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GyeongshinhaeGihwan T1 has Controlling Effects on the Factors Associated with Obesity

Introduction

GyeongshinhaeGihwan T1 has Controlling Effects on the Factors Associated with Obesity

Hyo-Jin AN ¹,², In-Young CHOI ¹,², Yang-Sam JUNG ³, Ki-Hyeon YOON ³, Hyung-Min KIM¹, Seung-Heon HONG²*, Soon-Shik SHIN³*

Abstract- GyeongshinhaeGihwan T1 (GGT1) is a newly developed oriental medicine to help control weight. To evaluate the anti-obesity effect of GGT1, we investigated a possible effect of GGT1 on macrophage-related obesity reactions; nitric oxide production and cytokine secretion in mouse peritoneal macrophages. According to recent reports, macrophages are participated in fat accumulation and closely related with obesity. In this study, using mouse peritoneal macrophages, we have examined whether GGT1 affects the production of nitric oxide (NO), tumor necrosis factor-a (TNF-a), and interleukin (IL)-12 by interferon-g and lipopolysaccharide (LPS). GGT1 inhibits LPS-induced NO production in a dose-dependent manner. The decrease in NO synthesis was reflected as a decreased amount of inducible NO synthase protein. We also found that GGT1 inhibits pro-inflammatory cytokines, TNF-a and IL-12 production. In mouse embryo preadipocyte 3T3-L1, GGT1 reduced the viability in a dose-dependent manner. These findings mean that GGT1 can be used in preventing and controlling adipogenesis and obesity.

Keywords GyeongshinhaeGihwan T1, peritoneal macrophages, preadipocyte, obesity, nitric oxide, cytokine

INTRODUCTION

Obesity is one of public health dilemma, especially in developed countries, and has steadily increased at an alarming rate in recent years. Morbid obesity increases the risk of hypertension, coronary artery disease, diabetes mellitus, cancer, sleep apnea, and osteoarthritis (Balsiger et al., 2000).
GyeongshinhaeGihwan T1 (GGT1) is an oriental medicine consisted of a mixture of different herbs. It is invented for weight loss and will be used for weight loss. However, it has not been cleared GGT1 is effective in an experimental model.
In 2000, Kapur et al. reported that nitric oxide (NO) is a new player in the modulation of energy metabolism (Kapur et al., 2000). NO is a highly reactive molecule produced from a guanidine nitrogen of NO synthase (NOS). Three isoforms of NOS have been identified and are classified into two major categories, namely constitutive and inducible NOS. Neuronal and endothelial NOSs, which are constitutively expressed, are activated by calcium and calmodulin and are called constitutive NOSs (Nathan, 1992). Of the three NO synthases, inducible NOS (iNOS), the high-output isoform, is the most widely expressed in various cell types after its transcriptional activation (Xie et al., 1992). Most importantly, iNOS is highly expressed in lipopolysaccharide (LPS)-activated macrophages (Petros et al., 1991).
The effect of experimental hyperlipidemia on functional activity of macrophages was studied in CBA and C57Bl/6 mice resistant. Two-month atherogenic diet increased the content of cholesterol in the serum and cells of peritoneal exudate in mice of both strains. In parallel, production of nitrites and 5'-nucleotidase activity in peritoneal macrophages increased (Kiseleva et al., 2002). Furthermore, expression analysis of macrophage and nonmacrophage cell populations isolated from adipose tissue demonstrates that adipose tissue macrophages are responsible for almost all adipose tissue tumor necrosis factor (TNF)-a expression and significant amounts of iNOS and interleukin (IL)-6 expression. Adipose tissue macrophage numbers increase in obesity and adipose tissue macrophages participate in inflammatory pathways that are activated in adipose tissues of obese individuals (Weisberg et al., 2003).
In relation with this concept, we studied the inhibitory effects of LPS-induced NO, TNF-a and IL-12 by GGT1 in mouse peritoneal macrophages. In addition, using preadipocyte, we showed that GGT1 inhibits the preadipocyte proliferations.


MATERIALS AND METHODS

Materials
Murine recombinant interferon (rIFN)-g (1 ´ 107 U/ml) was purchased from R&D Systems (Minneapolis, MI, USA). N- (1-naphtyl)-ethylenediamine dihydrochloride, LPS, sodium nitrite were purchased from Sigma (St. Louis, MO, USA). Rabbit polyclonal antisera to iNOS were obtained from Transduction Laboratories (Lexington, KY, USA). Recombinant (r) TNF-a and IL-12, biotinylated anti-murine TNF-a and IL-12, anti-murine TNF-a and IL-12 were purchased from R & D System Inc, USA. Thioglycollate (TG) was purchased from Difco Laboratories (Detroit, MI, USA). Dulbecco's Modified Eagle's Medium (DMEM) containing L-arginine (84 mg/l), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL (Grand Island, NY, USA).

Animals
Male C57BL/6J mice were purchased from Orient Co., LTD (Sungnam, Gyeonggi-do, Republic of Korea). They were housed five to ten per cage in a laminar air-flow room maintained at a temperature of 22 ± 1℃ and relative humidity of 55 ± 10% throughout the study.

Cell cultures
TG-elicited macrophages were harvested 3 - 4 days after i.p. injection of 2.5 ml TG to the mice and isolated, according to a procedure reported elsewhere (Chung et al., 2004). Using 8 ml of HBSS containing 10 U/ml heparin, peritoneal lavage was performed. The cells were then distributed in DMEM, which was supplemented with 10% heat-inactivated FBS, in 4-well tissue culture plates (2.5 ´ 105 cells/well) incubated for 3 h at 37°C in an atmosphere of 5% CO2. They were washed three times with HBSS to remove non-adherent cells, and equilibrated with DMEM that contained 10% FBS before treatment. 3T3-L1 cells, obtained from the Korean Cell Line Bank (Seoul, Republic of Korea), were maintained in DMEM medium supplemented with 10% FBS.

Preparation of GGT1
Extract of GGT1 was prepared by decocting the dried prescription of herbs with boiling distilled water. The extraction decocted for approximately 3 h has been filtered, lyophilized and stored at 4℃. The ingredients 25.2 g of GGT1 include as follows: Ophiopogonis Radix 7.5 g, Plantycodi Radix 7.5 g, Coicis Semen 7.5 g, Scutellariae Radix 3.75 g, Raphani Semen 3.75 g, Ephedrae Herba 2.62 g etc. These plant materials were obtained from Professor S. S. Shin, College of Oriental Medicine, Dongeui University.

MTT assay
Cell aliquots (2.5 × 105 cells/well) were seeded in microplate wells and incubated with 20 ml of a MTT solution (5 mg/ml) for 4 h at 37°C under 5% CO2 and 95% air. Consecutively, 250 ml of dimethylsulfoxide was added to extract the MTT formazan and the absorbance of each well at 510 nm was read by an automatic microplate reader.

Measurement of nitrite concentration

Peritoneal macrophages (2.5 ´ 105 cells/well) were cultured with various concentrations of GGT1. The cells were then stimulated with rIFN-g (20 U/ml). After 6 hours, the cells were finally treated with LPS (10 mg/ml). NO synthesis in cell cultures was measured by a microplate assay method after 24 h and 48 h, as previously described (Jeong et al., 2004). To measure nitrite, 100 ml aliquots were removed from conditioned medium and incubated with an equal volume of Griess reagent (1% sulfanilamide/0.1% N - (1-naphtyl)-ethylenediamine dihydrochloride/2.5% H3PO4) at room temperature for 10 min. The absorbance at 540 nm was determined by an automatic microplate reader. NO2- was determined by using sodium nitrite as a standard. The cell-free medium alone contained 5 to 9 mM of NO2-. This value was determined in each experiment and subtracted from the value obtained from the medium with peritoneal macrophages.

Western blot analysis

Peritoneal macrophages (5 ´ 106 cells/well) were pretreated with GGT1 (1 mg/ml). The cells were then incubated for 6 h with rIFN-g (20 U/ml). They were finally stimulated with LPS (10 mg/ml) for 12 h. Whole cell lysates were made by boiling peritoneal macrophages in a sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 10% 2-mercaptoethanol). Proteins in the cell lysates were then separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. The membrane was then blocked with 5% skim milk in phosphate-buffered saline (PBS)-tween-20 (Sigma, St. Louis, MO, USA) for 1 h at room temperature and then incubated with anti-iNOS antibodies. After washing in PBS containing 0.05% tween-20 three times, the blot was incubated with secondary antibody for 30 min and the antibody-specific proteins were visualized by an enhanced chemiluminesence detection system according to the recommended procedure (Amersham Corp. Newark, NJ, USA).

Assay of cytokine production

Peritoneal macrophages (2.5 ´ 105 cells/well) were incubated with rIFN-g (20 U/ml), GGT1, rIFN-g plus LPS (10 mg/ml), and rIFN-g plus at various concentrations of GGT1 for 24 h. The amount of TNF-a and IL-12 secreted by the cells were then measured by a modified enzyme-linked immunosorbent assay (ELISA), as described previously (Kim et al., 2004). ELISA TNF-a and IL-12 were carried out in duplicate in 96-well ELISA plates. The plates were then coated with each of 100 ml aliquots of anti-mouse TNF-a and IL-12 monoclonal antibodies at 10 mg/ml in PBS at pH 7.4, and subsequently incubated overnight at 4°C. The plates were washed in PBS containing 0.05% Tween-20 and blocked with PBS containing 1% BSA, 5% sucrose and 0.05% NaN3 for 1 h. After additional washes, sample or TNF-a and IL-12 standards were added and incubated at 37°C for 2 h. After 2 h incubation at 37°C, the wells were washed and then each of 0.2 mg/ml of biotinylated anti-mouse TNF-a and IL-12 were added and the plates were incubated at 37°C for 2 h. After washing the wells, avidin-peroxidase was added and the plates were incubated for 20 min at 37°C. Wells were again washed and ABTS substrate was added. Color development was measured at 405 nm using an automated microplate ELISA reader. A standard curve was run on each assay plate using recombinant TNF-a and IL-12 in serial dilutions.

Statistical analysis
Results were expressed as the mean ± SE of independent experiments, and statistical analyses were performed by one-way analysis of variance (ANOVA) with Tukey, and Duncan post hoc test to express the difference among the groups. All statistical analyses were performed using SPSS v10.0 statistical analysis software. A value of p < 0.05 was considered to indicate statistical significance.