Dihydrocapsaicin down-regulates apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells

Abstract

Background: Apolipoprotein M (apoM), as a novel apolipoprotein which is mainly expressed in liver and kidney tissues, is associated with development and progression of atherosclerosis and diabetes. Our group have recently shown that Dihydrocapsaicin(DHC)can significantly decrease atherosclerotic plaque formation in apoE-/- mice. However, the effect and possible mechanism of DHC on apoM expression remain unclear.

Methods: HepG2 cells were treated with 0 μM, 25 μM, 50 μM and 100 μM DHC for 24 h or were treated with 100 μM DHC for 0, 6, 12, and 24 h, respectively. The mRNA levels and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively.

Results: We found that DHC markedly decreased expression of apoM at both mRNA and protein level in HepG2 cells in a dose-dependent and time-dependent manner. Expression of Foxa2 was decreased while expression of LXRα was increased by DHC treatment in HepG2 cells. In addittion, overexpression of Foxa2 markedly compensated the inhibition effect induced by DHC on apoM expression. LXRα small interfering RNA significantly abolished the inhibition effect which induced by DHC on apoM expression. The liver of C57BL/6 mice treated with DHC had significantly lower expression of apoM. Furthermore, the liver had lower expression of Foxa2 while had higher expression of LXRα.

Conclusions: DHC could down-regulate apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells.

Figure 1

Effect of DHC on apoM expression in HepG2 cells. (A and B) HepG2 cells were divided into four groups and cultured in medium at 37°C containing 0 μM, 25 μM, 50 μM and 100 μM DHC for 24 h, respectively. ApoM transcript levels and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively. (C and D) HepG2 cells were treated with 100 μM DHC for 0, 6, 12, and24 h, respectively. ApoM transcript levels and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively. All results are presented as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 vs. the control group.

Figure 2

Effect of DHC on Foxa2 and expression in HepG2 cells. (A and B) HepG2 cells were divided into four groups and cultured in medium at 37°C containing 0 μM, 25 μM, 50 μM and 100 μM DHC for 24 h, respectively. Foxa2 transcript levels and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively. (C) HepG2 cells were transfected with negative control or Foxa2 siRNA. And then protein samples were measured by western blot analysis. (D) HepG2 cells were transfected with Foxa2 or control siRNA and then incubated with 100 μM DHC for 24 h. And then protein samples were measured by western blot analysis. (E) HepG2 cells were transfected with pcDNA3.1-mock (control group) or pcDNA-Foxa2 (pcDNA group). And then protein samples were measured by western blot analysis. (F) HepG2 cells were transfected with pcDNA-Foxa2 or pcDNA-control and then incubated with 100 μM DHC for 24 h. Protein samples were measured by western blot analysis. All results are presented as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 vs. the control group.

Figure 3

Effect of DHC on LXRα and expression in HepG2 cells. (A and B) HepG2 cells were divided into four groups and cultured in medium at 37uC containing 0 μM, 25 μM, 50 μM and 100 μM DHC for 24 h, respectively. LXRα transcript levels and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively. (C) HepG2 cells were transfected with negative control or LXRα siRNA. And then protein samples were measured by western blot analysis. (D) HepG2 cells were transfected with LXRα or control siRNA and then incubated with 100 μM DHC for 24 h. And then protein samples were measured by western blot analysis. (E) HepG2 cells were transfected with pcDNA3.1-mock (control group) or pcDNA-LXRα(pcDNA group). And then protein samples were measured by western blot analysis. (F) HepG2 cells were transfected with pcDNA-LXRα or pcDNA-control and then incubated with 100 μM DHC for 24 h. Protein samples were were measured by western blot analysis. All results are presented as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 vs. the control group.

Figure 4

Effect of DHC on hepatic apoM, Foxa2 and LXRα expression. C57BL/6 Mice were randomized into the control group or the DHC group, and treated with either vehicle (cholesterol-free vegetable oil) or DHC (3.0 mg/kg body weight) daily by oral gavage for 1 week. The protein expression of apoM, Foxa2 and LXRα was measured by western blot. All results are presented as mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05 vs. the control group.