Dihydrocapsaicin Down-regulates ApoM Expression Through Inhibiting Foxa2 Expression And Enhancing LXRα Expression In HepG2 Cells

IntroductionCapsaicinoids, including capsaicin and dihydrocapsaicin(DHC), which together typically represent 85-90% of the total capsaicinoid content in ’hot chilli peppers’ extract, and its minor components include nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin. These spice principles have been proved to augment carbohydrate metabolism, energy expenditure and lipid metabolism in rodents and/or humans, and have been successfully used in the treatment of diverse clinical conditions such as relief the pain of peripheral neuropathies, sympomatic treatment of rheumatoid arthritis and osteoarthritis. Previous studies have demonstrated that regular consumption of chilli increases the resistance of oxidation of serum lipoproteins in vitro and attenuate postprandial hyperinsulinemia, actions that may help in reducing cardiovascular diseases (CVD) risk. Adams et al. have demonstrated that capsaicin and DHC could inhibit in vitro platelet aggregation and the activity of clotting factors VIII and IX, a property which may contribute to the prevention of the onset and/or treatment of CVD. Furthermore, our group have recently proved that DHC could significantly decrease atherosclerotic plaque formation involving in a PPARy/LXRa pathway in apoE-/-mice fed a high-fat/high-cholesterol die. These reports support the notion that capsaicinoids associate with CVD, such as atherosclerosis and coronary heart disease in particular.Apolipoprotein M (apoM) was first described by Xu and Dahlback in 1999. ApoM is a member of the lipocalin protein superfamily, whose members exhibit diverse properties such as lipid binding, transport, and immunological functions. ApoM, mainly expressed in hepatocytes and in the tubular epithelial cells of the kidney, is mainly associated to HDL (96% is bound to HDL), but also binds to low density lipoprotein (LDL), very low density lipoprotein (VLDL) and chylomicrons. It has been proved that apoM plays an important role in formation of pre-P-HDL and cholesterol efflux to HDL, which further influences the HDL cholesterol concentration in plasma. Moreover, the silencing of apoM expression was associated with the absence of pre-P-HDL particles in plasma. In addition, plasma apoM is modestly reduced in patients with diabetes compared to controls. Futhermore, 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. These observations support the notion that apoM is linked to cholesterol metabolism and diabetes.FOXA genes, formerly termed HNF3 (hepatocyte nuclear factors), is transcription factor involved in glucose homeostasis and lipid metabolism in liver. Foxa2 is phosphorylated and excluded from the nucleus when pasma insulin levels increase. A binding site for Foxa2 in the APOM promoter is at position-474. It had been proved that obese mice had decreased apoM expression and plsma pre-P-HDL levels due to inactivation of Foxa2 in the hyperinsulinemic state. Treatment wild-type mice and ob/ob mice with an adenovirus containing phosphorylation-defective Foxa2 not only improved glucose and lipid homeostasis but also incresed hepatic apoM mRNA expression. In contrast, haploinsufficient Foxa2+/-mice exhibited decreases in hepatic apoM expression and in plasma pre-P-HDL and HDL levels. Together, these results suggest that Foxa2 regulates APOM transcription.Liver X Receptor a (LXRa) is a major transcriptional regulator of cholesterol homeosasis and also regulates lipid and glucose metabolism. LXRa is more restricted and mainly found in liver, intestine, fat tissue,macrophages, kidney and gonads, suggesting their important function in the control of cholesterol homeostasis, whereas LXRβ is expressed in most cell types. Zhang et al. demonstrated that LXR agonist, TO901317, could decrease hepatic apoM expression in the vivo and in vitro. They showed that serum apoM of mice treated with 100 mg/kg/d of TO901317 decreased significantly as compared to control mice. In cultured HepG2 cells, TO901317 caused a downregulation of apoM expression, indicating that apoM is another target gene of LXR. The combination of 9-cis-retinoic acid (RA) and T0901317 showed additive effects, which suggests that apoM expression is modulated by the LXR/RXR pathway.However, the relation between the DHC and apoM in HepG2 cells remain unclear.whether Foxa2 and LXRα-dependent pathway involving in this progress has not yet been explored.In the present study, we demonstrated that DHC could markedly down-regulate apoM expression through inhibiting Foxa2 expression and enhancing LXRα expression in HepG2 cells.Materials and Methods1. ChemicalsDHC was initially dissolved in 100% ethanol to make a stock solution of 1 mol/L and then diluted for further experiments in normal buffered saline. The ethanol concentration of the dilutions was 0.1%. Normal buffered saline containing 0.1% ethanol was used as the solvent in the control cells. Dilutions were prepared from stock solutions shortly before the experiment.2. Cell cultureHuman hepatocytes (HepG2) was purchased from the American Type Culture Collection(ATCC, Manassas, VA, USA). The HepG2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Cells were incubated at 37℃ in an atmosphere of 5% CO2. Cells were seeded in 6-or12-well plates or 60-mm dishes and grown to 60%-80% confluence before use.3. AnimalsEight-week-old, female C57BL/6 mice (Laboratory Animal Center of Peking University, Beijing, China) with a mean body mass of 20 g were randomly divided into two groups(n=5/group):(1) Control group were treated with cholesterol-free vegetable oil by oral gavage (0.2 mL per mouse) for one week. (2) DHC group were treated with cholesterol-free vegetable oil containing DHC (3.0 mg/kg body weight) daily by oral gavage(0.2 mL per mouse) for one week. All animals were housed five per cage at 25℃ on a 12-h/12-h light/dark cycle with unlimited access to chow and water. The animal procedures were approved by the Animal Experimental Committee at Nanfang Hospital (Guangdong, China).4. RNA Isolation and Real-time Quantitative PCR Analysis(qPCR)Total RNA from mouse tissues or cultured cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on an ABI 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA,USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value wasconsidered for comparative analysis. Quantitative measurements were determined using the △△Ct method and GAPDH expression was used as the internal control.5. Western blot analysesProteins were extracted from mouse tissues or cultured cells using RIPA buffer (Biocolor Ltd., Belfast, Northern Ireland, UK), quantified using the BCA protein assay kit (KeyGen Biotechnologies,Nanjing, China), and then subjected to Western blot analyses (10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 30μg protein per lane) using rabbit polyclonal anti-APOM antibodies (BD Bio-sciences, San Jose, CA, USA), rabbit polyclonal anti-Foxa2 antibodies(Epitomics.,CA,USA), and rabbit polyclonal anti-LXRα (Proteintech group, Inc., Chicago, IL, USA) and P-actin-specific antibodies (Abcam Inc.,Cambridge, MA, USA). The proteins were visualized using a chemiluminescence method (ECL Plus Western blot Detection System; Amerisham Biosciences, Foster City, CA, USA). 6. Transfection with small interfering RNA (siRNA)The siRNas against Foxa2 and LXRa and an irrelevant 21-nucleotide control siRNA (Negative Control) were purchsed from Ribo Biotechnology. Cells (2×106 cells/well) were transfected using Lipofectamine2000 transfection reagent for 48 h according to the manufacturer’s instructions. After 48 h of transfection, real-time RT-PCR and Western blotting were performed.7. Construction of recombinant plasmidsThe PIRES2-EGFP and PCR-XL-TOPO vectors (containing Foxa2 or LXRa which was ssembled by the chemically synthesized oligos through PCR) were purchased from Invitrogen. Segments of EcoRI-Foxa2 or LXRa and IRES-EGFP-XhoI were amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. EcoRI-Foxa2-IRES-EGFP-XhoI or EcoRI-LXRa-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested by EcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent E.coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-Foxa2 and pcDNA3.1-LXRa. The plasmid transfection process was performed using Lipofectamine2000 transfection reagent according to the manufacturer’s instructions.8. Statistical analysesData are reported as means ±S.D. The date were compared by one-way analysis of variance and the Student’s t-test, using the Statistical Package for the Social Sciences (version13.0) software (SPSS, Inc., Chicago, IL, USA). Statistical significance was obtained when p values were less than 0.05.Results1. DHC down-regulates apoM expression in HepG2 cells.ApoM is highly expressed in the liver and kidney in humans, mice, and pigs. In the liver, apoM is expressed in hepatocytes and mainly secreted into the plasma, where it becomes integrated in plasma lipoproteins. We first examined the effect of DHC on apoM expression in HepG2 cells by real-time quantitative PCR and Western blot assays. DHC obviously decreased apoM expression at both transcriptional levels and translational levels in a dose-dependent and time-dependent manner.2. Foxa2 is involved in DHC-induced down-regulation of apoM in HepG2 cells.Previously researches revealed that apoM expression is directly regulated by Foxa2, which is a transcription factor involved in hepatic development via the regulation of glucose homeostasis in liver. We next explore whether Foxa2 expression can be affected by DHC in HepG2 cells by real-time quantitative PCR and Western blot analysis. DHC obviously decreased Foxa2 mRNA and protein expression in a dose-dependent. We then examined the effect of Foxa2 siRNA on the down-regulation of apoM which was induced by DHC. In comparison to the control siRNA, treatment with siRNA targeting Foxa2 decreased Foxa2 protein expression by 87% in HepG2 cells. The down-regulation of apoM expression via DHC treatment was markedly accentated by Foxa2 siRNA treatment. Next, we observed the effects of recombinant plasmids over-expressing Foxa2 (pcDNA-Foxa2) on apoM expression following DHC treatment in HepG2 cells. Treatment with pcDNA-Foxa2 increased Foxa2 protein expression by 565% in HepG2 cells. Suppression of apoM expression by DHC was markedly compensated by treatment with pcDNA-Foxa2.3. DHC down-regulates apoM expression through enhancing LXRa expression in HepG2 cells.The previous study has proved that apoM mRNA levels were significantly lower in the presence of LXR agonists in HepG2 cell. A similar reduction was found in vivo. We next investigated whether LXRa involved in DHC included down-regulation of apoM. For this purpose, we performed gene and protein expression of LXRa in HepG2 cells following treatment with DHC. DHC obviously increased LXRa mRNA and protein expression in a dose-dependent manner. In comparison to the control siRNA, treatment with siRNA targeting LXRa decreased LXRa protein expression by 91% in HepG2 cells. The down-regulation of apoM expression by DHC treatment was markedly abolished by LXRa siRNA treatment. In addition, we observed the effects of recombinant plasmids over-expressing LXRa (pcDNA-LXRa) on apoM expression following DHC treatment in HepG2 cells. Treatment with pcDNA-LXRa increased LXRa protein expression by 617% in HepG2 cells. Suppression of apoM expression by DHC was markedly accentated by treatment with pcDNA-LXRa.4. Effect of DHC on hepatic tissue apoM expression.The liver is the major organ responsible for the production and degradation of apoM-containing lipoproteins. Therefore, we next analyzed the effect of DHC on apoM, Foxa2 and LXRa expression in the liver of C57BL/6 Mice by Western blot analyses. The control and DHC groups were treated with either vehicle (cholesterol-free vegetable oil) or with DHC (3.0 mg/kg body weight, dissolved in cholesterol-free vegetable oil) daily by oral gavage (0.2 mL per mouse) for one weeks and then protein levels in C57BL/6 Mice liver tissues were investigated by Western blot. The DHC group had significantly lower expression of apoM and Foxa2 than the control group while the DHC group had higher expression change of LXRa as compared to the control group.Conclusions1. DHC obviously decreased apoM expression at both transcriptional levels and translational levels in a dose-dependent and time-dependent manner.2. DHC obviously decreased Foxa2 mRNA and protein expression in a dose-dependent manner. The down-regulation of apoM expression via DHC treatment was markedly accentated by Foxa2 siRNA treatment. Suppression of apoM expression by DHC was markedly compensated by treatment with pcDNA-Foxa2.3. DHC obviously increased LXRa mRNA and protein expression in a dose-dependent manner. The down-regulation of apoM expression by DHC treatment was markedly abolished by LXRa siRNA treatment. Suppression of apoM expression by DHC was markedly accentated by treatment with pcDNA-LXRa.4. Oral gavage DHC can significantly decrease expression of apoM in the liver of C57BL/6 Mice. Meanwhile, DHC can markedly decresae expression of Foxa2 while increase expression of LXRa as compared to the control group in the liver of C57BL/6 Mice.