Effects of MOK, a pharmacopuncture medicine, on the TH1/TH2 immune response and antioxidation in Con A-stimulated primary mouse splenocytes

MOK 약침의 Con A로 활성화된 마우스 비장세포에서의 TH1/TH2 면역반응과 항산화에 대한 효과

Article information

Acupunct. 2017;34(2):39-48
Publication date (electronic) : 2017 May 20
doi : https://doi.org/10.13045/acupunct.2017081
Korean Medicine R&D Center, Dongguk University
*Corresponding author: Korean Medicine R&D Center, Dongguk University, Dongdaero 123, Gyeongju 38066, Republic of Korea., Tel: +82-54-770-2658, E-mail: jhbori@nate.com
Received 2017 April 6; Revised 2017 May 1; Accepted 2017 May 8.

Abstract

Objectives

In this study, we investigated the immunomodulatory and antioxidant effect of MOK, a pharmacopuncture medicine, in concanavalin A (Con A)-stimulated mouse splenocytes.

Methods

Primary splenocytes were isolated from ICR mice. The splenocytes were treated with MOK extract (1.25, 2.5, 5, 10, and 20 mg/mL) for 30 min and then stimulated with Con A (200 ng/mL) for the indicated times. Cell viability of the splenocytes was measured using an MTT assay. The mRNA expression of Th1/Th2 cytokines (IFN-γ, IL-4, IL-10, and Foxp3) and antioxidant enzymes (HO-1 and MnSOD) was measured by RT-PCR.

Results

Addition of MOK extract at 2.5, 5, and 10 mg/mL in Con A-stimulated splenocytes significantly decreased the production of IFN-γ and significantly increased the expression of IL-4, IL-10, and Foxp3 mRNA. MOK extract also increased the mRNA expression of HO-1 and MnSOD in splenocytes.

Conclusion

MOK extract modulated the Th1/Th2 immune response via the regulation of cytokine levels in splenocytes and exerted an antioxidant effect via the upregulation of antioxidant proteins.

Trans Abstract

목적

본 연구에서는 Con A 자극으로 활성화된 마우스(mouse) 비장세포에서 MOK 약침의 TH1/TH2 면역반응 및 항산화에 대한 효과를 확인하였다.

방법

ICR 수컷 마우스의 비장을 떼어내 비장세포(primary splenocytes)를 분리하여 배양하였다. 비장세포에 MOK 약침액(1.25, 2.5, 5, 10, 20 ㎎/㎖)을 처리하여 30분간 배양한 후 Con A (200 ng/mL)를 처리하여 일정 시간 배양하였다. 세포독성은 MTT assay 방법으로 측정하였으며, 세포배양액으로부터 Th1/Th2 사이토카인 (IFN-γ, IL-4, IL-10, Foxp3) 및 항산화 효소(HO-1, MnSOD)의 유전자 발현을 Reverse transcription PCR 방법으로 하였다.

결과

Con A 자극으로 활성화된 마우스 비장세포에 MOK 약침액을 처리하여 세포독성을 평가한 결과, 10 ㎎/㎖까지 나타나지 않았다. MOK 약침액은 IFN-γ의 유전자 발현을 유의적으로 억제하였고, IL-4, IL-10, Foxp3의 유전자 발현을 유의적으로 증가시켰다. 또한 MOK 약침액은 마우스 비장세포에서 항산화효소인 HO-1과 MnSOD의 유전자 발현을 유의적으로 증가시켰다.

결론

본 연구결과로부터 MOK 약침액은 Con A 유도 마우스 비장세포 활성화에서 TH1과 TH2 사이토카인 발현 조절 및 항산화 단백질의 발현 증가를 통해 TH1/TH2 면역반응에 대한 조절효과 및 항산화 효과를 나타내는 것을 알 수 있었다.

I. Introduction

In order to efficiently overcome infections, immunomodulation boosts the immune system through the coordination of many immune cells. Splenocytes consist of different white blood cell types, such as T and B lymphocytes, dendritic cells, and macrophages, which have different immune functions. In the body, the spleen is an important immune organ that contains a relatively homogenous fraction of B (60%) and T lymphocytes (40%). Thus, the immunomodulatory evaluation of splenocytes can provide an understanding of the effects of T and B cells1).

Cytokines produced by CD4+ helper T (Th) lymphocytes are known to regulate the functions of the immune system, including antibody production and cellular immune response. Th cells represent a functionally heterogeneous population, composed of distinct subsets termed Th1 and Th2, which are defined by their cytokine secretion profiles2). In general, cytokines produced by Th1 cells (e.g., IFN-γ and IL-2) promote the production of complement-fixing and opsonizing antibodies and macrophage activation. Cytokines produced by Th2 cells (e.g., IL-4, IL-5, IL-6, IL-10, andIL-13) have been reported to stimulate antibody production and promote mast cell and eosinophil granulocyte differentiation and activation3). However, Th1 cytokines antagonize Th2 cell generation, and Th2 cytokines antagonize Th1 cell generation. Therefore, Th1 cells (IFN-γ) can exacerbate Th1-mediated autoimmune diseases, such as nonobese diabetic (NOD) diseases, rheumatoid arthritis, and Crohn s disease, whereas Th2 cells (IL-4) can aggravate Th2-modulated disorders such as asthma4).

Pharmacopuncture therapy is a method for the stimulation of acupoints through the injection of herbal medicines at the same acupoints. This method is frequently used to regulate immune balance in clinical settings. MOK is one of the pharmacopuncture medicines that is used to treat the meridian of fire in nature and clinical symptoms related to heart and thyroid diseases5,6). MOK has been reported to exhibit anti-inflammatory and antioxidant effects in in vitro7). Among the constituents of MOK, Calculus Bovis from Bos taurus, Ursi Fel from Ursus arctos, and Hominis Placenta have been reported as herbs that may exert immunomodulatory effects-11). However, the scientific mechanism underlying the immunomodulatory effects of MOK is not fully known. Therefore, we investigated the immunomodulatory and antioxidant effects of MOK on Th1/Th2 imbalance and oxidative stress in primary Con A-stimulated mouse splenocytes.

II. Materials and Methods

1. Materials

1) Preparation of MOK extract

The MOK extract was manufactured at a Good Manufacturing Practice (GMP)- compliant facility at the Korea Immuno-Pharmacopuncture Association (Seoul, Korea). The quality of all the raw materials (Table 1) in MOK was approved by the Korea Food & Drug Administration (KFDA). MOK was extracted with dried herbs (106.2 g) in distilled water (1 L) for 3 h, mixed with ethanol a 1:1 ratio, filtered through a two-layer mesh, and concentrated under vacuum pressure. Freeze-dried MOK was dissolved in 1X PBS to yield MOK extract at a concentration of 53.1 mg/mL.

Constituents of MOK extract

2) Reagents

Concanavalin A (Con A), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), sulfanilamide, and N-1-napthylethylenediamine dihydrochloride (NED) were purchased from Sigma-Aldrich (St Louis, MO, USA). RPMI-1640, fetal bovine serum (FBS), and penicillin-streptomycin solution were purchased from GenDEPOT (Barker, TX, USA). TRIzol reagent was obtained from Bio-Rad Laboratories (Philadelphia, PA, USA). M-MLV reverse transcriptase was obtained from Promega (Madison, WI, USA), Taq-based PCR enzyme was purchased from Toyobo (Osaka, Japan), and thioglycollate broth was purchased from Difco Laboratories (Detroit, MI, USA).

3) Animals

Five-week-old male ICR mice (weight: 17–21 g) were obtained from Orient Bio Inc. (Gyeonggi-do, Korea). The animals were housed under controlled environmental conditions (ambient temperature, 23 ± 1 °C; relative humidity, 50 ± 10%; 12-h light/dark cycle) and permitted free access to food and water.

2. Methods

1) Isolation of splenocytes

Spleens were rapidly harvested from mice, minced, and passed through a stainless steel mesh to obtain a single cell suspension. The cell pellet was harvested after the splenocytes were resuspended in 1× PBS and centrifuged at 5,000 rpm for 5 min. Erythrocytes were removed by using RBC lysis buffer (Sigma-Aldrich). Splenocytes (2 × 106 cells/mL) were cultured in 3 mL RPMI-1640 medium supplemented with 10% FBS, penicillin (100 units/mL), streptomycin (100 μ g/mL), and β-mercaptoethanol (0.05 mM) at 37 °C in an atmosphere of 5% CO2 for 24 h to facilitate cell adherence. Subsequently, the non-adherent cells were removed.

2) MTT assay

An MTT-based colorimetric assay was used to determine the concentration at which MOK extract was toxic to splenocytes. Briefly, splenocytes (5 × 104 cells/well) were seeded in 96-well plates and treated with MOK extract at different concentrations for 24 h at 37 ° C in a 5% CO2 incubator. Subsequently, 10 μL MTT solution (5 mg/mL) was added to each well and incubated for 4 h. The resulting crystals were dissolved in 100 μL DMSO and the absorbance at 570 nm was measured using a microplate reader (GENios, TEKAN Instruments, Inc., Austria). Cell viability was calculated by using the following formula: (viable cells)% = (OD of MOK-treated sample/OD of untreated sample) × 100

3) Reverse Transcription (RT)-PCR assay

Splenocytes (1 × 106 cells/well) were seeded in 60- mm culture dishes, treated with MOK extract at 2.5, 5, and 10 mg/mL for 30 min, and then stimulated in the presence or absence of Con A for 5 h at 37 °C in a 5% CO2 incubator. The total RNA was isolated from each cell by using TRIzol reagent and cDNA was synthesized from total RNA by using a mixture of oligo-dT primer, 5× RT buffer (Promega Co., Madison, WI, USA), 0.5 mM dNTP, 3 mM MgCl2, RNase inhibitor, and Improm-IITM reverse transcriptase (2U) at 25 °C for 5 min and 42 ° C for 60 min. The reaction was terminated at 70 ° C for 10 min. PCR was performed using specific primers for the target genes and PCR mixture [2 μ L cDNA, 4 μ M5′ and 3′ primers, 10× buffer (10 mM Tris-HCl, pH 8.3), 50 mM KCl, 0.1% Triton X-100, 25 mM MgCl2, 250 μM dNTPs, and 1 U Taq polymerase] under the following incubation conditions: 30 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 57–61 °C, and extension for 1 min; followed by a final extension step of 10 min. The following primer sequences were used: IL-4, 5′-AGA TGG ATG TGC CAA ACG TCC TCA-3′ (sense) and 5′-AAT ATG CGA AGC ACC TTG GAA GCC-3′ (anti-sense); IL-10, 5′-GGA CAA CAT ACT GCT AAC CGA C-3′ (sense) and 5′-TGG ATC ATT TCC GAT AAG GCT TG-3′ (anti-sense); Foxp3, 5′-GGC CCT TCT CCA GGA CAG A-3′ (sense) and 5′-GCT GAT CAT GGC TGG GTT GT-3′ (anti-sense); IFN-γ, 5′-TCA ACA ACC CAC AGG TCC AG-3′ (sense) and 5′-CTT CCT GAG GCT GGA TTC CG-3′ (anti-sense); MnSOD, 5′-GTG ACT TTG GGT CTT TTG AG-3′ (sense) and 5′-GCT AAC ATT CTC CCA GTT GA-3′ (anti-sense); HO-1, 5′-AAG ATT GCC CAG AAA GCC CTG GAC-3′ (sense) and 5′-AAC TGT CGC CAC CAG AAA GCT GAG-3′ (anti-sense); and GAPDH, 5′-CTC GTG GAG TCT ACT GGT GT-3′ (sense) and 5′-GTC ATC ATA CTT GGC AGG TT-3′ (anti-sense), which was used as a control for PCR. The band intensity was quantified by automated densitometric analysis (ChemiDoc MP Imaging System (BioRad Laboratories, CA, USA).

4) Statistical Analysis

GraphPad Prism (GraphPad Software, Inc., San Diego) was used for statistical analysis. Data were expressed as means ± SEM (standard error of mean) of three independent experiments and were analyzed for statistical significance by analysis of variance (ANOVA) followed by Tukey’ s test for multiple comparisons. Null hypotheses of no difference were rejected if p-values were less than 0.05.

III. Results

1. Effects of MOK extract on cell viability in splenocytes

To investigate the cytotoxicity effects on splenocytes, the cells were treated with MOK extract between 1.25 mg/mL and 20 mg/mL and their viability was evaluated using an MTT assay. Cytotoxic effects were observed at 20 mg/mL MOK extract. However, no effects on the cell viability of splenocytes were observed after treatment with MOK extract in the range from 1.25–10 mg/mL (Fig. 1). Therefore, we used MOK extract at concentrations below 10 mg/mL for subsequent studies.

Fig. 1

Effects of MOK extract on cell viability in mouse primary splenocytes

Splenocytes were treated with MOK extract at 1.25, 2.5, 5, 10, and 20 mg/mL for 24 h. Cell viability was measured using the MTT assay. Data are expressed as means ± SEM of three independent experiments.

2. Effects of MOK extract on the expression of IFN-γ in Con A-stimulated splenocytes

To understand the modulatory effect of MOK on the Th1/Th2 immune response, we evaluated the expression of the Th1 cytokine, IFN-γ, in Con A-stimulated splenocytes by RT-PCR. As shown in Fig. 2, the expression of IFN-γ mRNA was significantly increased after Con A stimulation (p < 0.001). Con A-induced IFN-γ expression was significantly decreased by the treatment with MOK extract at 2.5, 5 and 10 mg/mL (p < 0.05, p < 0.05, and p < 0.001, respectively). These data indicate that MOK modulated the Th1 response via the downregulation of IFN-γ expression in activated splenocytes.

Fig. 2

Effects of MOK extract on the expression of IFN-γ mRNA in Con A-stimulated primary splenocytes

Spenocytes were treated with MOK extract at 2.5, 5, and 10 mg/mL for 30 min, and then stimulated with or without Con A (200 ng/mL) for 5 h. (A) The expression of IFN-γ mRNA was evaluated by RT-PCR assay, with GAPDH used as an internal control. (B) The histogram represents the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. cells only; p < 0.05, ††p < 0.01, and †††p< 0.001 vs. Con A only.

3. Effects of MOK extract on the expression of IL-4, IL-10, and Foxp3 in Con A-stimulated primary splenocytes

To understand the modulatory effect of MOK on the Th1/Th2 immune response, we evaluated the expression of the Th2 cytokines, IL-4, IL-10, and Foxp3, in Con A-stimulated splenocytes by RT-PCR. The results indicated that the treatment with 2.5, 5, and 10 mg/mL MOK extract dose-dependently increased the expression of IL-4, IL-10, and Foxp3 in Con A-stimulated splenocytes (Fig. 3). In particular, a significant increase in IL-4, IL-10, and Foxp3 was observed at 10 mg/mL MOK extract. These results indicated that MOK modulated the Th2 response via the upregulation of IL-4, IL-10, and Foxp3 expression in activated splenocytes.

Fig. 3

Effects of MOK extract on the expression of IL-4, IL-10, and Foxp3 mRNA in Con A-stimulated primary splenocytes

Spenocytes were treated with MOK extract at 2.5, 5, and 10 mg/mL for 30 min, and then stimulated in the presence or absence of Con A (200 ng/mL) for 5 h. (A) The expression of IL-4, IL-10, and Foxp3 mRNA was evaluated by RT-PCR assay, with GAPDH used as an internal control. (B) The histogram represents the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. cells only; p < 0.05, ††p < 0.01, and †††p < 0.001 vs. Con A only.

4. Effects of MOK extract on oxidative stress in primary splenocytes

To investigate the antioxidant effects of MOK extract in splenocytes, we analyzed the mRNA expression of antioxidant enzymes by using RT-PCR. The treatment of splenocytes with 10 mg/mL MOK extract significantly decreased the expression of the antioxidant enzymes, HO-1 and MnSOD mRNA (Fig. 4). These data indicated that MOK may exert an antioxidant effect in splenocytes through the upregulation of antioxidant enzymes.

Fig. 4

Effects of MOK extract on the expression of HO-1 and MnSOD mRNA in Con A-stimulated primary splenocytes

Splenocytes were treated with 10 mg/mL MOK extract for 30 min and then stimulated in the presence or absence of Con A (200 ng/mL) for 5 h. (A) The mRNA expression of HO-1 and MnSOD was determined by RT-PCR assay, with GAPDH used as an internal control. (B) The histogram represents the means ± SD of three independent experiments. *p < 0.05 vs. cells alone.

IV. Discussion

Acupuncture has been used to treat a variety of inflammatory conditions in the body. Growing evidence has indicated that acupuncture effectively improves various inflammatory diseases through immune regulation and anti-inflammatory effects12,13). Pharmacopuncture is a new acupuncture treatment method in Traditional Korean Medicine (TKM). This method is often used in clinics because it can directly target tissues without involving the digestive system5,6). MOK is one of the pharmacopuncture medicines that has been used to treat the meridian of fire in nature, clinical symptoms related to heart and thyroid diseases, and the Korean somatization disorder Hwa-Byung, which is a mental illness associated with the inability to control anger6). MOK consists of ten herbs, including Hominis Placenta, Moschus, Ursi Fel, Bovis Calculus, Scutellariae radix, Phellodendri Cortex, Pulsatilla koreana, Sophorae subprostratae radix, Saussurea lappa, and Aquilaria agallocha. The main constituents are Moschus, Bovis Calculus (Bos taurns), Ursi Fel (Ursus arctos), and Hominis Placenta. The immunomodulatory function of these herbs has been reported in modern pharmacological research along with antioxidant effects9,1419). However, comparisons of the biological activity of MOK with that of single medicines have been rarely reported. We have previously reported the anti-inflammatory and antioxidant effects of MOK extract in activated peritoneal macrophages7). In the present study, we investigated the effects of MOK extract on the Th1/Th2 immune imbalance and oxidative stress in primary splenocytes isolated from mouse spleen. The treatment of Con A-stimulated splenocytes with MOK extract decreased the expression of the Th1 cytokine, IFN-γ, and increased the expression of the Th2 cytokines, IL-4, IL-10, and Foxp3. These changes indicated that MOK extract was able to control immune imbalance via the regulation of the Th1/Th2 response in splenocytes.

Biological experiments are usually performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from in vivo cells by several important aspects20). In the present study, we used primary splenocytes isolated from mouse spleen instead of T cell lines. T cells and natural killer T cells are thought to be primarily activated by Con A and Th2 cytokines, including IL-4, IL-10, FoxP3, which are produced by the activation of T cells2123). In the present study, we investigated the effects of MOK on the Th1/Th2 immune response by the evaluation of the gene expression of Th1 and Th2 cytokines in primary splenocytes. Th1 cells secrete Th1 cytokines such as IL-2, IFN-γ, IL-12 and TNF-α, whereas Th2 cells secrete Th2 cytokines such as IL-4, IL-10, and Foxp3. The communication network between Th1 and Th2 cytokines may be synergistic or antagonistic toward lymphocyte proliferation and differentiation3,4). IFN-γ is a major Th1 cytokine, which plays an important role in the coordination of protective immunity against infection with intracellular parasites and bacteria. It is produced by NK T cells during the innate immune response and by CD4+ and CD8+ T cells during the adaptive immune response to a variety of pathogens2426). In the present study, the treatment of Con A-stimulated splenocytes with MOK significantly inhibited the expression of IFN-γ mRNA. IL-4 and IL-10 are the main Th2 cytokines; they support antibody production, promote mast cell growth and eosinophil differentiation and activation, which results in humoral or allergic responses. In the present study, MOK extract significantly increased the expression of IL-4 and IL-10 mRNA in Con A-stimulated splenocytes.

Natural regulatory T (Treg) cells are constitutively produced in the thymus and express very high levels of CD25. Treg cells produce IL-1027,28); their suppressive function is closely related to the expression of Forkhead box transcription factor 3 (Foxp3)29). The role of Treg cells (CD4+CD25+FoxP3+) has been reported in the prevention of autoimmune diseases and immunopathology for all types of infections30,31). In the present study, treatment with MOK significantly increased the expression of Foxp3 mRNA in Con A-stimulated splenocytes. This result suggested that MOK extract may control the autoimmune response through the activation of Treg cells. Autoimmune diseases may be caused by an imbalance of Thl/Th2 cytokines and regulatory cytokines. In further studies, we will investigate the effects of MOK extract on Treg cell number and function in thyroid autoimmune diseases.

Oxidative stress induced by ROS overproduction, which results in damaged cellular lipids, proteins, and DNA, is thought to be implicated in a variety of diseases or pathological conditions including cancers, cardiovascular disease, acute inflammatory problems, complications of diabetes mellitus, chronic inflammatory diseases, central nervous system disorders, neurodegenerative disorders, and age-related disorders32,33). The endogenous components of the antioxidant system include enzymes such as SOD, glutathione peroxidase (GSPx), and HO-134,35). We previously reported that the antioxidant effects of MOK extract in LPS-stimulated mouse peritoneal macrophages occurred via an increase in the expressions of MnSOD and HO-17). In the present study, the treatment of splenocytes with MOK increased the mRNA expression of MnSOD and HO-1. These results indicated the antioxidant potential of MOK extract.

V. Conclusions

In conclusion, MOK extract inhibited the mRNA expression of the Thl cytokine IFN-γ, induced the expression of Th2 cytokines (IL-4, IL-10, and Foxp3), and also induced the mRNA expression of the antioxidant enzymes SOD and HO-1 in Con A-stimulated mouse splenocytes. This suggested that MOK extract exerted immunomodulatory and an tioxidant effects in splenocytes through the regulation of Th1/Th2 cytokines and antioxidant enzymes.

References

1. Cesta MF. Normal structure, function, and histology of the spleen. Toxicol Pathol 2006;34(5):455–65.
2. Romagnani S. Biology of human TH1 and TH2 cells. J Clin Immunol 1995;15(3):121–9.
3. Murphy KM, Reiner SL. The lineage decisions of helper T cells. Nat Rev Immunol 2002;2(12):933–44.
4. Abbas AK1, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383(6603):787–93.
5. Korean Acupuncture & Moxibution Soc. The Acupuncture and Moxibution Medicine Paju: Hanmi medical publishing co; 2016. p. 204–8.
6. Jung C, Jung JH, Lee MS. Immune Pharmacopuncturology Chungnam: Kyungrak medical publishing co; 2011. p. 127–33.
7. Hwang JH, Hwang MS, Park YK. MOK, a Pharmacopuncture Medicine, Reduces Inflammatory Response through Inhibiting the Proinflammatory Cytokine Production in LPS-stimulated Mouse Peritoneal Macrophages. The Acupunct 2017;34(1):11–21.
8. Kim HJ, Gwan R, Han JW, et al. Analysis of physioactivities on OK yakchim. J Immuno-Pharmacopuncture 2013;2(1):9–16.
9. The National College of Oriental Medicine Herbology Classroom. Herbology Seoul: Youngrimsa; 2016. p. 216–8. p. 221–3. p. 250–3. p. 257–8. p. 273–4. p. 395–6. p. 560–2. p. 617–8.
10. Kim HJ, Gwan R, Han JW, Jung C. Anti-oxidant and anti-inflammatory effects of V yakchim. J Immuno-Pharmacopuncture 2013;2(1):1–8.
11. Hwang JH, Cho HS, Lee HJ, et al. Effect of Inhibition Macrophage Migration Inhibitory Factor Activation by Hominis Placenta Herbal Acupuncture on Rheumatic Arthritis. The Acupunct 2008;25(3):41–51.
12. Zijlstra FJ, van den Berg-de Lange I, Huygen FJ, Klein J. Anti-inflammatory actions of acupuncture. Mediators Inflamm 2003;12(2):59–69.
13. Cho SY, Yang SB, Shin HS, et al. Anti-inflammatory and immune regulatory effects of acupuncture after craniotomy: study protocol for a parallel-group randomized controlled trial. Trials 2017;18:10.
14. Feng Y, Siu K, Wang N, et al. Bear bile: dilemma of traditional medicinal use and animal protection. J Ethnobiol Ethnomed 2009;5(2):1–45.
15. Li X, Xu Y, Zhang C, et al. Protective Effect of Calculus Bovis Sativus on Dextran Sulphate Sodium-Induced Ulcerative Colitis in Mice. Evid Based Complement Alternat Med 2015;2015:469506.
16. Jang SY, Park JW, Bu Y, Kang JO, Kim J. Protective effects of hominis placenta hydrolysates on radiation enteropathy in mice. Nat Prod Res 2011;25(20):1988–92.
17. Park SY, Phark S, Lee M, Lim JY, Sul D. Antioxidative and anti-inflammatory activities of placental extracts in benzo[a]pyrene-exposed rats. Placenta 2010;31(10):873–9.
18. De D, Datta Chakraborty P, Mitra J, et al. Ubiquitin-like protein from human placental extract exhibits collagenase activity. PLoS One 2013;8(3):e59585.
19. Park JY, Lee JY, Jeong MS, et al. Effect of Hominis Placenta on cutaneous wound healing in normal and diabetic mice. Nutr Res Pract 2014;8(4):404–9.
20. Pan C, Kumar C, Bohl S, Klingmueller U, Mann M. Comparative Proteomic Phenotyping of Cell Lines and Primary Cells to Assess Preservation of Cell Type-specific Functions. Mol Cell Proteomics 2009;8(3):443–50.
21. Iwashima S, Ozaki T, Maruyama S, et al. Novel culture system of mesenchymal stromal cells from human sub-cutaneous adipose tissue. Stem Cells Dev 2009;18(4):533–43.
22. Kubo N, Narumi S, Kijima H, et al. Efficacy of adipose tissue-derived mesenchymal stem cells for fulminant hepatitis in mice induced by concanavalin A. J Gastroenterol Hepatol 2012;27(1):165–72.
23. Mizuhara H, Kuno M, Seki N, et al. Strain difference in the induction of T-cell activation-associated, interferon gamma-dependent hepatic injury in mice. Hepatology 1998;27(2):513–9.
24. Sun JC, Lanier LL. NK cell development, homeostasis and function: Parallels with CD8+ T cells. Nat Rev Immunol 2011;11(10):645–57.
25. Takada K, Jameson SC. Naive T cell homeostasis: From awareness of space to a sense of place. Nat Rev Immunol 2009;9(12):823–32.
26. O’ Garra A, Robinson D. Development and function of T helper 1 cells. Adv Immunol 2004;83:133–62.
27. Barthlott T, Moncrieffe H, Veldhoen M, et al. CD25+CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production. Int Immunol 2005;17:279288.
28. Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 2005;201(5):723–35.
29. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 2007;445(7129):766–70.
30. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6(4):345–52.
31. Belkaid Y. Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol 2007;7(11):875–88.
32. Davies KJ. Oxidative stress: the paradox of aerobic life. Biochem Soc Symp 1995;61:1–31.
33. Sareen SG, Jack LS, James LG. The antioxidant nutrients, reactive species, and disease. Advanced nutrition and human metabolism 4th edited California, USA: Thomson Wadsworth; 2004. p. 368–77.
34. Leopold JA, Loscalzo J. Oxidative mechanisms and atherothrombotic cardiovascular disease. Drug Discov Today Ther Strateg 2008;5(1):5–13.
35. Frankel D, Mehindate K, Schipper HM. Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia. J Cell Physiol 2000;185(1):80–6.

Article information Continued

Fig. 1

Effects of MOK extract on cell viability in mouse primary splenocytes

Splenocytes were treated with MOK extract at 1.25, 2.5, 5, 10, and 20 mg/mL for 24 h. Cell viability was measured using the MTT assay. Data are expressed as means ± SEM of three independent experiments.

Fig. 2

Effects of MOK extract on the expression of IFN-γ mRNA in Con A-stimulated primary splenocytes

Spenocytes were treated with MOK extract at 2.5, 5, and 10 mg/mL for 30 min, and then stimulated with or without Con A (200 ng/mL) for 5 h. (A) The expression of IFN-γ mRNA was evaluated by RT-PCR assay, with GAPDH used as an internal control. (B) The histogram represents the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. cells only; p < 0.05, ††p < 0.01, and †††p< 0.001 vs. Con A only.

Fig. 3

Effects of MOK extract on the expression of IL-4, IL-10, and Foxp3 mRNA in Con A-stimulated primary splenocytes

Spenocytes were treated with MOK extract at 2.5, 5, and 10 mg/mL for 30 min, and then stimulated in the presence or absence of Con A (200 ng/mL) for 5 h. (A) The expression of IL-4, IL-10, and Foxp3 mRNA was evaluated by RT-PCR assay, with GAPDH used as an internal control. (B) The histogram represents the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. cells only; p < 0.05, ††p < 0.01, and †††p < 0.001 vs. Con A only.

Fig. 4

Effects of MOK extract on the expression of HO-1 and MnSOD mRNA in Con A-stimulated primary splenocytes

Splenocytes were treated with 10 mg/mL MOK extract for 30 min and then stimulated in the presence or absence of Con A (200 ng/mL) for 5 h. (A) The mRNA expression of HO-1 and MnSOD was determined by RT-PCR assay, with GAPDH used as an internal control. (B) The histogram represents the means ± SD of three independent experiments. *p < 0.05 vs. cells alone.

Table 1

Constituents of MOK extract

Herbal name Scientific name Ratio (mg/mL)
Hominis Placenta Hominis Placenta 2
Moschus Moschus berezovskii 0.5
Ursi Fel Ursus actos 0.3
Bovis Calculus Cow bezoar Bos taurus 0.3
Scutellariae radix Scutellaria baicalensis 10
Phellodendri Cortex Phellodendron amurense 10
Pulsatilla koreana Pulsatila koreana 10
Sophorae subprostratae radix Sophora tonkinensis 10
Aucklandiae radix Aucklandia lappa 5
Aquilaria agallocha Aquilaria agallocha 5