The anti-obesity effects of Tongbi-san in a high-fat diet-induced obese mouse model

Abstract

Background: Recently, it has been noted that natural herbal medications may be effective in treating obesity. Tongbi-san (TBS) is a traditional medicine usually used for dysuria (i.e., painful urination), containing three herbs, Cyperus rotundus L., Citrus unshiu Markovich, and Poria cocos. In this study, we aimed to examine whether TBS can inhibit high-fat diet (HFD)-induced adipogenesis in the liver and epididymal adipose tissue of obese mice.

Methods: Male C57BL/6 N mice were fed a normal diet, an HFD, an HFD plus orlistat 10 or 20 mg/kg, or an HFD plus TBS 50 or 100 mg/kg for 11 weeks. Body weight was checked weekly and histological tissue examinations were investigated. An expression of genes involved in adipogenesis was also assessed.

Results: Oral administration of TBS significantly reduced body weight and decreased epididymal and visceral white adipose tissue (WAT) weight. In addition, we found that TBS enhanced the expression of the adenosine monophosphate-activated protein kinase (AMPK) and inhibited the expression of transcription factors, such as CCAAT/enhancer-binding proteins (C/EBPs), sterol regulatory element-binding protein 1 (SREBP1), and peroxisome proliferator-activated receptor γ (PPARγ) in the liver and epididymal WAT as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Conclusion: These findings demonstrate that the anti-obesity effects of TBS may be linked to the activation of AMPK.

Keywords: AMPK; Adipogenesis; High-fat diet; Obesity; Tongbi-san.

Fig. 1

Effect of TBS on adipose tissue size and body weight in high-fat diet-induced obesity in C57BL/6 N mice. (a) Macroscopic mouse body and WAT size analysis. (b) Body weight and (c) body weight gain were assessed every week. (d) Food intake was recorded two or three times per one week. (e) The levels of serum TC were determined using enzymatic methods. CON: normal diet group; HFD: high-fat diet group; Orlistat: HFD plus orlistat (10 or 20 mg/kg) group; TBS: HFD plus TBS (50 or 100 mg/kg) group. The values represent the mean ± S.D. ^#p < 0.05 and ^###p < 0.001 vs. the control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. HFD group

Fig. 2

Effect of TBS on adipose tissue weight in high-fat diet-induced obesity in C57BL/6 N mice. (a) The epididymal adipose tissue weight, (b) relative epididymal adipose tissue weight ratio, (c) visceral adipose tissue weight, (d) relative visceral adipose tissue weight ratio, (e) total adipose tissue weight, and (f) total adipose tissue weight ratio were measured after 11 weeks of diet treatments in mice. The values represent the mean ± S.D. ^###p < 0.001 vs. the control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. HFD group

Fig. 3

Effects of TBS on lipid accumulation in epididymal white adipose tissue. (a) The epididymal white adipose tissue (WAT) from representative mice in each group was fixed, embedded in paraffin, and stained with H&E. Images are shown at the original magnification of 100x. (b) The average diameter of adipocytes in epididymal WAT of each group. CON: normal diet group; HFD: high-fat diet group; Orlistat: HFD plus orlistat (10 or 20 mg/kg) group; TBS: HFD plus TBS (50 or 100 mg/kg) group. The values represent the mean ± S.D. of three independent experiments. ^###p < 0.001 vs. the control group; ^**p < 0.01 and ^***p < 0.001 vs. HFD group. Scale bar is 200 μm

Fig. 4

Effects of TBS on AMPK, PPARγ, C/EBPα, and SREBP1 mRNA expression in epididymal white adipose tissue. Total protein and RNA were prepared from epididymal white adipose tissue (WAT), and the protein expression of (a) p-AMPK and the mRNA levels of (b) AMPK, (c) PPARγ, (d) C/EBPα, and (E) SREBP1 were determined by western blot analysis and quantitative RT-PCR (qRT-PCR). Densitometric analysis was performed using ImageJ ver. 1.50i. The values represent the mean ± S.D. of three independent experiments. ^##p < 0.01 and ^### p < 0.001 vs. the control group; ^**p < 0.01 and ^***p < 0.001 vs. HFD group

Fig. 5

Effects of TBS on liver tissue morphology and lipid accumulation. (a) Macroscopic analysis of mouse liver tissue. (b) Liver tissue from representative mice in each group were fixed, embedded in paraffin, and stained with H&E solution. Images are shown at the original magnification of 100x. CON: normal diet group; HFD: high-fat diet group; Orlistat: HFD plus orlistat (10 or 20 mg/kg) group; TBS: HFD plus TBS (50 or 100 mg/kg) group. Scale bar is 100 μm

Fig. 6

Effects of TBS on liver tissue the expression of p-AMPK, AMPK, PPARγ, C/EBPα, and SREBP1. Total protein and RNA prepared from liver tissue were used to measure the expression of protein of (a) p-AMPK and the levels of mRNA of (b) AMPK, (c) PPARγ, (d) C/EBPα, and (e) SREBP1 by western blotting and qRT-PCR. Densitometric analysis was performed using ImageJ ver. 1.50i. The values represent the mean ± S.D. of three independent experiments. ^#p < 0.05 and ^###p < 0.001 vs. the control group; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. HFD group

Fig. 7

The mechanism of TBS. TBS injections significantly reduced lipid accumulation by regulating the expression of AMPK, PPARγ, C/EBPα, and SREBP1 in the liver and epididymal white adipose tissue of an HFD-induced obese mouse model