Stimulation of Innate Immune Function by Panax ginseng after Heat

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Stimulation of innate immune function by Panax ginseng after heat processing Myoung-Sook Shin, Ji Hoon Song, Pilju Choi, Jong Hun Lee, SongYi Kim, Kwang Soon Shin, Jungyeob Ham, and Ki Sung Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00152 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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Journal of Agricultural and Food Chemistry

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Running title: Immune function of ginseng

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Stimulation of innate immune function by Panax ginseng after heat

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processing

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Myoung-Sook Shina,1,, Ji Hoon Songb,1, Pilju Choia, Jong Hun Leec, Song-Yi Kimd, Kwang-Soon

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Shine, Jungyeob Hama,§ and Ki Sung Kangd,§

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a

Institute of Natural Products, Korea Institute of Science and Technology (KIST), Gangneung 210-

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340, Korea

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b

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c

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Gyeonggi 443-742, Korea

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d

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e

Department of Medicine, University of Ulsan College of Medicine, Seoul 05505, Korea

Department of Food Science and Biotechnology, College of Life Science, CHA University,

College of Korean Medicine, Gachon University, Seongnam 13120, Korea

Department of Food Science and Biotechnology, Kyonggi University, Suwon 443-760, Korea

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FOOTNOTES

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1. These two authors contributed equally to the works described in this study.

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2.

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PHONE: 82-31-750-5402. FAX: 82-31-750-5416. E-MAIL: [email protected] (Kang KS)

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PHONE: 82-33-650-3502. FAX: 82-33-650-3508. E-MAIL: [email protected] (Ham J)

§

Corresponding authors;

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ACS Paragon Plus Environment


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ABSTRACT

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Panax ginseng Meyer has been used for the treatment of immune diseases and for the strengthening

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the immune function. In this study, we evaluated the innate immune stimulating functions and action

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mechanisms of white ginseng (WG) and heat-processed ginseng (HPG) in RAW264.7 cells.

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According to LC-MS analysis results, WG contained typical ginsenosides, such as Rb1, Rc, Rb2, Rd,

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and Rg1, whereas HPG contained Rg3, Rk1, and Rg5 as well as typical ginsenosides. HPG, not WG

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enhanced NF-κB transcriptional activity, cytokine production (IL-6 and TNF-α), and MHC class I

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and II expression in RAW264.7 cells. In addition, HPG phosphorylated MAPKs and NF-kB

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pathways. In experiments with inhibitors, the ERK inhibitor completely suppressed the effect of

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HPG on IL-6 and TNF-α production. HPG-induced c-Jun activation was suppressed by an ERK

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inhibitor, and partially suppressed by JNK, p38, and IκBα inhibitors. Collectively, these results

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suggested that HPG containing Rg3, Rg5 and Rk1 increased macrophage activation which regulated

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by ERK/c-Jun pathway in RAW264.7 cells.

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KEYWORDS: Ginseng, heat processing, macrophage cells, ERK/c-Jun pathway

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1. INTRODUCTION

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Innate immunity plays a crucial role in the defense system against various infections by foreign

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organisms or harmful invaders.1.2 Macrophages are able to distinguish between self (host) and non-

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self (foreign materials or pathogens) entities. The membrane receptors, which include pattern

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recognition receptors (PRRs), also play a role in sensing non-self entities. Macrophages bind foreign

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materials (activators) or pathogens via Toll-like receptor (TLR)-4, complement receptor 3, CD14,

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dectin-1, the mannose receptor, or the scavenger receptor.3 These receptors are able to trigger the

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cascades of intracellular signaling molucules, such as NF-κB and the MAPK (JNK, ERK, p38)

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pathways, which results in gene transcription and the secretion of cytokines. 4-6

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NF-κB, a well-characterized transcription factor that exists predominantly in cells as p65 and p50

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heterodimers, plays an essential role in innate immune system-related gene expression.7,8 AP-1 is a

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downstream transcription factor of MAPKs, which are a group of basic leucine zipper proteins

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consisted of heterodimers such as c-Fos, c-Jun, ATF, and JDF.9 The major forms of AP-1 are Fos/Jun

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heterodimers that have high affinity for protein binding to AP-1 sites. AP-1 induces gene expression

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involved in cell differentiation and proliferation.9,10 Reactive oxygen species (ROS), IL-6, TNF-α

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and nitric oxide (NO) are well-known factors secreted by activated macrophages.11

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The root of ginseng (Panax ginseng Meyer) is traditional herbal medicine that has been used for

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the maintenance of immune homeostasis and to enhance resistance to illnesses by affecting the

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immune system.12,13 Asian ginseng stimulates the innate immune function of J774A.1 macrophage

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cells, but Siberian ginseng does not significantly increase innate immune function.14 Unlike other

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plants, P. ginseng contains ginsenosides Rg1, Rb1, Rb2, Rc, and Rd (Figure 1), and a variety of P.

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ginseng products such as red ginseng is available.15 Generally, ginsenosides are denatured by heat

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and acidic conditions. It is well-known that red ginseng exerts better biological activities than that by

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raw ginseng, which may result from the chemical transformation of ginsenosides during the heat

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treatment.16,17 In red ginseng production processing, the contents of converted ginsenosides such as 3



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Rg2, Rh1, and Rg3 increase, and the contents of the natural ginsenosides such as Rg1, Re, Rb1, Rc

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and Rd decrease.18 The heat processing is known to induce the chemical change of ginsenosides and

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to improve the pharmacological activities of ginseng. Several ginsenosides have been reported to

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have immunogenic effects, but the effects and mechanisms of heat-treated ginseng remain largely

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unknown. In the present study, therefore, we evaluated the innate immune stimulating functions and

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mechanisms of white ginseng (WG) and heat- processed ginseng (HPG) in RAW264.7 macrophages.

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2. MATERIALS AND METHODS

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2.1 Cell culture

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The RAW264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul,

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Republic of Korea) and maintained in high glucose DMEM supplemented with 10% fetal bovine

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serum (FBS, heat-inactivated at 56 °C for 30 min) in the presence of 1% penicillin-streptomycin in a

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humidified atmosphere at 37 °C and containing 5% CO2.

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2.2 Chemicals and Reagents

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Antibodies against JNK (FL), ERK1 (C-16), p38 (C-20), IκBα (C-21), p65 (C-20), MAPKAPK-2

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(H-66) and β-actin (I-19) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).

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Phospho-specific antibodies against p65 (Ser-536), JNK (Thr-183/Tyr-185), ERK1/2 (Thr-202/Tyr-

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204), p38 (Thr-180/Tyr-182), MAPKAPK-2 (Thr-222), and c-Jun (Ser-73), in addition to c-Jun was

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purchased

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histocompatibility complex (MHC) class I (H-2Db) FITC (clone 28-14-8), MHC class II (I-A/I-E)

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PE (clone M5/114.15.2), and the isotype-matched control antibodies were purchased from

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eBioscience (San Diego, CA, USA). The chemical inhibitors SB203580, SP600125, U0126, and

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BAY 11-7082 were obtained from Calbiochem (Darmstadt, Germany). All chemical inhibitors were

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prepared in DMSO to give a final DMSO concentration of 0.998). 5



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2.5 Cytotoxicity assay

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RAW264.7 macrophages were treated with HPG or WG at various concentrations (25–1000

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µg/mL) for 24 h. After incubation, 20 µL CCK-8 solution was added, the cells were incubated for a

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further 3 h, and the absorbance values were measured at 450 nm. The percentage of cell viability was

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measured by comparing with that of the control group.

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2.6 NF-κB transcriptional activity

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RAW264.7 macrophages were transfected with the pNiFty2-Luc plasmid, an NF-κB-inducible

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reporter plasmid purchased from InvivoGen (San Diego, CA, USA), by using Lipofectamine LTX

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(Invitrogen, CA, USA) as instructed by the manufacturer’s protocol. After transfection for 24 h, the

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cells were treated with Pam3CSK4 (200 ng/mL), HPG, or WG at the indicated concentrations for 20 h.

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NF-κB activation was subsequently measured by using the Luciferase Assay System as instructed by

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the manufacturer’s guidelines, and measurement using FilterMax F5 multi-mode microplate reader

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(Molecular Devices, USA). All experiments were performed in duplicate.

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2.7 Preparation of cell lysates and immunoblotting

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RAW264.7 cells were treated with HPG or WG at the indicated concentrations for the indicated

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periods. After treatment, the cells were washed with PBS and lysed in cold RIPA buffer (T&I, Seoul,

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Korea) supplemented with 1 mM PMSF, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM sodium

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orthovanadate, and protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA).

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The cells were scrapped by rubber policeman and then collected supernatants by centrifugation at

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13000 rpm, 15 min at 4 °C. The protein concentration of each sample was adjusted to be constant

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after measured with BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Samples were

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mixed with 4x Laemmli Sample Buffer containing 2-mercaptoethanol (Bio-Rad, Hercules, CA, 6


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USA), then boiling for 5 min using DRY BATH block for heating. Samples were electrophoresed by

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10% SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA,

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USA). The membrane was then incubated in 5% skim milk in Tris-buffered saline with 0.05% Tween

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20 (TBS-T) overnight at 4 °C. The primary antibodies were added and the membranes were

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incubated for 2 h with shaking at room temperature. The membranes were washed with TBS-T three

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times, treated with secondary antibodies (horseradish peroxidase-conjugated) for 1 h, and washed

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again with TBS-T three times. The membranes were visualized with the ECL system (Thermo

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Scientific, MA, USA) using an LAS-4000 (GE Healthcare Life Sciences, PA, USA).

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2.8 Flow cytometric analysis

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RAW264.7 macrophages were incubated with 100 or 200 µg/mL HPG for 24 h. After treatment,

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the cells were washed in PBS and then collected from the culture dish by scraping, centrifugation at

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300 g for 5 min, and resuspension of the resultant pellet in cold wash buffer (2% bovine calf serum

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in PBS). Subsequently, the cells were diluted to 1×106 cells/mL, the wash buffer was removed by

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centrifugation, and the cells were stained with FITC-conjugated anti-MHC class I or PE-conjugated

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anti-MHC class II antibody for 30 min on ice in the dark. Finally, the cells were washed three times

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with cold FACS buffer three times and then analyzed by FACSVerse flow cytometer system (BD

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Biosciences, CA, USA).

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2.9 ELISA assay for cytokines

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The RAW264.7 macrophage cells were incubated with HPG at the various concentrations. After 20

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h of incubation, the levels of TNF-α and IL-6 level were measured by using a sandwich ELISA kit

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according to the manufacturer’s instructions. All experiments were repeated in triplicate.

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2.10 Statistical analysis 7



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The results were expressed as the mean ± standard deviation of duplicate or triplicate experiments.

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Statistical analysis was performed using Student t-test with #P < 0.05 or

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statistically significant.

##

P < 0.01 accepted as

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3.RESULTS AND DISCUSSION

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3.1 Chemical properties of HPG and WG

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The HPLC chromatograms of all ginseng extract are presented in Figure 1. WG contained typical

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ginsenosides, such as Rb1, Rc, Rb2, Rd, and Rg1 (Fig. 1A). The HPLC chromatogram shown in Fig.

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1B was obtained from the analysis of ginsenosides in HPG. As shown in Fig. 1 and Table 1, when

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WG was heat-processed at 120 ºC, the ginsensides Rb1, Rb2, Rc, Rd, and Rg1 were converted to

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Rg3, Rg5, and Rk1. Additionally, the remaining ginsenoside in WG without a completely dissociated

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glycoside was detected between 5 min and 10 min.

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3.2 Effect of HPG and WG on the viability of RAW264.7 cells

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To understand the role of HPG and WG in macrophage stimulation, we first confirmed the

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cytotoxicity of HPG and WG in RAW264.7 cells. RAW264.7 cells were incubated with various

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concentrations (25–1000 µg/mL) of HPG or WG for 24 h. As shown in Fig. 2, treatment with low

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concentrations of HPG or WG were not cytotoxic, resulting in slightly increased cell proliferation at

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400–500 µg/mL. However, 1000 µg/mL HPG or WG was toxic to RAW264.7 cells. Therefore,

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subsequent experiments were conducted within a low concentration range (25–500 µg/mL) of HPG

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or WE.

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3.3 Effects of HPG and WG on NF-κB activity in transfected RAW264.7 cells

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To investigate changes in NF-κB transcriptional activity by HPG or WG, we treated HPG or WG

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for 20 h to transiently transfected RAW264.7 cells. As shown in Fig. 3, WG did not affect NF-κB 8


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transcriptional activity, whereas HPG strongly enhanced transcriptional activity of NF-κB in a

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concentration-dependent manner. Pam3CSK4 is a bacterial lipoprotein that is a potent activator of

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NF-κB.21 From these results it was determined that, HPG, but not WG, had the ability to trigger the

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signaling cascade that led to the activation of NF-κB transcriptional activity.

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3.4 Effects of HPG and WG on MHC class-I and class-II expression in RAW264.7 cells

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The three functions of macrophages that induce immune activation include phagocytosis for

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removal of foreign materials, processing of foreign materials, and presentation to membranes as

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antigen complexes with major histocompatibility complexes (MHCs).22-24 These antigen-MHCs are

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recognized by T-receptors, which initiate the acquired immune system.25 We therefore investigated

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MHC-class I expression by HPG or WG in RAW264.7 cells using flow cytometry system. Treatment

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of HPG (50 µg/mL), not WG (50 µg/mL) strongly increased MHC-class I expression in RAW264.7

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cells (data not shown). Next, we investigated MHC class-I and -II expression by HPG treatment. As

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shown in Fig. 4A and 4B, treatment with HPG enhanced MHC class I and II expression in

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concentration dependent manner in RAW264.7 macrophages. These results suggested that HPG

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might induce the activation of acquired immune systems such as T cell proliferation and

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differentiation.

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3.5 Effects of HPG and WG on the phosphorylation of MAPKs and NF-κB in RAW264.7 cells

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To understand the underlying signaling pathways by the HPG, we analyzed its effect on the

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phosphorylation of MAPKs and NF-κB in RAW264.7 cells. As shown in Fig. 5A, HPG induced the

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phosphorylation of MAPKs (ERK, JNK, and p38) in a concentration-dependent manner. However,

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WG did not cause the phosphorylation of MAPKs. The total amounts of ERK, JNK, and p38 were

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not affected by HPG or WG treatment for 30 min. In the NF-κB pathway, HPG induced the

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degradation of IκBα and phosphorylated p65 at Ser-536. In contrast, WG did not affect the 9



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degradation of IκBα or phosphorylation of p65 (Fig. 5B). The total amounts of p65 was not affected

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by HPG or WG treatment for 30 min. Collectively, these results indicated that HPG, not WG served

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as an activator of macrophages and resulted in the phosphorylation of the MAPK and NF-κB

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pathways. Therefore, we used HPG for next experiments.

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3.6 Effects of HPG on IL-6 and TNF-α productions in RAW264.7 macrophages

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TNF-α has been characterized as a cytotoxic factor produced by macrophages that induces

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apoptotic cell death, inflammation, and tumor inhibition. IL-6 is secreted from macrophage cells in

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response to specific microbial molecules such as pathogen-associated molecular patterns (PAMPs)

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and also participates in fighting infection. Therefore, we treated RAW264.7 cells with different

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concentrations of HPG (25–500 µg/mL) for 20 h. As shown in Fig. 6A, HPG strongly enhanced

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TNF-α production in a concentration dependent manner; the optimal concentration for the production

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of TNF-α was 400 µg/mL. However, HPG stimulated IL-6 production at 200 µg/mL and the optimal

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concentration for IL-6 production was 500 µg/mL (Fig. 6B). Collectively, these results suggested

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that HPG might possess immunostimulatory activity in RAW264.7 cells.

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3.7 ERK/c-Jun is a major signaling pathway for TNF-α and IL-6 production by HPG in RAW264.7

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macrophages

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Although many natural products, including ginseng extract, exert immune enhancing activity, the

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underlying mechanisms are still unclear.26-29 Therefore, we tried to identify the molecular

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mechanisms of HPG action using chemical inhibitors in RAW264.7 macrophages. Therefore, we

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first confirmed phosphorylation of MAPKs and NF-κB after treatment of chemical inhibitors in

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RAW264.7 cells. As shown in Fig.7A, treatment of SB (SB203585), SP (SP600125), U (U0126) and

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BAY (BAY 11-7082), a specific inhibitor of p38, JNK, MEK1/2 and IκBα respectively, suppressed

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HPG-induced phosphorylation of p38, JNK, ERK1/2 and p65. Since SB is an inhibitor of p38 10


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pathway, phosphorylation of MAPKAPK-2, a direct target molecule of p38 MAPK, was also

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inhibited in the same manner as phosphorylation of p38. The phosphorylation of JNK completely

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inhibited by SP treatment, because SP is a direct inhibitor for phosphorylation of JNK. MEK1/2 are

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upstream molecules of ERK1/2, treatment of U inhibited phosphorylation of ERK1/2. BAY is an

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inhibitor for degradation of IκBα, therefore p65 phosphorylation was inhibited. These data suggested

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that specific inhibitors did not affect other protein kinase activation. Next, we confirmed the

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phosphorylation of c-Jun, a downstream molecule of MAPKs after treatment of HPG. As shown in

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Fig. 7B, HPG significantly phosphorylated c-Jun for 30 min. In contrast, treatment with the specific

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MEK1/2 inhibitor (U) suppressed the HPG-induced phosphorylation of c-Jun. Further, specific

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inhibitors for p38 (SB), JNK (SP) and IκBα (BAY) also inhibited the phosphorylation of c-Jun. From

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these data, we speculated ERK1/2, JNK and NF-κB pathways are essential for macrophages

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activation by HPG.

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Based on the above results, next we determined HPG-induced TNF-α and IL-6 production signal

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pathways. As shown in Fig 7C, treatment with U completely suppressed HPG-induced TNF-α

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production, and treatment of SB and SP slightly inhibited TNF-α production. Similar result is shown

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in Fig. 7D; the treatment with U inhibited HPG-induced IL-6 production, SP and SB were partially

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involved in HPG-induced IL-6 production. However, treatment of BAY (BAY 11-7082), an inhibitor

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of IκBα phosphorylation, did not affect HPG-induced TNF-α and IL-6 production (Fig. 7C, 7D).

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These results suggested that ERK1/2-c-Jun pathway is a major regulator of HPG-induced TNF-α and

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IL-6 production, and JNK or p38 partially regulated HPG-induced IL-6 production in RAW234.7

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cells. Collectively, these results suggested that TNF-α and IL-6 production by HPG occurred

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primarily through the mainly ERK/c-Jun pathway in RAW264.7 cells (Fig. 7).

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The less polar ginsenosides Rg3, Rg5, and Rk1 are major components of HPG. From the analysis

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of the production of TNF-α in the presence of less polar ginsenosides than Rb1, HPG-induced TNF-α

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production was deduced to be related to ginsenosides Rg3, Rg5, and Rk1 (Fig. 8A). In addition, we 11



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investigated whether immune enhancing activity exhibit in non-ginsenoside fractions such as

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polysaccharides in RAW264.7 cells. As shown in Fig. 8B, polysaccharides fraction of ginseng

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increased TNF-a production in concentration dependent manner, and ginsenosides fraction also

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produced TNF-α. Further, HPG-treated group showed higher TNF-α production than polysaccharides

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or ginsenosides treated group. These results suggested that immunostimulatory activity of HPG can

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be attributed not only to less polar ginsenosides to polysaccharides (Fig. 8B).

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In summary, HPG enhanced cytokine production, NF-κB transcription activity, and MHC class I

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and II expression in RAW264.7 cells. HPG strongly activated the MAPKs and NF-κB (IκBα-p65)

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pathways. In specific inhibitor experiments, the MEK1/2 inhibitor (U) completely suppressed the

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effect of HPG on IL-6 and TNF-α productions, and JNK and p38 inhibitors partially suppressed IL-6

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production (Fig. 9). In addition, HPG-induced c-Jun activation was completely suppressed by an

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ERK inhibitor, whereas JNK (SP), p38 (SB), and IκBα (BAY) inhibitors partially contributed.

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Therefore, these results suggested that HPG possessed ginsenoside Rg3, Rg5 and Rk1 increased

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macrophage activation which regulated by mainly the ERK/c-Jun pathway in RAW264.7 cells. The

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synergistic action of ginsenoside with other ginseng ingredients will be carried out in our future

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research theme.

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Acknowledgments

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This study was supported by the National Research Foundation of Korea (NRF) grant supported by

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the Korea government (MSIP) (NRF-2014R1A1A3050928). This work was also funded by the

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Korea Institute of Science and Technology institutional program (2Z04930).

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Conflict of interest

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All the authors have no conflicts of interest to declare.

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24. Mosser, D.M. The many faces of macrophage activation. J. Leuko. Biol. 2003, 73(2), 209-212.

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25. Iwasaki, A.; Medzhitov, R. Regulation of adaptive immunity by the innate immune

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system. Science. 2010, 327(5963), 291-295.

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26. Azike, C.G.; Charpentier, P.A.; Hou, J.; Pei, H.; Lui, E.M.K. The Yin and Yang actions of North

350

American ginseng root in modulating the immune function of macrophages. Chin. med. 2011,

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6(1), 21.

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27. Kim, B.G.; Shin, K.S.; Yoon, T.J.; Yu, K.W.; Ra, K.S.; Kim, J.M.; Suh, H.J. Fermentation of

353

Korean red ginseng by Lactobacillus plantarum M-2 and its immunological activities. Appl.

354

Biochem. Biotechnol. 2011, 165(5-6), 1107-1119.

355

28. Shin, M.S.; Hwang, S.H.; Yoon, T.J.; Kim, S.H.; Shin, K.S. Polysaccharides from ginseng leaves

356

inhibit tumor metastasis via macrophage and NK cell activation. Int. J. Biol. Macromol. 2017,

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103, 1327–1333.

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29. Hwang, S.H.; Shin, M.S.; Yoon, T.J.; Shin, K.S. Immunoadjuvant activity in mice of

359

polysaccharides isolated from the leaves of Panax ginseng C.A. Meyer. Int. J. Biol. Macromol.

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2018, 107, 2695-2700.

361 362 363

15



364

Legends for figures

365 366

Table 1. Comparison of ginsenoside contents between WG and HPG (mg/g extract). Data are

367

presented as the means ± SD of three independent experiments. ##P < 0.01 vs. the WG group.

368 369

Figure 1. Chromatograms of ginseng extracts. HPLC chromatogram of ginsenoside analysis (Rg1,

370

Rb1, Rc, Rb2, and Rd) in WG (A). HPLC chromatogram of ginsenoside analysis (Rg1, Rb1, Rc, Rb2,

371

Rd, 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5) in HPG (B). Data are presented as the means ± SD of

372

three independent experiments. ##P < 0.01 vs. the WG group.

373 374

Figure 2. Effect of HPG and WG on the viability of RAW264.7 cells. RAW264.7 cells (1.0 × 105

375

cells/well, 96-well plates) were treated with HPG or WG at a series of concentrations (25–1000

376

µg/mL) for 24 h and cytotoxicity was determined using a CCK-8-based colorimetric assay. Data are

377

presented as the means ± SD of three independent experiments. #P < 0.05 vs. the control group.

378 379

Figure 3. Effect of HPG and WG on NF-κB transcriptional activity.

380

Transiently transfected RAW264.7e cells (4 × 105 cells/well, 24-well plate) were treated with HPG or

381

WG at 50 µg/mL and 100 µg/mL for 20 h. NF-κB transcriptional activity was measured using

382

Luciferase Assay System. Data are presented as the means ± SD of three independent experiments.

383

##

P < 0.01 or #P < 0.05 vs. the control group.

384 385

Figure 4. Effects of HPG on MHC class-I and class-II expression in RAW264.7 cells. RAW264.7

386

cells (2.0 × 106 cells/6-cm dish) were treated with the indicated concentrations (100 µg/mL, 200

387

µg/mL) of HPG for 24 h. The cells were stained with FITC-conjugated anti-MHC class I (A) or PE-

388

conjugated MHC class II antibody (B), then analyzed with FACSVerse flow cytometry system. The 16


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389

bar chart displays the median of the fluorescence intensity (MFI) for each histogram. Data are

390

presented as the means ± SD of three independent experiments. ##P < 0.01 vs. the MHC-I control or

391

MHC-II control group.

392 393

Figure 5. Effect of HPG and WG on the phosphorylation of MAPK and NF-κB in RAW264.7

394

cells. RAW264.7 cells (2.5 × 106 cells/6-cm dish) were treated with the indicated concentrations (25

395

µg/mL, 50 µg/mL) of HPG or WG for 30 min. Whole-cell lysates were then immunoblotted with the

396

specific antibodies indicated on the left side of each panel. The phosphorylation of ERK, JNK, and

397

p38 was determined by using phospho-specific antibodies and total ERK1/2, JNK, and p38 were

398

detected by specific antibodies (A). RAW264.7 cells (2.5 × 106 cells/6-cm dish) were treated with the

399

indicated concentrations (25 µg/mL, 50 µg/mL) of HPG or WG for 30 min. Whole-cell lysates were

400

then immunoblotted with the specific antibodies. IκBα, the phosphorylation of p65, and total p65

401

were detected by specific antibodies (B). β-Actin served as an internal loading control. All

402

immunoblot bands obtained from the same cell lysates (A, B).

403 404

Figure 6. Effects of HPG on TNF-α and IL-6 production in RAW264.7 cells. RAW264.7 cells (4

405

× 105 cells/well, 24-well plate) were treated with HPG at the indicated concentration for 20 h. The

406

supernatant was collected and the concentrations of TNF-α and IL-6 were measured by sandwich

407

ELISA assay kit. Data are presented as the means ± SD of three independent experiments. ##P < 0.01

408

or #P < 0.05 vs. the control group.

409 410

Figure 7. ERK/c-Jun axis is a major signaling pathway for TNF-α and IL-6 production by

411

HPG in RAW264.7 cells. RAW264.7 macrophages (2.5 × 106 cells/6-cm dish) were pretreated with

412

SB203580 (10 µM), SP600125 (10 µM), U0126 (10 µM), or BAY11-7082 (5 µM) for 30 min and

413

then stimulated with HPG (50 µg/mL) for 30 min. Whole-cell lysates were immunoblotted with the 17



414

specific antibodies indicated on the left side of each panel. The phosphorylation of c-Jun was

415

determined by using a phospho-specific antibody and total c-Jun was detected by a specific antibody.

416

β-Actin served as an internal loading control (A, B). RAW264.7 macrophages (4 × 105 cells/mL, 24-

417

well plate) were pretreated with SB203580 (10 µM), SP600125 (10 µM), U0126 (10 µM), or

418

BAY11-7082 (5 µM) for

419

30 min and then stimulated with HPG (500 µg/mL) for 20 h. The resulting release of TNF-α and IL-

420

6 into the culture media was quantified by using ELISA kits). The bar chart displays the intensity of

421

phospho-c-Jun after normalized by c-Jun using Image J software. Data are presented as the means ±

422

SD of three independent experiments. ##P < 0.01 or #P < 0.05 vs. the HPG treatment group.

423 424

Figure 8. HPG-containing ginsenosides contribute TNF-α production in RAW264.7 cells

425

RAW264.7 cells (4 × 105 cells/well, 24-well plate) were treated with the indicated ginsenosides at

426

the indicated concentrations (25 µg/mL ~ 100 µg/mL) for 18 h. The supernatant was collected and

427

the concentration of TNF-α was measured by sandwich ELISA assay kit (A). RAW264.7 cells (1 ×

428

105 cells/well, 96-well plate) were treated with the indicated samples at the indicated concentrations

429

(12.5 µg/mL ~ 50 µg/mL) for 22 h. The supernatant was collected and the concentration of TNF-α

430

was measured by sandwich ELISA assay kit (B). Data are presented as the means ± SD of three

431

independent experiments. ##P < 0.01 vs. the control group.

432 433

Figure 9. Signal pathways underlying the HPG-regulated immune effects.

434 435 436 437 438 18


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Page 19 of 29


439 440

Table 1. Comparison of ginsenoside contents between WG and HPG (mg/g extract). Data are

441

presented as the means ± SD of three independent experiments. ##P < 0.01 vs. the WG group.

Rg1

Rb1

Rc

Rb2

S-

R-

Rg3

Rg3

Rd

Rk1

Rg5

Total

WG

22.4

67.2

56.0

32.1

17.9

N.D.

N.D.

N.D.

N.D.

195.6

HPG

13.6

32.9

21.3

13.5

12.4

21.5##

12.3##

15.6##

16.4##

159.5

442

19



Figure 1. Chromatograms of ginseng extracts. HPLC chromatogram of ginsenoside analysis (Rg1, Rb1, Rc, Rb2, and Rd) in WG (A). HPLC chromatogram of ginsenoside analysis (Rg1, Rb1, Rc, Rb2, Rd, 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5) in HPG (B). Data are presented as the means ± SD of three independent experiments. ##P < 0.01 vs. the WG group. 133x175mm (96 x 96 DPI)


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Figure 2. Effect of HPG and WG on the viability of RAW264.7 cells. RAW264.7 cells (1.0 × 105 cells/well, 96well plates) were treated with HPG or WG at a series of concentrations (25–1000 µg/mL) for 24 h and cytotoxicity was determined using a CCK-8-based colorimetric assay. Data are presented as the means ± SD of three independent experiments. #P < 0.05 vs. the control group. 96x118mm (96 x 96 DPI)



Figure 3. Effect of HPG and WG on NF-κB transcriptional activity. Transiently transfected RAW264.7e cells (4 × 105 cells/well, 24-well plate) were treated with HPG or WG at 50 µg/mL and 100 µg/mL for 20 h. NF-κB transcriptional activity was measured using Luciferase Assay System. Data are presented as the means ± SD of three independent experiments. ##P < 0.01 or #P < 0.05 vs. the control group. 80x84mm (96 x 96 DPI)


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Figure 4. Effects of HPG on MHC class-I and class-II expression in RAW264.7 cells. RAW264.7 cells (2.0 × 106 cells/6-cm dish) were treated with the indicated concentrations (100 µg/mL, 200 µg/mL) of HPG for 24 h. The cells were stained with FITC-conjugated anti-MHC class I (A) or PE-conjugated MHC class II antibody (B), then analyzed with FACSVerse flow cytometry system. The bar chart displays the median of the fluorescence intensity (MFI) for each histogram. Data are presented as the means ± SD of three independent experiments. ##P < 0.01 vs. the MHC-I control or MHC-II control group. 161x205mm (96 x 96 DPI)



Figure 5. Effect of HPG and WG on the phosphorylation of MAPK and NF-κB in RAW264.7 cells. RAW264.7 cells (2.5 × 106 cells/6-cm dish) were treated with the indicated concentrations (25 µg/mL, 50 µg/mL) of HPG or WG for 30 min. Whole-cell lysates were then immunoblotted with the specific antibodies indicated on the left side of each panel. The phosphorylation of ERK, JNK, and p38 was determined by using phosphospecific antibodies and total ERK1/2, JNK, and p38 were detected by specific antibodies (A). RAW264.7 cells (2.5 × 106 cells/6-cm dish) were treated with the indicated concentrations (25 µg/mL, 50 µg/mL) of HPG or WG for 30 min. Whole-cell lysates were then immunoblotted with the specific antibodies. IκBα, the phosphorylation of p65, and total p65 were detected by specific antibodies (B). β-Actin served as an internal loading control. All immunoblot bands obtained from the same cell lysates (A, B). 110x203mm (96 x 96 DPI)


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Figure 6. Effects of HPG on TNF-α and IL-6 production in RAW264.7 cells. RAW264.7 cells (4 × 105 cells/well, 24-well plate) were treated with HPG at the indicated concentration for 20 h. The supernatant was collected and the concentrations of TNF-α and IL-6 were measured by sandwich ELISA assay kit. Data are presented as the means ± SD of three independent experiments. ##P < 0.01 or #P < 0.05 vs. the control group. 166x111mm (96 x 96 DPI)



Figure 7. ERK/c-Jun axis is a major signaling pathway for TNF-α and IL-6 production by HPG in RAW264.7 cells. RAW264.7 macrophages (2.5 × 106 cells/6-cm dish) were pretreated with SB203580 (10 µM), SP600125 (10 µM), U0126 (10 µM), or BAY11-7082 (5 µM) for 30 min and then stimulated with HPG (50 µg/mL) for 30 min. Whole-cell lysates were immunoblotted with the specific antibodies indicated on the left side of each panel. The phosphorylation of c-Jun was determined by using a phospho-specific antibody and total c-Jun was detected by a specific antibody. β-Actin served as an internal loading control (A, B). RAW264.7 macrophages (4 × 105 cells/mL, 24-well plate) were pretreated with SB203580 (10 µM), SP600125 (10 µM), U0126 (10 µM), or BAY11-7082 (5 µM) for 30 min and then stimulated with HPG (500 µg/mL) for 20 h. The resulting release of TNF-α and IL-6 into the culture media was quantified by using ELISA kits). The bar chart displays the intensity of phospho-c-Jun after normalized by c-Jun using Image J software. Data are presented as the means ± SD of three independent experiments. ##P < 0.01 or #P < 0.05 vs. the HPG treatment group.


Page 26 of 29

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163x238mm (96 x 96 DPI)



Figure 8. HPG-containing ginsenosides contribute TNF-α production in RAW264.7 cells RAW264.7 cells (4 × 105 cells/well, 24-well plate) were treated with the indicated ginsenosides at the indicated concentrations (25 µg/mL ~ 100 µg/mL) for 18 h. The supernatant was collected and the concentration of TNF-α was measured by sandwich ELISA assay kit (A). RAW264.7 cells (1 × 105 cells/well, 96-well plate) were treated with the indicated samples at the indicated concentrations (12.5 µg/mL ~ 50 µg/mL) for 22 h. The supernatant was collected and the concentration of TNF-α was measured by sandwich ELISA assay kit (B). Data are presented as the means ± SD of three independent experiments. ##P < 0.01 vs. the control group.

173x127mm (96 x 96 DPI)


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Figure 9. Signal pathways underlying the HPG-regulated immune effects. 87x107mm (96 x 96 DPI)



TOC 177x159mm (96 x 96 DPI)


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Stimulation of Innate Immune Function by Panax ginseng after Heat

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