Effect of keishibukuryogan on genetic and dietary obesity models

Abstract

Obesity has been recognized as one of the most important risk factors for a variety of chronic diseases, such as diabetes, hypertension/cardiovascular diseases, steatosis/hepatitis, and cancer. Keishibukuryogan (KBG, Gui Zhi Fu Ling Wan in Chinese) is a traditional Chinese/Japanese (Kampo) medicine that has been known to improve blood circulation and is also known for its anti-inflammatory or scavenging effect. In this study, we evaluated the effect of KBG in two distinct rodent models of obesity driven by either a genetic (SHR/NDmcr-cp rat model) or dietary (high-fat diet-induced mouse obesity model) mechanism. Although there was no significant effect on the body composition in either the SHR rat or the DIO mouse models, KBG treatment significantly decreased the serum level of leptin and liver TG level in the DIO mouse, but not in the SHR rat model. Furthermore, a lower fat deposition in liver and a smaller size of adipocytes in white adipose tissue were observed in the DIO mice treated with KBG. Importantly, we further found downregulation of genes involved in lipid metabolism in the KBG-treated liver, along with decreased liver TG and cholesterol level. Our present data experimentally support in fact that KBG can be an attractive Kampo medicine to improve obese status through a regulation of systemic leptin level and/or lipid metabolism.

Figure 1

Analysis by three-dimensional HPLC of major chemical compounds included in keishibukuryogan extract.

Figure 2

Effect of KBG on body weight changes and tissue weight in obesity models. (a, b) SHR rats were administered with control water or KBG (500 mg/kg, p.o., daily) for 8 weeks. Body weight changes (a) or tissue weight (b, liver and fat, at 8 weeks after daily treatment) of SHR rats is shown. (c, d) C57BL/6 mice were fed with normal diet (ND) or high-fat diet (HFD) for 10 weeks and then administered with control water or KBG (500 mg/kg, p.o., daily) for 12 weeks under the same feeding condition. After 8 weeks of control or KBG treatment, mice fed with HFD were changed to ND until the termination of experiment with maintaining KBG treatment for another 4 weeks. Body weight changes (a) or tissue weight (b, liver and fat at 12 weeks after daily treatment) of DIO mouse is shown. Data are mean ± SEM (n = 7–15).

Figure 3

Effect of KBG on serum levels of leptin and insulin in obesity models. (a, b) SHR rats were administered with control water or KBG (500 mg/kg, p.o., daily) for 8 weeks. Serum samples were collected upon the termination and levels of leptin (left) or insulin (right) were measured. (b, c) C57BL/6 mice were fed with normal diet (ND) or high-fat diet (HFD) for 10 weeks and then administered with control water or KBG (500 mg/kg, p.o., daily) for 12 weeks under the same feeding condition. After 8 weeks of control or KBG treatment, mice fed with HFD were changed to ND until the termination of experiment with maintaining KBG treatment for another 4 weeks. Serum samples were collected before the time for food changing (b) or upon the termination (c) and levels of leptin (left) or insulin (right) were measured by using specific ELISA assay. Data are mean ± SEM (n = 7–15). ^* P < 0.005; ^** P < 0.001.

Figure 4

Effect of KBG on liver lipid levels in obesity models. (a) SHR rats were administered with control water or KBG (500 mg/kg, p.o., daily) for 8 weeks. Liver tissue samples were collected upon the termination and levels of triglyceride (TG, left), total cholesterol (cholesterol, middle), and free fatty acid (FAA, right) were measured. (b) C57BL/6 mice were fed with normal diet (ND) or high-fat diet (HFD) for 10 weeks and then administered control water or KBG (500 mg/kg, p.o., daily) for 12 weeks under the same feeding condition. After 8 weeks of control or KBG treatment, mice fed with HFD were changed to ND until the termination of experiment with maintaining KBG treatment for another 4 weeks. Liver tissue samples were collected upon the termination and levels of triglyceride (TG, left), total cholesterol (cholesterol, middle), and free fatty acid (FAA, right) were measured. Data are mean ± SEM (n = 7–15). ^* P < 0.005.

Figure 5

Histopathological evaluation of fat and liver tissue in DIO mice. C57BL/6 mice were fed with normal diet (ND) or high-fat diet (HFD) for 10 weeks and then administered with control water or KBG (500 mg/kg, p.o., daily) for 12 weeks under the same feeding condition. After 8 weeks of control or KBG treatment, mice fed with HFD were changed to ND until the termination of experiment with maintaining KBG treatment for another 4 weeks. (a) Representative images (×200) of H&E staining of liver (upper panels) and white adipose tissue (lower panels) of DIO mice are shown. (b) Steatosis scores of DIO mice. N.D.: not detected. (c) Observed number of adipocyte counts of WAT in DIO mice. Data are mean ± SEM (n = 7–15). ^** P < 0.001.

Figure 6

Effect of KBG on obesity-associated gene expression in liver tissue of DIO mice. C57BL/6 mice were fed with normal diet (ND) or high-fat diet (HFD) for 10 weeks and then administered with control water or KBG (500 mg/kg, p.o., daily) for 12 weeks under the same feeding condition. After 8 weeks of control or KBG treatment, mice fed with HFD were changed to ND until the termination of experiment with maintaining KBG treatment for another 4 weeks. Liver tissue samples were collected upon the termination and the relative expression of obesity-associated genes (PPARg, PPARa, and Srebf1) was determined by RT-PCR and shown as relative expression to HFD group (HFD = 1). Data are mean ± SEM (n = 7–15). ^** P < 0.001.