RAW264.7 cells were determined by sandwich ELISA using the BD PharMingen mouse or human ELISA set (Pharmingen, San Diego, CA, USA). Briefly, plates were coated with capture antibody in ELISA coating buffer (Sigma, Louis, MO, USA) and incubated overnight at 4 °C. Plates were washed with PBS-Tween 20 (0.05%) and subsequently blocked (10% FBS in PBS) for 1 h at 20 °C. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates and plates were incubated for 2 h at 20 °C. After washing, biotin-conjugated antimouse IL-6 and SAv-HRP (streptavidin-horseradish peroxidase conjugate) were added to the plates and plates were incubated for 1 h at 20 °C. Finally, tetramethylbenzidine (TMB) substrate solution was added to the plates and after 15 min incubation in the dark, a 2 N H2SO4 solution was added to stop the reaction. Optical densities were measured at 450 nm on an automated ELISA reader (Versa Max, Molecular Devices, Sunnyvale, CA, USA). IFN-γ and IL-12 levels were also measured in plasma by sandwich ELISA using the BD PharMingen mouse ELISA set. Next procedure followed the same protocol as described above.

3.2. NGE-stimulated RAW264.7 macrophage cells elevate cytokines levels and NO production Next, it was determined whether NGE has a role in the regulation of cytokines and NO. It is known that LPS interacts with the cell surface protein Toll like receptor4 (TLR4), and stimulates RAW264.7 macrophage cells (Takeda et al., 2003). As expected, LPS-mediated stimulation of RAW264.7 cells significantly promoted the expression of IL-6 and TNF-α and NO production. RT-PCR analysis showed that iNOS, IL-6 and TNF-α mRNA levels were increased by NGE treatment in RAW264.7 macrophage cells (Fig. 2A). Moreover, the up-regulation of immune-stimulatory mRNA level of immune-enhancing-related immune-stimulatory cytokines IL-6, IL-12 and NO production was observed in the RAW264.7 macrophage cells treated with NGE compared with the control group (Fig. 2B). These results indicate that NGE enhance the activation of mRNA (iNOS, IL-6 and TNF-α) level, cytokine level and NO production in RAW264.7 macrophage cells.

2.12. RT-PCR RNA was isolated from the mouse spleen tissues and cell using easyblue RNA extraction kit (iNtRON Biotech, Sungnam, Korea) according to the manufacturer's instructions. Isolated RNA content was measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). 2 μg of total cellular RNA from each sample was reverse transcribed using cDNA synthesis kit (TaKaRa, Otsu, Shinga, Japan). PCR was conducted out in a 20 μL reaction mixture consisting of DNA template, 10 pM of each gene-specific primer, 10 × Taq buffer, 2.5 mM dNTP mixture, and 1 unit of Taq DNA polymerase (Takara, Otsu, Shinga, Japan). PCR was performed using the specific primer and primer sequences for mouse iNOS, IL-6, TNF-α and GAPDH are shown in Table 1.

3.3. NGE-stimulated RAW264.7 macrophage cells activate MAPKs and NFκB signaling pathways

2.13. Statistical analysis To determine the mechanism by which NGE activates cytokines (IL6, IL-12 and TNF-α) and up-regulates NO production, we checked MAPKs and NF-κB signaling pathway. It was reported that LPS treatment resulted in the activation of all three MAPKs and NF-κB pathways (Dauphinee & Karsan, 2006). When RAW264.7 macrophage cells were treated with 25, 100 and 500 μg/mL of NGE for 24 h, ERK phosphorylation was marginally increased after concentration of 500 μg/mL NGE treatment. Furthermore, we also found that NGE activates phosphorylation of JNK and p38. JNK and p38 phosphorylation was marginally increased after concentration of 500 μg/mL NGE treatment (Fig. 3A). NGE induced the phosphorylation of ERK, JNK, and p38 in a dose-dependent manner. Accordingly, NGE treatment resulted in nuclear accumulation of phosphorylated form of NF-κB (Fig. 3B). Moreover, NGE treatment greatly increased expression of both iNOS and COX-2 (Fig. 3A). Our findings indicate that NGE induces the activation of MAPK and NF-κB pathways as like LPS.

All experiment results were expressed as the means ± standard deviations (SD) or means ± SEM of at least three separate tests. Table 1 The sequence of PCR primer. Primer type

Primer name

Primer sequence

Moues

IL-6

F: 5′-CAA GAG ACT TCC ATC CAG TTG C-3′ R: 5′-TTG CCG AGT TCT CAA AGT GAC-3′ F: 5′-AAT GGC AAC ATC AGG TCG GCC ATC ACT3′ R: 5′-GCT GTG TGT CAC AGA AGT CTC GAA CTC3′ F: 5′-ATG AGC ACA GAA AGC ATG ATC-3′ R: 5′-TAC AGG CTT GTC ACT GGA ATT-3′ F: 5′-GAG GGG CCA TCC ACA GTC TTC-3′ R: 5′-CAT CAC CAT CTT CCA GGA GCG-3′

iNOS

TNF-a GAPDH

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Fig. 1. NGE activates murine macrophage RAW264.7 cells. (A) RAW264.7 cells (1 × 104 cell/mL) were treated with various concentrations of NGE or LPS (1 μg/mL) and then incubated for 24 h. After treatment, cell viability was measured using WST assay. (B) Cells were seeded into 60 mm culture dish at a density of 3x104 cell/dish. The next day, cells were treated with NGE for 24 h. RAW264.7 cell surface was observed by taking a photograph using a camera attached to a microscope. Data are presented as mean ± SEM. ∗∗∗ P < 0.001 compared with non-stimulated cells.

each WBCs cell number to similar extent. Furthermore, we measured the levels of IL-6, IL-12 and IFN-γ in serum of immunosuppressed shortterm mice treated with different concentrations of NGE (Fig. 4B). NGE reversed the decrease of IL-6, IL-12, IFN-γ) level induced by CP (Fig. 4B). These results suggest that NGE effectively encouraged immune-enhancing by promoting the CP-stimulated decrease of the number of WBCs, neutrophils, monocytes, lymphocytes and cytokine (IL-6, IL-12, IFN-γ) level in serum.

3.4. NGE encourages the immuno-enhancing effects in immunosuppressed short-term mice model To investigate the immune-enhancing effects of dose-dependent NGE under physiological conditions, an in vivo study was performed on short-term. NGE was orally administered every day to cyclophosphamide (CP)-immunosuppressed mice and the mice were intraperitoneally injected with CP or G-CSF twice in one week (Supplementary Fig. 1A). We also monitored the body weight changes and food intake every day of the week throughout the study. We found that CP treatment did not show any changes of food intake but decreased body weight by 15% compared with vehicle-treated mice (Supplementary Fig. 1B, C). Moreover, we found that spleen weight was significantly decreased by CP and it was markedly induced by application of NGE or G-CSF (Supplementary Fig. 1D). Next, we determined the number of white blood cells (WBSs) in the blood of mice using HAMAVET950 hematology analyzer. In the immunosuppressed shortterm mice applied with CP, the total number of WBCs was significantly decreased compared with vehicle group mice, but this number of WBCs was dramatically induced by NGE or G-CSF (Fig. 4A). Counting each subtypes of WBCs including neutrophils, monocytes, and lymphocytes showed that NGE and G-CSF encouraged the CP-induced decrease in

3.5. NGE or Red ginseng encourages the immuno-enhancing effects in immunosuppressed long-term mice model To gain the immune-enhancing effects of NGE compared with Red ginseng (RG), an in vivo study was achieved on long-term. RG has been used as a representative traditional remedy in oriental medicine. Moreover, RG has been reported to have immune-stimulatory effects via functional activation of macrophages (Byeon et al., 2012). RG enhanced the phagocytic activity of macrophages (Matsuda, Hasegawa, & Kubo, 1985) and stimulated the generation of inflammatory mediators like nitric oxide (NO) (Choi et al., 2008). NGE or RG was pre-administered orally every day to mice since one week before sensitization with CP. On the other hand, NGE or RG was post-administered orally every day 4

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Fig. 2. NGE upregulates immune-stimulatory mRNA, cytokines, and NO production in murine macrophage RAW264.7 cells. Murine macrophage RAW264.7 cells were stimulated NGE (25, 100, or 500 mg/mL) or LPS for 24 h. (A) The iNOS, IL-6 and TNF-α mRNA levels were measured by RT-PCR analysis in murine macrophage RAW264.7 cells. The bar graphs represent the quantitation of RT-PCR data. (B) The level of IL-6, IL-12 and NO production in cell culture supernatants were measured by sandwich ELISA. Data are presented as mean ± SEM. ∗P < 0.05, **P < 0.01 and ***P < 0.001 as compared to non-stimulated cells, respectively.

increased as compared with CP group by NGE or RG administration in spleen tissue of CP-induced immunosuppressed mice (Fig. 6B). Our data clearly show that NGE increased significantly the leukocytes (WBC, neutrophils, lymphocytes, monocytes, eosinophils and basophils) levels, mRNA expression (iNOS, TNF-α, IL-6) and immune-enhancing cytokines (IL-6, IL-12, IFN-γ) reduced by CP in mice. Furthermore, pretreatment group (NGE or RG) maintained leukocytes numbers, immune-stimulatory mRNA levels and cytokines levels related with immune-enhancing activity without decrease induced by CP. Post-treatment group also recovered leukocytes numbers, mRNA levels and cytokines levels related with immune-enhancing activity as like pretreatment group. It should be noted that NGE showed stronger immuneenhancing activity than RG.

to CP-induced immunosuppressed mice. BALB/c mice were intraperitoneally injected with CP or G-CSF twice a week (Supplementary Fig. 2A). We determined the amount of toxicity and body weight three times a week to check stress caused by the NGE or RG. Food intake and body weight were monitored throughout the study. As a result, we found that vehicle group shows higher weight as compared to other groups. We also found that CP-injected group did not show any changes of food intake but exhibited a slight decrease (15%) in body weight compared with vehicle group (Supplementary Fig. 2B, C). In addition, we determined the WBCs and each subtypes of WBCs including granulocytes (neutrophils, basophils, eosinophils), monocytes, and lymphocytes. Our data showed that NGE or RG encouraged the CP-induced decrease in blood cell number like G-CSF (Fig. 5). Besides, we further tested whether NGE or RG regulates expression of cytokines at transcriptional level (Fig. 6). ELISA assay showed that IL-6, IL-12 and IFN-γ cytokine levels were induced in NGE or RG administered group as compared with CP group in serum of CP-induced immunosuppressed mice (Fig. 6A). Specially, the NGE pre-administered group considerably induced IL-6 level. RT-PCR analysis showed that iNOS, TNF-α and IL-6 mRNA levels were reduced by agonist treatment and significantly

4. Discussion Chemotherapeutics are often used to inhibit the proliferation of cancer cells. However, they can also harm healthy cells and cause side effects such as immunosuppression (H. Wang et al., 2011). Traditional oriental medicines long used in Asia, may be beneficial candidates for Fig. 3. NGE-induced MAPK and NF-κB signaling activation in RAW264.7 macrophages. Cells were stimulated with NGE (25, 100, or 500 mg/mL) or LPS (1 μg/mL). (A) Phosphorylated ERK1/2, p38, JNK and isolated iNOS, COX2 from cell lysates were determined by immunoblotting analysis. GAPDH was used as an internal control. (B) Cytoplasmic extracts (upper panel) and nuclear extracts (lower panel) were isolated from cells stimulated with NGE or LPS. Level of NF-κB was determined by immunoblotting analysis. Tubulin and lamin were used as loading controls for the cytoplasmic and nuclear fractions, respectively.

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2014). Moreover, WBCs fight against infection causing phagocytosis and deliver the pathogens to phagosomes, where pathogens are destroyed (Parham & Janeway, 2009). Lymphocytes are reflected as an important parameter which plays a crucial role in regulating immune system through the differentiation and proliferation of T-cells and Bcells (Vivier, Ugolini, Blaise, Chabannon, & Brossay, 2012). In order to stimulate the state of a weakened immune system we used mice treated with CP and GM-CSF. The spleen is important immune organs and immune-potentiators could increase the weight of the spleen (Pang et al., 2007). The spleen functions as a secondary lymphoid tissue where lymphocytes are activated with antigens derived from an infection (Pozo, Godfrey, & Bowles, 2009), suggesting that the increase in spleen weight was possibly due to the greater activity of this organ (Liu et al., 2015). In our model, the effect of CP treatment was indicated by reduced spleen weight, since CP decreases the capacity of the immune system. However, the relative spleen weights in NGE administration groups were higher than CP treatment groups. The present study shows the increase in WBCs and each subtypes of WBCs including granulocytes (neutrophils, basophils, eosinophils), monocytes, and lymphocytes by NGE or RG which encourage CP-induced decrease like G-CSF. Notably, we found that NGE enhanced the production of NO, secretion of immune-stimulatory cytokine (IL-6, IL-12, IFN-γ) in serum and the expression of immune-stimulatory mRNA (iNOS, TNF-α, IL-6) in spleen These results support the conclusion that NGE stimulates the immune system.

the alleviation of immune diseases. In recent years, many studies have shown that extract from Cervus nippon mantchuricus (NGE) and Red ginseng (RG) had wide pharmacologic effects and are immune-modulator (Choi et al., 2013; Lee, Hwang, et al., 2014). The objective of this study was to characterize the immune-enhancing activity of NGE performing the experiments in the murine macrophage cells and CP-induced immunosuppressed mice. Lipopolysaccharide (LPS) has been reported to synergize with TLR4 for cytokine production in macrophages (Poltorak et al., 1998). Moreover, it is now evident that mammalian Toll-like receptor family (TLRs) plays a prominent role in the direct activation of host defense mechanisms. The activation of TLRs stimulates an innate immune response (Medzhitov, 2007), which involves the production of direct antimicrobial effector molecules, including NO, and increases an adaptive immune response by inducing the production of IL-1β, IL-6, TNF-a, IFNγ and IL-12 that augment both cell-mediated and immune responses (Modlin, 2002). Since macrophages produce colony stimulating factor (CSF), an activation of macrophages is important for preventing neutropenia and secondary diseases (Gordon, 2003). The first objective of this study was to characterize the immuneenhancing activity of NGE. For that purpose, we observed cell differentiation and measured immune-stimulatory mRNA, cytokine and NO production levels. Our results demonstrate that NGE increased the differentiation, the production of cytokines, such as IL-6 and IL-12, and effector molecules such as NO in macrophage. In the present study, NGE upregulated the mRNA levels of iNOS, TNF-α and IL-6 in a dose-dependent manner in murine macrophage. NGE and LPS seem to have similar effects on cell differentiation and cytokine production in macrophages in a dose-dependent manner. The stimulation of TLRs leads to the activation of several MAPK pathways, resulting in the increase of antibody and cytokine production in macrophages (Zhang et al., 1999). Previous studies have shown that MAPK pathway has a crucial role in innate immune response signaling (Dong, Davis, & Flavell, 2002). The three major families of MAPK pathway, which mediate innate immune response signaling, include ERK1/2, p38 and c-Jun N-terminal kinase (JNK) (Rincon, Flavell, & Davis, 2000). The activation of MAPK pathway subsequently induces gene expressions by activating several transcription factors, including NF-κB (Ninomiya-Tsuji et al., 1999). NF-κB activity in macrophages can be triggered by a range of different stimuli, including LPS (Muzio, Natoli, Saccani, Levrero, & Mantovani, 1998). Within the nucleus, these p65-containing transcription factors regulate the expression of genes associated with the immune response, including TNF-α (Shakhov, Collart, Vassalli, Nedospasov, & Jongeneel, 1990) and inducible nitric oxide synthase (iNOS) (Hughes et al., 2008). We found that NGE activated MAPKs (ERK1/2, P38 and JNK). Therefore, the downstream regulators of NF-κB signaling pathway might be the target for NGE. In contrast, NGE markedly decreased the responses induced in macrophages by expression of immune-enhancing cytokines through modulation of MAPK and NF-κB transcription factors. Moreover, NGE treatment greatly increased expression of both iNOS and COX-2. Our results demonstrate that NGE has a typical specificity to selectively and efficiently activate macrophages. To estimate the immune-stimulatory activity of the NGE on immune system, we used cyclophosphamide (CP)-treated immunosuppressed mice model. CP is an important chemotherapeutic drug that adverses immune system of organism and leads to immunosuppression (H. Wang et al., 2011). Granulocyte macrophage-colony stimulating factor (GMCSF) is synthesized and secreted by a variety of activated cells including macrophages, T lymphocytes and cytokine-activated endothelial cells (Strieter et al., 1989). The immuno-stimulatory activity of GM-CSF have been broadly implicated in neutropenic patients after chemotherapy and the patients showed major increase in total white blood cell (WBC) and neutrophil counts after GM-CSF treatment (Roberts, 2005). Neutropenia is associated with the treatment of different disease conditions such as cancer chemotherapy (Shabbir, Shahzad, Ali, & Zia-ur-Rehman,

5. Conclusion Our purpose of the study is to know whether NGE has an immuneenhancing effect. NGE enhanced the activity of macrophages, and expression of immune-stimulatory mRNA and increased levels of cytokines and NO. More importantly, NGE contributed to its immune-enhancing effect by stimulating macrophages through up-regulation of the MAPK or NF-κB signaling pathways. In CP-induced immunosuppressed mice, NGE encouraged the impairment in leukocyte, cytokine, NO production and expression of immune-stimulatory mRNA. Hence, we conclude that NGE is helpful for alleviating an effective immunosuppression. It should be noted that NGE showed stronger immune-enhancing activity than RG. The present results support the possibility that NGE are good candidate compounds for the activation of TLR signaling and subsequently for the treatment of infectious diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2017.06.053. Conflict of interests The authors have no conflict of interest to declare. Acknowledgments This work was supported by Nongshim Corporation (no. 20121086), and a grant from Korean Medicine R & D Project of the Ministry of Health and Welfare (HI12C1889 and HI13C0530). References Aderem, A., & Ulevitch, R. J. (2000). Toll-like receptors in the induction of the innate immune response. Nature, 406(6797), 782–787. http://dx.doi.org/10.1038/ 35021228. Agarwal, S., Piesco, N. P., Johns, L. P., & Riccelli, A. E. (1995). Differential expression of Il-1-beta, Tnf-alpha, Il-6, and Il-8 in human monocytes in response to lipopolysaccharides from different microbes. Journal of Dental Research, 74(4), 1057–1065. Byeon, S. E., Lee, J., Kim, J. H., Yang, W. S., Kwak, Y. S., Kim, S. Y., ... Cho, J. Y. (2012). Molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from Korean red ginseng. Mediators of Inflammation, 2012, 732860. http://dx.doi. org/10.1155/2012/732860. Choi, H. S., Kim, S. R., Hong, S. H., Ku, J. M., Kim, M. K., Seo, H. S., ... Ko, S. G. (2013).

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