KBH-1, an herbal composition, improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats

Abstract

Background: KBH-1 is an herbal mixture of Saururus chinensis, Curcuma longa and Polygala tenuifolia. Each herb has been reported to have various pharmaceutical activities; however, the synergistic effect of this herbal composition on obesity has not yet been determined. We investigated the alleviation effect of KBH-1 and its possible molecular mechanism in obesity-induced hepatic steatosis and leptin resistance in the hypothalamus.

Methods: We used HepG2 cells, primary neuronal cells and a high-fat diet (HFD)-induced obesity rat model to determine the effect of KBH-1 in vitro and in vivo on hepatic steatosis and leptin resistance accompanied by obesity. To identify the alleviation effect on lipid accumulation, HepG2 cells stimulated by FFA were stained with Oil Red O; in addition, immunoblotting and qPCR were performed to determine the effect of KBH-1 on the activation of proteins and nuclear enzymes in HepG2 cells and the steatotic liver of HFD-induced obesity rats. To examine the effect of KBH-1 on the leptin resistance of the hypothalamus and its possible molecular mechanism, we examined the effect of KBH-1 on the activation of the leptin resistance-related protein in primary cultured cortical neuron cells and the hypothalamus of an HFD-induced obesity rat model. In addition, we used HPLC analysis to identify the standard compound of KBH-1.

Results: KBH-1 not only suppressed the lipid deposition in HepG2 cells exposed to free fatty acids (FFA) but also significantly down-regulated major factors in lipogenesis and up-regulated major factors in lipolysis. Similarly, in a HFD-induced obesity model, KBH-1 improved hepatic steatosis by alleviating the effects on lipogenic genes and kinases. In addition, KBH-1 significantly improved the leptin-mediated signals impaired by obesity or FFA in the obesity model and primary cultured cortical neuron cells. In addition, KBH-1 was analyzed to include six standard compounds using HPLC analysis, among these compounds, onji-saponin B and curcumin were potently suppressed the level of triglycerides.

Conclusions: KBH-1 exhibits alleviating effects by improving hepatic steatosis and leptin resistance by up-regulating the activation of AMPK and suppressing the expression of PPARγ. These findings show the potential of KBH-1 as a functional food supplement or preventive agent in the treatment of obesity.

Keywords: Curcuma longa; Hepatic steatosis; KBH-1; Leptin resistance; Polygala tenuifolia; Saururus chinensis.

Figures

Fig. 1

Effect of KBH-1 on lipid accumulation and the activation of lipogenic enzymes in HepG2 cells. HepG2 cells cultured in DMEM/F12 supplemented with 10% fetal bovine serum, then exposed to a mixture for FFA (oleic acid/palmitic acid at 2:1) at a final concentration of 1 mM for 24 h, and the effect of various concentrations of KBH-1 (10–90 μg/ml) was monitored. (a) Fat droplets in cells were stained with Oil Red O dye. (b) The FFA-induced phosphorylation of AMPK and ACC was measured using SDS-PAGE and immunoblotting. ACC, AMPK, and β-actin were used for normalization (n = 3). (c) The FFA-induced mRNA expression of major lipogenic transcription factors (SREBP-1c, SCD-1, CD36, ACC, ACOX1, CPT-1 and PPARα) was analyzed by quantitative real-time PCR. The results are expressed as a relative density after normalization to the β-actin mRNA level. The data are expressed as the mean ± SEM. Significant differences from each control (no KBH-1 treatment) are indicated by **p < 0.001

Fig. 2

Effect of KBH-1 on hepatic steatosis of the HFD-induced obesity model. Animals were subdivided into 5 groups: ND, HFD, PC (treated with 200 mg/kg of green tea extract), KBH-1 150 mg/kg, and KBH-1 300 mg/kg. (a) The body weight change of each group in an HFD-induced obesity model. (b) Representative hematoxylin-eosin staining. (c) The changes of hepatic TG and TC and liver weight. The data are expressed as the mean ± SEM. Significant differences from the HFD group are indicated by *p < 0.05 or **p < 0.001

Fig. 3

Anti-lipogenic effect of KBH-1 on hepatic steatosis. (a) Liver tissue was homogenized and the lysates were subjected to western blotting for AMPK, ACC phosphorylation and PPARγ expression. (b). Liver tissue was homogenized and SREBP-1c, ACC and HMGCR were analyzed by quantitative real-time PCR. (c) Serum and hepatic MDA, TNFα and IL-1 β was measured with a commercial TBAR assay kit and ELISA kit. The data are expressed as the mean ± SEM. Significant differences from the HFD group are indicated by *p < 0.05 or **p < 0.001

Fig. 4

Effect of KBH-1 on the leptin resistance of the hypothalamus. (a) Hypothalamus tissue was homogenized and (A) serum leptin was measured with an ELISA kit. The feed efficiency ratio (%) was calculated as weight gain/food intake × 100. (b) The phosphorylation of ERK, AKT and AMPK was measured using western blotting. The data are expressed as the mean ± SEM. Significant differences from the HFD group are indicated by *p < 0.05 or **p < 0.001

Fig. 5

Effect of KBH-1 on leptin-resistant cortical neuron cells. Cells were cultured in neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine, and 25 μM glutamate. After 24 h, the culture medium was replaced with neurobasal medium without glutamate. For the resistance experiments, primary neurons were pretreated with 0.5 mM FFA (oleic acid:palmitic acid = 2:1) overnight prior to leptin treatment. For treatment studies, neurons were treated with KBH-1 for 1 h, then treated with 10 nM leptin. (a) Cell viability was determined by the CCK assay. (b) The activation of AMPK, RAS, cRaf, ERK, STAT3 and JAK2 were measured using SDS-PAGE and immunoblotting. β-actin was used for normalization (c, n = 3). The data are expressed as the mean ± SEM. Significant differences from each control (no KBH-1 treatment) are indicated by *p < 0.05 or **p < 0.001

Fig. 6

HPLC profile of KBH-1 and identification of active compound. The HPLC chromatogram of the six standard compounds was monitored at 280 nm. Black, green and red lines represent KBH-1 used in vitro, KBH-1 used in vivo with diluting agent and a mixture of six single compounds (a). HepG2 cells cultured in DMEM/F12 supplemented with 10% fetal bovine serum, then exposed to a mixture for FFA (oleic acid/palmitic acid at 2:1) at a final concentration of 1 mM for 24 h, and the effect of various concentrations (1-20 μg/ml) of six standard compounds (quercitrin, onji-saponin B, BDMC, DMC, curcumin and sauchinone) was monitored. (b) The TG level in cells was stained with Adipore. The data are expressed as the mean ± SEM. Significant differences from each control (no KBH-1 treatment) are indicated by *p < 0.05 or **p < 0.001