Increased Th1 cytokine secretion by chiisanogenin and chiisanoside isolated from <i>Acanthopanax chiisanensis</i>

DOI 10.17480/psk.2017.61.6.344

Increased Th1 cytokine secretion by chiisanogenin and chiisanoside isolated from Acanthopanax chiisanensis

Chang-Eui Hong

, Seung-Yeon Han

, Hwan-Gyu Kim

, and Su-Yun Lyu

*

Department of Biology, Chonbuk National University, 567, Baekje-daero, deokjin-gu, Jeonju-si, Jeonbuk, 561-756, Korea

**

Department of Pharmacy, Sunchon National University, 255, Jungangno, Suncheon, Jeonnam, 540-742, Korea

***

Suncheon Research Center for Natural Medicines, Suncheon, Jeonnam, 540-950, Korea (Received December 15, 2017; Revised December 21, 2017; Accepted December 22, 2017)

Abstract — Acanthopanax species (Araliaceae) is used traditionally in Korea, Japan, and China, and is known for its ginseng- like activities and among them, Acanthopanax chiisanensis Nakai is one of the most abundant. In this study, we investigated the effects of chiisanoside and chiisanogenin isolated from A. chiisanensis on the secretion of interleukin (IL)-2, IL-12, tumor necrosis factor alpha (TNF- α), and interferon gamma (IFN-γ) by human lymphocyte cell line U937. Our results show an increase in Th1 cytokine (IL-2, IL-12, TNF- α, and IFN-γ) secretion which suggests that chiisanogenin and chiisanoside from A. chiisanensis may influence the immune response towards a predominance of Th1 cytokines in the immune system. Fur- ther studies on Th2 cytokine secretion may be needed to confirm this conclusion.

Keywords Acanthopanax chiisanensis, Chiisanogenin, Chiisanoside, Immuno-stiumlation

Introduction

Acanthopanax species (Araliaceae) are used traditionally in Korea, Japan, and China, and is known for its ginseng-like activities.

Approximately 15 species of this genus are known to be grown in Korea, and among them, Acanthopanax chiisan- ensis Nakai is one of the most abundant. It has been taken widely to treat rheumatism, diabetes, and hypertension.

Previously, Hahn et al isolated 3,4-seco-lupane type triter- penes from A. chiisanensis which included chiisanoside, chiisa- nogenin, and isochiisanoside.

Chiisanoside has been reported to have anti-hepatotoxic activity, anti-diabetic activity, and effects on mitogen-induced proliferation of lymphocytes.

Also, chiisanogenin inhibited H

/K

-ATPase which may lead to protection from ulcer of the stomach.

However, no infor- mation on the immunostimulatory effect of chiisanoside and chiisanogenin has been reported.

Lymphocytes play a major role in host defense as part of the innate and adaptive immunity system

and can be divided into T lymphocytes, B lymphocytes, and natural killer (NK) cells.

T-cells and B-cells are the major cellular component of the adaptive immune response.

Among these two cells, T lym- phocytes are classified into two subsets, Th1- and Th2-type cells, based on the types of cytokines they produce. Th1 cells mainly secrete IL-2, interferon-gamma (IFN- γ), and TNF-α, whereas Th2 cells secrete IL-3, IL-4, IL-5, IL-6, and IL-10.

Th1 cells are responsible for cell-mediated immunity, cytotox- icity, and macrophage activation and Th2 cells are responsible for humoral immune response.

In this study, we investigated the effects of chiisanoside and chiisanogenin isolated from A. chiisanensis on the secretion of IL-2, IL-12, TNF- α, and IFN-γ by human lymphocyte cell line U937. The goal of this study was to identify and provide addi- tional insight immuno-stimulatory cytokines induced in this cell by chiisanoside and chiisanogenin.

Experiment methods

Preparation of chiisanoside and chiisanogenin from A.

chiisanensis

#

Corresponding Author Su-Yun Lyu

Department of Pharmacy, Sunchon National University, 255 Jun- gangno, Suncheon, Jeonnam, 540-742, Korea

Tel.: 061-750-3759 Fax.: 061-750-3708 E-mail: [email protected]

†

These authors contributed equally to this work

Short Report

The plant was collected from Jiri-mountain in Jeonbuk Prov- ince, and the leaves were separated. Chiisanoside (Fig. 1A) and chiisanogenin (Fig. 1B) were isolated from the leaves and stem bark of A. chiisanensis and provided by Dr. Duk-Ryong Hahn at Susin Ogapy Co. (Chunahn, Chungnam, Korea) as reported previ- ously.

Resulting solutions were filtered step-by-step with var- ious membrane filters (0.8, 0.45, and 0.22 μm; Merck Millipore, Temecula, CA, USA). Samples were stored in −80

C until used.

Cell culture

Human U937 lymphocyte cell line was obtained from Korea Cell Bank (Seoul, Korea) and was maintained in RPMI medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100 U/mL peni- cillin (Sigma, St. Louis, MO, USA), and 100 μg/mL streptomycin (Sigma) at 37

C in a humidified atmosphere containing 5% CO

.

Determination of the cell density

Cells were counted with a hematocytometer using trypan blue (Sigma) dye exclusion. Twenty microliters of trypan blue solution was mixed with an equal amount of cell suspension and was loaded onto a hematocytometer. Cells that excluded the dye were counted within 5 min after mixing of the dye and cell suspension.

Cell cytotoxicity test

The cytotoxic effect was determined by using MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt; MTS, Promega, UK) and an elec- tron coupling reagent (phenozine methosulphate; PMS, Promega). Cells were maintained at a temperature of 37

C in a 5% CO

humidified incubator (Sanyo, Japan) for 48 hr. The for- mazan production was determined by measuring the absor- bance at 450 nm with a spectrophotometric reader (Molecular Devices, Sunnyvale, CA, USA).

Enzyme-linked immunosorbent assay (ELISA) for measuring cytokine release

Various concentrations of chiisanoside and chiisanogenin were added to the cells and incubated at 37

C in a 5% CO

humidified incubator (Sanyo) for 48 hr, and 200 μL of the cul- ture supernatant were removed for the measurement of Th2 cytokines; IL-2, IL-12, TNF- α, and IFN-γ. To measure the cytokine secretion, 96-well ELISA plates (SPL Life Sciences, Korea) were coated with purified anti-mouse monoclonal anti- body (BD Biosciences, San Jose, CA, USA) in coating buffer (0.1 M sodium carbonate, pH 9.5) and incubated overnight at 4

C. Wells were aspirated and washed 3 times with 300 μL of wash buffer containing phosphate buffered saline (PBS, Sigma) with 0.05% Tween-20 (Sigma). Afterwards, plates were blocked with 200 μL of 1% (w/v) bovine serum albumin (BSA, Sigma) at room temperature for 1 h. Following 3 washes, sam- ples and standards (BD Biosciences) were added and incu- bated at room temperature for 2 h. After 4 washes, 100 μL of biotinylated detection antibody (BD Biosciences) diluted in blocking buffer was added and incubated at room temperature for 1 h. Following 5 washes, the presence of biotinylated anti- bodies was detected with streptavidin-peroxide (BD Biosci- ences) by incubating the plate at room temperature for 1 h. At the end of the incubation, the plate was washed 7 times and developed using tetramethylbenzidine (TMB) substrate reagent set (BD Biosciences) and the reaction was stopped with 50 μL of 1 M H

PO

. The plate was read at 450 nm within 30 min of the stopping reaction with a spectrophotometric reader (Molecular Devices).

Statistical analysis

Statistical analysis was performed with MINITAB (MINITAB, Fig. 1 − Structure of chiisanoside (A) and chiisanogenin (B) isolated

from the leaves and stem bark of Acanthopanax chiisanensis.

Minitab Inc., State College, PA, USA) using Student’s t-test. Prob- ability values ( P-value) of <0.001, <0.01, or <0.05 were consid- ered significant with 99.9, 99, or 95% of confidence, respectively.

Experimental results

Cytotoxic effects of chiisanogenin and chiisanoside on U937 cells

Human U937 lymphocytes were treated with chiisanogenin and chiisanoside for 48 hr at various concentrations and the viability was determined by MTS assay. As a result, more than 90% of the cells survived at concentrations lower than 100 μg/

ml (Fig. 2) when treated with chiisanogenin or chiisanoside.

The results obtained showed that the chosen concentrations of chiisanogenin and chiisanoside that were used for cytokine secretion assay had no major cytotoxic effect on U937 cells, compared to the controls.

Effect of chiisanogenin and chiisanoside on IL-2 secretion U937 cells were treated with chiisanogenin and chiisanoside to determine whether these components from A. chiisanensis have an effect on IL-2 secretion. As a result, when cells were treated with 10

– 10

mg/ml, chiisanogenin shifted the IL-12 secretion towards a more immunostimulative response. On the other hand, when the cells were treated with 10

– 10

mg/

ml, there was no significant change compared to the control (Fig. 3A). In the case of chiisanoside, the most significant

change was observed at 10

mg/ml, showing a 2.72 fold increase. However, there was no change of IL-2 secretion in the concentration range of 10

– 10

mg/ml, compared to the control (Fig. 3B).

Effect of chiisanogenin and chiisanoside on IL-12 secretion U937 cells were treated with chiisanogenin and chiisano- side, and their effects on IL-12 secretion were observed. When cells were treated with 10

– 10

mg/ml of chiisanogenin, there was a significant fold change in IL-12 secretion, the most significant increase shown at 10

mg/ml (Fig. 4A). In case of chiisanoside, there was a significant increase of IL-12 secre- tion when treated at 10

– 10

mg/ml, the most prominent increase shown at 10

mg/ml. However, there was no change of IL-12 secretion at 10

– 10

mg/ml, compared to the con- trol (Fig. 4B).

Fig. 2 − Cell viability of U937 human lymphocytes treated with chiisanogenin and chiisanoside determined by MTS assay.

Cells were maintained at a temperature of 37

C in a 5%

CO

humidified incubator for 48 hrr and the formazan production was determined by measuring the absorbance at 450 nm with a spectrophotometric reader.

Fig. 3 − Increase of IL-2 secretion in U937 human lymphocytes by

chiisanogenin and chiisanoside. Cells were treated with

various concentrations of chiisanogenin and chiisanoside

for 48 hr. Probability values of <0.001, <0.01, or <0.05

were considered significant with 99.9%, 99% or 95% of

confidence, respectively.

Effect of chiisanogenin and chiisanoside on TNF- α secretion

We further studied the effects of chiisanogenin and chiisano- side on TNF- α secretion, which is related to anti-cancer activi- ties, in U937 cells. As a result, chiisanogenin significantly increased the TNF- α secretion as high as 2.06-fold at the con- centration of 10

– 10

mg/ml. On the other hand, there was no change below the concentration of 10

mg/ml (Fig. 5A). In the case of chiisanoside, no significant increase of TNF- α secre- tion was observed in U937 cells, compared to control (Fig. 5B).

Effect of chiisanogenin and chiisanoside on IFN- γ secretion

Chiisanogenin induced IFN- γ secretion in U937 cells at 10

– 10

mg/ml, and the most significant change was seen at 10

mg/ml, showing a 2.96-fold change (Fig. 6A). When the cells were treated with chiisanoside, we could see a significant increase at the concentration of 10

and 10

– 10

mg/ml.

However, IFN-γ secretion started to decrease when the cells were treated with the concentration lower than 10

mg/ml (Fig. 6B) The most significant change was when cells were treated with 10

mg/ml of chiisanoside (2.94-fold change).

Discussion

The plants belonging to Acanthopanax species have been traditionally used in Korea.

We wondered whether chiisano- genin and chiisanoside from A. chiisanensis could act as an immunomodulator which could lead to improved cell-mediated immunity. Cytokines are produced by variety of immune cells such as B cells, T cells, macrophages, and monocytes. They have a key role in immune modulation in organisms such as in regulating cell survival, cell death and inflammation.

Selected cytokines that were measured in this study were IL- 2, IL-12, TNF- α, and IFN-γ, which are all type 1 cytokines.

Th1 cells are responsible for cell-mediated immunity: phago- Fig. 4 − Increase of IL-12 secretion in U937 human lymphocytes by

chiisanogenin and chiisanoside. Cells were treated with various concentrations of chiisanogenin and chiisanoside for 48 hr. Probability values of <0.001, <0.01, or <0.05 were considered significant with 99.9%, 99% or 95% of confidence, respectively.

Fig. 5 − Increase of TNF-α secretion in U937 human lymphocytes by

chiisanogenin and chiisanoside. Cells were treated with

various concentrations of chiisanogenin and chiisanoside for 48

hr. Probability values of <0.001, <0.01, or <0.05 were

considered significant with 99.9%, 99% or 95% of confidence,

respectively.

cyte-dependent host response, cytotoxicity, and macrophage activation. On the other hand, Th2 cells are responsible for the humoral immune response: activation of eosinophilic cells, inhibition of macrophage function and mast cell growth.

IL-2 regulates lymphocyte differentiation, immune response and homeostasis, controls regulatory T cells and differentiates T helper cell subsets including Th1, Th2 and Th17 cells.

In specific, IL-2 signals influence the differentiation of effector CD4

cells into Th1 or Th2 cells. Polarization to the Th1 cell phenotype depends on IL-12 signals, which lead to STAT4- mediated induction of the Th1 cell signature transcription fac- tor, T-bet.

In this study, it was shown that IL-2 and IL-12 secretion was dose-dependently increased in U937 human lym- phocytes by chiisanogenin and chiisanoside. Since contact with IL-2 promotes Th1 cell generation by increasing the levels of the IL-12 receptor β2 subunit (IL-12Rβ2) and of T-bet

, we can conclude that the increase of IL-2 led to the promotion of IL-12 secretion in our study.

IL-12 has the ability to induce IFN- γ production by T and NK cells to enhance the cytolytic response and to promote Th1 cell generation.

Since our study showed an increase in IL-2 and IL-12 secretion, it may be possible to assume that this led to the promotion of IFN- γ production, because recent data suggested that that IL-2 promotes Th1 cell differentiation via the induction of T-bet expression and the upregulation of the IL-12R β2 subunit which results in enhance IFN-γ produc- tion.

In addition, TNF- α is a significant regulator of the inflammatory and immune responses. It is released in many cell types including lymphocytes, monocytes, and macro- phages and is considered to be an important molecule during regulation of the immune response against tumors.

Since TNF- α and IL-2 act synergistically with IL-12 to augment IFN- γ production

, we can assume that the increased secretion of TNF- α by chiisanogenin and chiisanoside helped trigger the production of IFN- γ in our experiment.

The analysis and expression of cytokines have been a com- monly used technique in research and clinical laboratories and is clearly important in expanding our comprehension of many immunological functions.

Taken together, our results show an increase in Th1 cytokine secretion which suggests that chii- sanogenin and chiisanoside from A. chiisanensis may influence the immune response towards a predominance of Th1 cyto- kines in the immune system. Further studies on Th2 cytokine secretion may be needed to confirm this conclusion.

Conclusion

Oriental medicines have been shown to have biological activi- ties that can enhance the hose defense system and have been applied for cancer immunotherapy.

Since cancer patients in Asia tend to lean on traditional medicines for complementary effects during anti-cancer therapy, it might be interesting to fur- ther study the immunomodulatory effects of chiisanogenin and chiisanoside from Acanthopanax chiisanensis because cancer patients tend to show decreased inflammatory responsiveness.

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