Effect of Gambisan on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes

Abstract

This study was conducted to explore the antiadipogenic effect and possible mechanism of Gambisan on 3T3-L1 cells. For quality control, Gambisan was standardized by HPLC and the standard compounds ephedrine, epigallocatechin-3-gallate, and caffeine were screened. Cultured 3T3-L1 cells that had been induced to differentiate were treated with various concentrations of Gambisan or its major component extracts (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.) for 72 hours for MTT assay to determine cell viability or 10 days for LDH assay, triglyceride assay, DNA content measurement, Oil red O staining, RT-PCR, and western blot. Gambisan significantly inhibited adipogenesis in 3T3-L1 cells by reducing triglyceride contents and lipid accumulation in a dose-dependent manner without obvious cytotoxicity. Viability and DNA content in 3T3-L1 cells treated with Gambisan were significantly higher than cells treated with the major component extracts at every concentration. The anti-adipogenic effects of Gambisan appeared to be mediated by a significant downregulation of the expression of lipoprotein lipase mRNA and PPAR γ , C/EBP α , and SREBP-1 protein apart from the expression of hormone-sensitive lipase. Gambisan could act as a possible therapeutic agent for obesity. However, further studies including in vivo assays and clinical trials are needed to confirm the efficacy, safety and mechanisms of the antiobesity effects of Gambisan.

Figure 1

HPLC profiles of Gambisan and its standard compounds of ephedrine, EGCG, and caffeine. (a), (b), and (c) are profiles of standard ephedrine, EGCG, and caffeine, respectively. (d), (e), and (f) are profiles of ephedrine, EGCG, and caffeine extracted from Gambisan. Detection was done at 210 nm (about 27 min), 280 nm (about 7.5 min), and 280 nm (about 11.5 min), respectively.

Figure 2

Effect of Gambisan and its major component extracts on 3T3-L1 cell viability. 3T3-L1 cells were treated with various doses (0.01–1000 μg/mL) of Gambisan (A) or its major components (Ephedra intermedia Schrenk (B), Atractylodes lancea DC. (C) and Thea sinensis L. (D)). Cell viability was measured by the MTT assay after 72 hr (a) and by the LDH assay after 10 days (b). The percentage of viable cells was calculated by defining the cell viability without treatment as 100%. Values are expressed as mean ± SEM of six independent experiments. **P < 0.01, ***P < 0.001 compared with control (expressed by t-test). EIS: Ephedra intermedia Schrenk, ALL: Atractylodes lancea DC., TSL: Thea sinensis L., MTT assay: methyl thiazol tetrazolium assay, LDH assay: lactate dehydrogenase assay.

Figure 3

Effect of Gambisan and its major component extracts on the TG deposition and DNA contents in 3T3-L1 cells. 3T3-L1 cells were treated with various doses (0, 100, 250, and 500 μg/mL) of Gambisan or its major components (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.). After 10 days, the cellular TG contents (a) were measured with TG determination kit at 550 nm. The DNA contents in 3T3-L1 cells (b) were measured as internal control. Values are expressed as mean ± SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control (expressed by t-test). EIS: Ephedra intermedia Schrenk, ALL: Atractylodes lancea DC., TSL: Thea sinensis L.

Figure 4

Effect of Gambisan on lipid accumulation in 3T3-L1 cells. 3T3-L1 cells were treated with various doses (0, 100, 250, and 500 μg/mL) of Gambisan. After 10 days, cells were fixed and stained with Oil red O dye. Images of representative cells (a) scanned, (c) captured with a microscope (200x magnification), and (b) quantified by the i-solution program using a CCD camera are shown. The extent of lipid accumulation was expressed as the percentage of control. Values are expressed as mean ± SEM of five independent experiments. ***P < 0.001 compared with control (expressed by t-test).

Figure 5

Effect of Gambisan on adipocyte-specific gene and major adipogenic transcriptional factor expression in 3T3-L1 cells. 3T3-L1 cells were treated with various doses (0, 10, 100, 250, and 500 μg/mL) of Gambisan. After 10 days, the mRNA expression levels of HSL and LPL were measured using RT-PCR, and the protein expression levels of PPARγ, C/EBPα, and SREBP-1 were measured using western blot analysis. Insets show representative RT-PCR band (a) and western blot analysis band (c) used for quantification, and bar graphs represent the band intensities of PCR products (b) and of western blots (d) adjusted by β-actin used as the internal control. Values are expressed as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control (expressed by t-test). HSL: hormone-sensitive lipase, LPL: lipoprotein lipase, RT-PCR: reverse transcription-polymerase chain reaction, PPARγ: peroxisome proliferator-activated receptor gamma, C/EBPα: CCAAT/enhancer binding protein alpha, SREBP-1: sterol regulatory element binding protein-1.