Capsaicinoids, including capsaicin and dihydrocapsaicin, are the major pungent constituents of ‘hot chilli peppers’ of the Capsicum genus. In addition to food additive uses in our diet, the other biological properties and medical applications of capsaicin make this compound very popular. Previous studies have demonstrated that capsaicin is able to stimulate the release of calcitonin gene-related peptide (CGRP) by activating transient receptor potential channel vanilloid type 1 (TRPV1) and therefore it has potential benefits for cardiovascular function . Furthermore, capsaicinoids can also contribute to their beneficial effects on the cardiovascular system through their antioxidant properties [30, 31]. Our group have recently shown that DHC can significantly decrease atherosclerotic plaque formation involving in a PPARγ/LXRα pathway in apoE−/− mice fed a high-fat/high-cholesterol diet . In the present study, we demonstrated that apoM expression was regulated by DHC in HepG2 cells in a dose-dependent and time-dependent manner and that inhibition of apoM expression by DHC was mediated by Foxa2 and LXRα.
ApoM is a recently discovered apolipoprotein that is important for pre-β-HDL formation [32–35]. Overexpression of apoM in mice increases plasma HDL-cholesterol, and apoM-deficiency decreases HDL-cholesterol . Moreover, apoM-enrichment of HDL augments the ability of HDL to mobilize cholesterol from macrophage-derived foam cells [32, 36]. Here, we found that DHC could decrease apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells. ApoM is predominantly associated with HDL (96% is bound to HDL), but only 5% of the plasma HDL particles contain an apoM molecule . Although it has been proved that apoM-deficient led to the appearance of plasma HDL particles that were larger than those of normal mice,which impaired in their ability to transport cholesterol out of macrophages , the effect of apoM-deficient on HDL is limited since just 5% of the plasma HDL particles contain an apoM molecule. In addition, apoM is a negative acute response protein that decreases during inflammation. The level of apoM in mouse serum decreased following LPS administration and decrease of its expression in the liver occurs rapidly . Inflammation could increase the risk for atherosclerosis including alterations in lipid and lipoprotein metabolism. Besides, HDL isolated during inflammaion is poorer at removing cholesterol from macrophages than normal HDL . Zhang et al. showed that high glucose could inhibit apoM expression in HepG2 cells. Serum apoM concentrations and hepatic apoM mRNA levels were significantly reduced in the hyperglycemic rats, indicating that the low expression levels of apoM in these diabetic animals could be ascribed to hyperglycemia . Similarly, plasma apoM is modestly reduced in patients with diabetes compared to controls . These observations suggest that apoM is link to the development and progression of atherosclerosis and diabetes. In the present study, we found that DHC could decrease apoM expression by inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells. The interesting thing was that our previous study revealed that DHC could significantly decrease atherosclerotic plaque formation through enhancing expression of PPARγ and LXRα in apoE−/− mice fed a high-fat/high-cholesterol diet . Here, we demonstrated that DHC could enhance LXRα expression, which was consistent with our previous study. However, it is important to consider the inhibition of apoM expression by DHC when it was used as the therapeutic drug for atherosclerosis and diabetes. Thus, more experiments should be performed to prove DHC might be potentially used to treat the patients with atherosclerosis and diabetes.
Foxa2, a key regulator of hepatic lipid metabolism, which is transcriptionally active in the fasted state and induces expression of enzymes involved in fatty acid oxidation, ketogenesis, and VLDL secretion and bile acid metabolism, is regulated via insulin/PI3K/Akt-mediated phosphorylation at a single conserved threonine (Thr156) residue . Previous studies have shown that apoM expression and plasma pre-β-HDL levels are decreased in obese mice due to inactivation of Foxa2 in the accompanying hyperinsulinemic state . Treatment of wild-type mice and ob/ob mice with an adenovirus containing phosphorylation-defective Foxa2 not only improved glucose and lipid homeostasis but also increased hepatic apoM mRNA expression. In contrast, haplo-insufficient Foxa2+/−mice exhibited decreases in hepatic apoM expression and in plasma pre-β-HDL and HDL levels. A binding site for Foxa2 in the APOM promoter, at position −474, was identified . In the present study, we showed that Foxa2 expression was significantly decreased by DHC treatment in HepG2 cells. The down-regulation of apoM expression via DHC treatment was markedly accentated by Foxa2 siRNA treatment. Moreover, the suppression of apoM expression by DHC was markedly compensated by treatment with pcDNA-Foxa2. These suggested that Foxa2 was involved in apoM expression regulation by DHC in HepG2 cells.
LXRα is expressed mainly in the liver, intestine, adipose tissue and macrophages . LXRs are involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity [26, 40]. Zhang et al. demonstrated that TO901317 downregulates apoM expression in the liver. They showed that mice treated with 100 mg/kg/d of TO901317 had significantly lower apoM plasma levels as compared to control mice. In cultured HepG2 cells, TO901317 caused a downregulation of apoM expression,which was in line with the in vivo findings . Thus, the agonist significantly decreases apoM mRNA expression in vivo and in vitro, indicating that APOM is another target gene of LXR. In the present study, we showed that the down-regulation of APOM expression via DHC treatment was markedly abolished by LXRα siRNA treatment. Moreover, the suppression of apoM expression by DHC was markedly accentated by treatment with pcDNA-LXRα, suggesting that LXRα is also involved in apoM expression regulation by DHC in HepG2 cells.
The liver is the major organ responsible for the production and degradation of apoM-containing lipoproteins . We showed that oral gavage DHC can significantly decrease expression of apoM. Meanwhile, DHC can markedly decrease expression of Foxa2 than the control group while increase expression of LXRα as compared to the control group in the liver of C57BL/6 Mice. In vivo studies were consistent with the in vitro findings, demonstrating DHC down-regulates apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression.