Dansameum regulates hepatic lipogenesis and inflammation in vitro and in vivo

Abstract

Although the clinical guidelines for nonalcoholic fatty liver disease (NAFLD) therapy recommended hepato-protection and exercise to reduce body weight, no established medication exists for NAFLD treatment. Thus, the effect of a candidate substance, dansameum (DSE), using an in vitro and NAFLD mouse model (that is, apolipoprotein E-Knockout mice), were investigated. The molecular pathways for lipogenesis and inflammation were evaluated using Nile staining, Western blotting, reverse transcription-polymerase chain reaction, and immunohistochemistry. It was shown that DSE significantly ameliorated the production of lipogenesis-related factors, including liver X receptor-α, peroxisome proliferator-activated receptor-γ, sterol regulatory element binding protein-1, fatty acid synthase, acetyl-CoA carboxylase 1, and CD36. In addition, DSE significantly reduced the production of inflammation factors, including interleukin-1β, interleukin-6, and nuclear factor kappa B. Furthermore, DSE significantly reduced the phosphorylation of c-Jun amino terminal kinase. Taken together, this suggests that DSE may be a functional food candidate for regulating NAFLD, based on its effects.

Keywords: Functional foods; Inflammation; Lipogenesis; Nonalcoholic fatty liver disease; Steatosis; Traditional Chinese medicine.

Fig. 1

Effects of Dansameum (DSE) on the accumulation of intracellular lipids. (A) HepG2 cells were pretreated using DSE for 16 h, and then incubated with 0.5 mM palmitic acid (PA) for 8 h before staining using Nile red. (B) Statistical results obtained from panel (A). Intracellular lipid droplets were evaluated using fluorescence microscopy (magnification × 400). Data are presented as mean ± standard error (n = 6). *p < 0.05 versus the PA-stimulated response in the absence of DSE treatment. (Color figure online)

Fig. 2

Effects of Dansameum (DSE) on lipogenesis pathways in HepG2 cells. HepG2 cells were pretreated using DSE for 16 h, and then stimulated using 0.5 mM palmitic acid (PA) for 8 h. RT-PCR and gel electrophoresis were used to evaluate mRNA expression levels. (A) The mRNA expressions of lipogenesis-related receptors: liver X receptor-α (LXR-α) and peroxisome proliferator-activated receptor gamma (PPAR-γ). (B and C) The mRNA expressions of LXR-α and PPAR-γ, relatively toPA-stimulated cells without DSE pretreatment (n = 6). (D) The mRNA expressions of lipogenesis-related transcription factors: sterol regulatory element binding protein-1 (SREBP-1), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD)-1, and CD36. (E–I) The protein expressions of SREBP-1, ACC1, FAS, SCD-1, and CD36, relative to PA-stimulated cells without DSE pretreatment (n = 6). Data are presented as mean ± standard error. *p < 0.05 versus the PA-stimulated response in the absence of DSE treatment

Fig. 3

Protective effect of Dansameum (DSE) on lipogenesis pathways in the liver of ApoE^−/− mice that received a high-fat (HF) diet. Photomicrographs of the untreated ApoE^−/− mice, mice that received only the HF diet for 12 weeks, and mice that received 240 mg/kg of DSE at 12 weeks after the HF diet. (A) Hematoxylin and eosin staining (bar = 100 µm), the red lines indicates the inflammation and damaged areas. (B) Liver tissue sections that were stained using Oil Red O (bar = 100 µm). (C) The graph indicates the intensity of Oil Red O stain from panel (B). (D) Immunohistochemical staining for LXR, PPAR-γ, SREBP-1, and CD36. (E–H) The expressions of LXR-α, PPAR-γ, SREBP-1, and CD36 in ApoE^−/− mice (n = 6). Data are presented as mean ± standard error. *p < 0.05 versus the HF-diet response in the absence of DSE treatment in ApoE^−/− mice

Fig. 4

Effects of Dansameum (DSE) on the phosphorylationof c-Jun amino terminal kinase (JNK) in HepG2 cells. (A) A total of 1 × 10⁵ HepG2 cells were pretreated using DSE for 16 h, and then incubated with 0.5 mM palmitic acid (PA) for 8 h. Whole-cell lysates were prepared and protein expressions were determined using Western blotting. (B) The relative expressions of phosphorylated JNK, relative to the untreated group (n = 6). Data are presented as mean ± standard error. *p < 0.05 versus the PA-stimulated response in the absence of DSE treatment

Fig. 5

Effects of Dansameum (DSE) on the expressions of pro-inflammatory cytokines and NF-κB activation in HepG2 cells. HepG2 cells were pretreated using DSE for 16 h, and then incubated with 0.5 mM palmitic acid (PA) for 8 h. (A) The mRNA expressions of IL-1β and IL-6 were determined using RT-PCR. (B and C) The protein expressions of IL-1β and IL-6, relative to PA-stimulated cells without DSE pretreatment (n = 6). (D and F) Whole-cell lysates and nuclear fractions were prepared to evaluate protein expressions using Western blot analysis. (E and G) The phosphorylation of NF-κB in whole cells and the nucleus, relative to PA-stimulated cells without DSE pretreatment (n = 6). Data are presented as mean ± standard error. *p < 0.05 versus the PA-stimulated response in the absence of DSE treatment

Fig. 6

Effects of Dansameum (DSE) on JNK, pro-inflammatory cytokines, and NF-κB activation in ApoE^−/− mice that received a high-fat (HF) diet. Photomicrographs of the untreated ApoE^−/− mice, mice that received only the HF diet for 12 weeks, and mice that received 240 mg/kg of DSE at 12 weeks after the HF diet. (A) the ROS marker indicates 8-oxoguanine (8-OXG), (bar = 100 µm) (B) The graph indicates the intensity of ROS stain from panel (A). (C) JNK expression in mouse liver tissues (bar = 100 µm). (D) The relative expressions of phosphorylated JNK in ApoE^−/− mice (n = 6). (E) Immunohistochemical staining for LXR, PPAR-γ, SREBP-1, and CD36. (F–I) The expressions of LXR-α, PPAR-γ, SREBP-1, and CD36 in ApoE^−/− mice (n = 6). Data are presented as mean ± standard error. *p < 0.05 versus the HF-diet response in the absence of DSE treatment in ApoE^−/− mice