Alpinetin enhances cholesterol efflux and inhibits lipid accumulation in oxidized low density lipoprotein-loaded human macrophages Zhengming Jiang, Haiqiang Sang, Xin Fu, Ying Liang, Ling Li*. Department of Cardiology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
*
Corresponding author.
Department of Cardiology, the First Affiliated Hospital of Zhengzhou University, No. 1 Jian she dong Road, Zhengzhou 450052, China Tel: 008637166913177 Fax: 008637166913178 Email: [email protected]
Running title: Alpinetin promotes cholesterol efflux.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1328. This article is protected by copyright. All rights reserved.
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Abstract
Alpinetin is a natural flavonoid abundantly present in the ginger family. Here, we investigated the effect of alpinetin on cholesterol efflux and lipid accumulation in oxidized low density lipoprotein (ox-LDL)-treated THP-1 macrophages and human peripheral blood monocyte-derived macrophages (HMDMs). After exposing THP-1 macrophages to alpinetin, cholesterol efflux was determined by liquid scintillator. The mRNA and protein levels of peroxisome proliferator-activated receptor-gamma (PPAR-γ), liver X receptor-alpha (LXR-α), ATP binding cassette transporter A1 (ABCA1), ABCG1 and scavenger receptor class B member 1, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Alpinetin promoted apolipoprotein A-I (ApoA-I)- and high-density lipoprotein (HDL)-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in a dose-dependent fashion in ox-LDL-treated THP-1 macrophages and HMDMs. Small-interfering RNA-mediated silencing of PPAR-γ or LXR-α, dose-dependently reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Taken together, our results reveal that alpinetin exhibits positive effects on cholesterol efflux and inhibits ox-LDL-induced lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.
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Key words: Alpinetin; cholesterol efflux; peroxisome proliferator-activated receptor-gamma (PPAR-γ); liver X receptor-alpha (LXR-α); ATP binding cassette transporter A1 (ABCA1), ABCG1.
Introduction
A critical event in the progression of atherosclerosis is the differentiation of monocytes into macrophages that accumulate lipoprotein-derived cholesterol and develop into foam cells to help plaque formation in arteries (1). Reverse cholesterol transport (RCT) is a process to transport excess cellular cholesterol from peripheral tissues to the liver for excretion, and thus prevents atherosclerosis (2). Major constituents of RCT include acceptors, such as high-density lipoprotein (HDL) and apolipoprotein A-I (ApoA-I), and enzymes, such as lecithin: cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) (3). A critical part of RCT is cholesterol efflux (3), in which lipid-poor ApoA-I, the major component of HDL, interacts with ATP-binding cassette A1 (ABCA1) to export free cholesterol (FC) and phospholipids (PL) from macrophages and serves as the first step in RCT (4-7). Additionally, accumulated cholesterol can be removed from macrophages by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1) (8, 9), caveolins (10) and sterol 27-hydroxylase (11), and collected by HDL and ApoA-I (3). Esterified cholesterol in the HDL is then delivered to the liver for excretion.
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Alpinetin (7-hydroxy-5-methoxyflavanone, molecular formula C16H14O4, molecular weight 270.28), is a natural flavonoid abundantly found in the ginger family, such as turmeric, cardamom, and radix curcumae (12-14). Oral administration of a turmeric extract has been shown to inhibit low-density lipoprotein (LDL) oxidation and exhibit hypocholesterolemic effects in rabbits with experimental atherosclerosis (15). Cardamom extract can protect platelets from aggregation and lipid peroxidation (16). It seems that the members of ginger family have anti-atherosclerosis effects. In the present study, we sought to investigate the effect of alpinetin on cholesterol efflux, an important process for preventing atherosclerosis.
Methods
Cell Culture Human monocytic THP-1 cells were bought from Shanghai Cells Bank (Shanghai, China). THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Invitrogen) in a humidified atmosphere of 5% CO2 at 37 °C. Differentiation of THP-1 monocytes into macrophages was induced by culturing the cells in the presence of 100 nM of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) for 72 h. Fresh human peripheral blood mononuclear cells were isolated from healthy volunteers with Ficoll density centrifugation according to the approval of the ethics committee of Zhengzhou University. Healthy volunteers gave their written informed consent. Monocytes were This article is protected by copyright. All rights reserved.
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prepared by negative selection using human monocyte enrichment set-DM (BD Biosciences, San Jose, CA). After 2h of incubation (37 °C, 5% CO2) for adherence, the medium was replaced with RPMI 1640 containing 20% autologous human serum (1). Adhered moncytes were cultured at 37 °C in 5% CO2 for 7 days to induce differentiation into macrophages with change of medium every 3 days (1). THP-1 macrophages or HMDMs were exposed to 50 μg/ml of ox-LDL for 24 h and then incubated with varying concentrations of alpinetin (the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; 50, 100, and 150 μg/ml) for 24 h.
Cholesterol efflux assay Percentage of cholesterol efflux was analyzed by liquid scintillation counting. Cells were labeled with 1.0 μCi/ml [3H] cholesterol. 10 μg/ml of ApoA-I or 50 μg/mL of HDL was also added to media. The percentage of cholesterol efflux was calculated by the following equation: [total media count/ (total cellular count + total media count)] × 100%.
Cell transfection Human PPAR-γ- and LXR-α-specific small interfering RNA (siRNA) and scrambled control RNA were obtained from Ambion Inc. (Austin, TX, USA). The transfection of siRNA was conducted with lipofectamine 2000 (Invitrogen). Briefly, THP-1 cells were stimulated with 100 nM PMA for 72 h and then exposed to 50 μg/ml of ox-LDL for another 24 h. Cells were incubated with 25 or 50 nM of control, PPAR-γ- or LXR-α-specific siRNA for 24 h and then indicated concentrations of alpinetin were added. Cells were collected 24 h after the treatment This article is protected by copyright. All rights reserved.
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with alpinetin. The oligonucleotide sequences used to construct siRNA in the present study were: 5’-GGAUGCAAGGGUUUCUUCCtt-3’ and 5’-GGAAGAAACCCUUGCAUCCtt-3’ for
PPAR-γ
siRNA;
and
5’-GGAGUGUGUCCUGUCAGAAtt-3’
and
5’-UUCUGACAGGACACACUCCtc-3’ for LXR-α siRNA.
Reverse transcription polymerase chain reaction Total RNA from cells was extracted with TRIzol reagent (Invitrogen) in according to the manufacturer’s instructions. RNA was reverse transcribed into complementary DNA (cDNA) by a Superscript First-Strand Synthesis System for RT-PCR kit (Invitrogen). The primer sequences used in this study were shown in Table 1. These primers were used to amplify cDNA fragments by PCR using Taq DNA polymerase (Invitrogen). The PCR cycles were conducted in a thermal cycler (Sigma). The PCR products were subjected to eletrophoresis in a 1% agarose gel.
Western blot analysis Cells were harvested and protein extracts were prepared with the ReadyPrep Protein Extraction kit (Total protein, Bio-Rad, Hercules, CA, USA). Protein samples were subjected to Western blot analyses (10% SDS-PAGE; 20 μg/lane) with anti-PPAR-γ, LXR-α, ABCA1, ABCG1, SR-B1, and β-actin (Santa Cruz, Santa Cruz, CA, USA) antibodies. The proteins were visualized by a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA, USA).
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Oil Red O staining After treatment, THP-1 cells were fixed with 10% neutral buffered formalin at room temperature for 1 h. Cells were washed with PBS and stained with 0.5% Oil Red O (in 60% isopropanol) for 1 h. Stained cells were washed with distilled water and observed under a microscope. The stained lipid droplets were extracted with isopropanol for quantification by measuring its absorbance at 490 nm.
Statistical analysis Quantitative data were represented as mean ± standard deviation (SD). Statistical significance of the data was analyzed by analysis of variance (ANOVA). P < 0.05 was considered significant. For nonquantitative data, results represent 3 independent experiments.
Results
Alpinetin enhances cholesterol efflux from ox-LDL-treated THP-1 macrophages and HMDMs. Dose-dependent enhancement of cholesterol efflux to ApoA-I was observed in response to alpinetin in THP-1 macrophages and HMDMs (Figure 1A). At 50, 100, and 150 μg/ml of alpinetin, the cholesterol efflux from THP-1 macrophages showed increases by 1.4-, 1.9-, and 2.6- fold of control group (with the treatment of ApoA-I only; Figure 1A), respectively. Similarly, alpinetin enhanced cholesterol efflux from HMDMs by 1.3-, 1.6-, and 2.3- fold of control at the concentration of 50, 100, 150 μg/ml (Figure 1A), respectively. This article is protected by copyright. All rights reserved.
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We also explored the effects of alpinetin on HDL-mediated cholesterol efflux (Figure 1B). Only 150 μg/ml of alpinetin, but not 50 or 100 μg/ml of alpinetin, enhanced HDL-induced cholesterol efflux in THP-1 macrophages and HMDMs (Figure 1A)
PPAR-γ
is
involved
in
alpinetin-induced
increase
of
cholesterol
efflux
from
ox-LDL-stimulated THP-1 macrophages and HMDMs. To determine how alpinetin increases cholesterol efflux, the expression of PPAR-γ, a key molecule involved in cholesterol efflux, was analyzed in ox-LDL loaded THP-1 macrophages and HMDMs. Treatment with alpinetin dose-dependently enhanced the mRNA levels in THP-1 macrophages and HMDMs, compared with control (Figure 2A). Consistent with mRNA data, the protein levels of PPAR-γ were also raised by alpinetin in a dose-dependent manner in both THP-1 macrophages and HMDMs (Figure 2B). PPAR-γ siRNA was used to analyze whether alpinetin-promoted cholesterol efflux is dependent on PPAR-γ. 25 and 50 nM of PPAR-γ siRNA suppressed PPAR-γ protein expression by up to 60% and 92%, respectively (Figure 2C). Transient transfection of THP-1 macrophages with PPAR-γ siRNA substantially abolished alpinetin-mediated induction of cholesterol efflux to ApoA-I and HDL. However, scrambled control siRNA had no effects on alpinetin-promoted cholesterol efflux to ApoA-I and HDL (Figure 2C). These results indicate that alpinetin enhances cholesterol efflux at least partially through PPAR-γ signaling pathway.
LXR-α confers alpinetin-induced increase of cholesterol efflux from ox-LDL-loaded THP-1 macrophages and HMDMs. This article is protected by copyright. All rights reserved.
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To analyze whether LXR-α expression is affected by alpinetin in ox-LDL-loaded THP-1 macrophages, RT-PCR and Western blot analysis were performed. As shown in Figure 3A and B, the expressions of LXR-α mRNA and protein were increased in a dose-dependent manner in ox-LDL–loaded THP-1 macrophages and HMDMs when cells were treated with alpinetin. We further examined the effect of LXR-α siRNA on alpinetin-induced cholesterol efflux. 25 and 50 nM of LXR-α siRNA decreased LXR-α protein expression by about 78% and 93%, respectively (Figure 3C). Cellular cholesterol efflux to ApoA-I and HDL was significantly reduced in THP-1 macrophages treated by the combination of LXR-α siRNA and alpinetin, compared with cells treated by alpinetin alone (Figure 3D). Thus, our results suggest that LXR-α is implicated in alpinetin-promoted cholesterol efflux.
Alpinetin decreases ox-LDL-induced lipid accumulation in THP-1 cells. Alpinetin inhibited ox-LDL-induced lipid accumulation by 16%, 40%, and 60% at the concentrations of 50, 100, and 150 μg/ml, respectively (Figure 4A). Alpinetin-mediated suppression of lipid accumulation in ox-LDL-treated THP-1 cells was almost completely reversed by 50 μg/ml of PPAR-γ siRNA (Figure 4B) or LXR-α siRNA (Figure 4C).
The expression of ABCA1 and ABCG1 is upregulated by alpinetin in ox-LDL-loaded THP-1 macrophages and HMDMs. The expression of ABCA1, ABCG1 and SR-B1 was also measured in THP-1 macrophages and HMDMs by RT-PCR and Western blot analysis (Figure 5). Alpinetin enhanced ABCA1 and ABCG1 mRNA (Figure 5A) and protein (Figure 5B) levels in a concentration-dependent This article is protected by copyright. All rights reserved.
9
manner in THP-1 macrophages and HMDMs, but not SR-B1.
The expression of LXR-α, PPAR-γ, ABCA1, and ABCG1 in PPAR-γ siRNA-transfected or LXR-α-transfect THP-1 cells. The expression of LXR-α, PPAR-γ, ABCA1 and ABCG1 proteins was also measured in alpinetin-stimulated THP-1 macrophages, which were transfected with PPAR-γ siRNA or LXR-α siRNA (Figure 6). Specific targeting of PPAR-γ decreased the expression of LXR-α, ABCA1 and ABCG1 at protein levels (Figure 6A). Similarly, specific silencing of LXR-α attenuated the expression of PPAR-γ, ABCA1 and ABCG1 (Figure 6B).
Discussion
Alpinetin promoted ApoA-I- and HDL-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in ox-LDL-treated THP-1 macrophages and HMDMs. Targeting of PPAR-γ or LXR-α, reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Our results indicate that alpinetin enhances cholesterol efflux and decreases ox-LDL-mediated lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.
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PPAR-γ, a transcription factor, is abundantly expressed in the foam cells of atherosclerotic lesions (17, 18). PPAR-γ has been shown to coordinate a complex physiologic response to ox-LDL in macrophages, which involves cholesterol uptake, processing and removal through ABCA1 (19). Alpinetin is a novel plant flavonoid derived from Alpinia katsumadai Hayata (20). It has been shown that alpinetin inhibits lipopolysaccharide (LPS)-induced inflammatory mediator response through activation of PPAR-γ in THP-1 macrophages (21). We hypothesized that alpinetin might have effects on cholesterol efflux in human macrophages. To test this hypothesis, we analyzed the effects of alpinetin on ApoA-I-induced cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. Interestingly, we found that alpinetin dose-dependently elevated cholesterol efflux in both THP-1 macrophages. Moreover, similar results were observed in ox-LDL-treated HMDMs. Both results indicate that alpinetin has positive effects on cholesterol efflux.
We found that alpinetin raised the expression of PPAR-γ at both mRNA and protein levels. Specific knock-down of PPAR-γ significantly reversed alpinetin-mediated positive effects on cholesterol efflux, indicating that to some extent, PPAR-γ is necessary for alpinetin-induced increase of cholesterol efflux. However, PPAR-γ inhibition does not completely suppress alpinetin-mediate effects, suggesting that additional pathways are involved. We also found that alpinetin suppressed ox-LDL-induced lipid accumulation, which was reversed by specific targeting of PPAR-γ. Our results suggest that PPAR-γ is a critical factor for alpinetin-mediated biology effects on THP-1 cells. Actually, many Chinese traditional medicines regulate cholesterol efflux or lipid accumulation through PPAR-γ signaling This article is protected by copyright. All rights reserved.
11
pathways, such as quercetin (22), resveratrol (23) and arctigenin (24). On the other hand, alpinetin induces the activation of PPAR-γ in THP-1 macrophages to inhibit LPS-induced inflammation (21). It seems that increasing the expression and promoting the activation of PPAR-γ are new strategies for alpinetin to regulate various cell behaviors under different cell contexts.
The nuclear receptors LXR-α is a direct target gene of PPAR-γ/retinoid X receptor (RXR) heterodimer (25). Previous work has documented that macrophage lipid loading induces ligand activation of PPAR-γ, which leads to primary induction of LXR-α, coupled with the upregulation of LXR-α target genes ABCA1 (19, 26). Here, we found that alpinetin enhanced the expression of LXR-α at both mRNA and protein levels in THP-1 macrophages and HMDMs. Silencing of LXR-α substantially inhibited alpinetin-mediated increase in cholesterol efflux and recovered alpinetin-mediated inhibition of ox-LDL-induced lipid accumulation
in
THP-1 macrophages,
indicating
the importance
of
LXR-α
in
alpinetin-mediated effect on THP-1 cells. ABCA1, a member of the ATP binding cassette (ABC) family, is a key contributor to modulate the cholesterol efflux and lipid uptake (27, 28). In addition to ABCA1, ABCG1 plays an important role in maintaining cholesterol homeostasis (29). ABCA1 regulates ApoA-I-medaited cholesterol efflux, but ABCG1 regulates HDL-induced cholesterol efflux (30, 31). SR-B1 increases bidirectional cholesterol exchange between cells and lipoprotein including HDL (32). In the present study, we found that alpinetin elevated ABCA1 and ABCG1 mRNA and protein expression, but not SR-B1 in both THP-1 macrophages and HMDMs, indicating that alpinetin enhances cholesterol efflux This article is protected by copyright. All rights reserved.
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and lipid accumulation through ABCA1 and ABCG1. Consistent with the previous studies (19, 25, 26), specific silencing of PPAR-γ decreased the expression of LXR-α, ABCA1 and ABCG1. Knockdown of LXR-α reduced the expression of PPAR-γ, ABCA1 and ABCG1. These results suggested that PPAR-γ and LXR-α, might be co-regulators, which modulate the expression of ABCA1 and ABCG1. How LXR-α silencing affects the expression of PPAR-γ is not clear yet. One possible mechanism might be through NF-κB signaling pathway. LXR-α agonist treatment reduces IκB-α degradation and NF-κB p65 activation in a mouse model of reperfusion
(SAO)
shock
(33).
On
the
other
hand,
the
activation
of
ERK
1/2-NF-κB-Nox4-dependent signaling pathway results in the reduction of PPAR-γ expression under hypoxic condition in human pulmonary artery smooth muscle cells (34). PPAR-γ expression is inhibited by the crosstalk between the canonical NF-κB and Notch signaling pathways, which promotes pancreatic cancer progression in mice (35). It seems that LXR-α can reduce the activation of NF-κB, which negatively regulates the expression of PPAR-γ. Alternatively, LXR-α silencing might induce the activation of NF-κB, which in turn attenuates the expression of PPAR-γ.
In sum, our observations reveal that alpinetin enhances cholesterol efflux and suppresses lipid accumulation through PPAR-γ/ LXR-α/ ABCA1/ABCG1 pathway in THP-1 macrophages and HMDMs.
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References
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Figure 1 Effects of alpinetin on cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. THP-1 macrophages or HMDMs were exposed to 50 μg/ml of ox-LDL for 24 h and then incubated with varying concentrations of alpinetin (50, 100, and 150 μg/ml) and 10 μg/ml of ApoA-I (A) or 50 μg/ml of HDL (B) for 24 h. Data represent mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 2 PPAR-γ is involved in alpinetin-promoted cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. (A) PPAR-γ mRNA expression in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (B) PPAR-γ protein expression in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (C) PPAR-γ protein expression in ox-LDL-loaded THP-1 macrophages transfected with PPAR-γ siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. (A-C) representative blots are from three independent experiments with similar results. THP-1 macrophages were exposed to 50 μg/ml of ox-LDL for 24 h and transfected with PPAR-γ siRNA or control siRNA. 24 h after transfection, cells were incubated with 150 μg/ml of alpinetin and 10 μg/ml of ApoA-I (D) or 50 μg/ml of HDL (E) for another 24 h. Data represent mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 3 LXR-α confers alpinetin-increased cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. (A) LXR-α mRNA levels in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (B) The expression of LXR-α protein in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (C) LXR-α protein expression in ox-LDL-loaded THP-1 macrophages transfected with LXR-α siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. (A-C) blots are from three independent experiments with similar results. THP-1 macrophages were exposed to 50 μg/ml of ox-LDL for 24 h and transfected with LXR-α siRNA or control siRNA. 24 h after transfection, cells were incubated with 150 μg/ml of alpinetin and 10 μg/ml of ApoA-I (D) or 50 μg/ml of HDL (E) for another 24 h. Data represent mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 4 Alpinetin inhibits ox-LDL-induced lipid accumulation in THP-1 macrophages. Cells were stained with Oil Red O for the presence of neutral lipids in ox-LDL-stimulated THP-1 macrophages (A), or ox-LDL-stimulated THP-1 macrophages transfected with PPAR-γ siRNA (B) or LXR-α siRNA (C). The absorbance of cell monolayers was spectrophotometrically determined at 490 nm after Oil Red O staining. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 5 Alpinetin enhances ABCA1 and ABCG1 mRNA and protein expression in ox-LDL-loaded THP-1 macrophages and HMDMs. (A) ABCA1, ABCG1 and SR-B1 mRNA expression in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (B) ABCA1, ABCG1 and SR-B1 protein levels in THP-1 macrophages and HMDMs. β-actin was used as an internal control. Blots represent three independent experiments with similar results`. Representative blots are from three independent experiments with similar results. Data represent mean ± SD (n = 3). **P < 0.01 versus indicated controls.
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Figure 6 PPAR-γ and LXR-α confer alpinetin-increased expression of ABCA1 and ABCG1 in THP-1 macrophages and HMDMs. (A) the protein levels of LXR-α, ABCA1 and ABCG1 in ox-LDL-loaded THP-1 macrophages transfected with PPAR-γ siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. (B) The protein levels of PPAR-γ, ABCA1 and ABCG1 in ox-LDL-loaded THP-1 macrophages transfected with LXR-α siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. Data represent mean ± SD (n = 3). **P < 0.01 versus LXR-α (A) or PPAR-γ controls (B); ^^P < 0.01 versus ABCA1 controls (A) and (B); ##P < 0.01 versus ABCG1 controls (A) and (B).
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Table 1 RT-PCR primer sequences. Gene
Primer sequences
PPAR-γ
Forward: 5’-CACAAGAACAGATCCAGTGGTTGCAG-3’; Reverse: 5’-AATAATAAGGTGGAGATGCAGGCTCC-3’
LXR-α
Forward:5′-AAGCCCTGCATGCCTACGT-3′; Reverse:5′-TGCAGACGCAGTGCAAACA-3′
ABCA1
Forward: 5′-CCCTGTGGAATGTACCTATGTG-3′; Reverse:5′-GAGGTGTCCCAAAGATGCAA-3′
ABCG1
Forward: 5′-CGGAGCCCAAGTCGGTGTG-3′; Reverse:5′-TTTCAGATGTCCATTCAGCAGGTC-3′
SR-B1
Forward: 5’-ATGATCGTGATGGTGCCGTC-3’ Reverse: 5’-GGAAGGAGATCCCTATCCCC-3’
β-actin
Forward: 5’-GTGGGGCGCCCCAGGCACCA-3’; Reverse: 5’-CTCCTTAATGTCACGCACGATTTC-3’
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*
Corresponding author.
Department of Cardiology, the First Affiliated Hospital of Zhengzhou University, No. 1 Jian she dong Road, Zhengzhou 450052, China Tel: 008637166913177 Fax: 008637166913178 Email: [email protected]
Running title: Alpinetin promotes cholesterol efflux.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1328. This article is protected by copyright. All rights reserved.
1
Abstract
Alpinetin is a natural flavonoid abundantly present in the ginger family. Here, we investigated the effect of alpinetin on cholesterol efflux and lipid accumulation in oxidized low density lipoprotein (ox-LDL)-treated THP-1 macrophages and human peripheral blood monocyte-derived macrophages (HMDMs). After exposing THP-1 macrophages to alpinetin, cholesterol efflux was determined by liquid scintillator. The mRNA and protein levels of peroxisome proliferator-activated receptor-gamma (PPAR-γ), liver X receptor-alpha (LXR-α), ATP binding cassette transporter A1 (ABCA1), ABCG1 and scavenger receptor class B member 1, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Alpinetin promoted apolipoprotein A-I (ApoA-I)- and high-density lipoprotein (HDL)-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in a dose-dependent fashion in ox-LDL-treated THP-1 macrophages and HMDMs. Small-interfering RNA-mediated silencing of PPAR-γ or LXR-α, dose-dependently reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Taken together, our results reveal that alpinetin exhibits positive effects on cholesterol efflux and inhibits ox-LDL-induced lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.
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Key words: Alpinetin; cholesterol efflux; peroxisome proliferator-activated receptor-gamma (PPAR-γ); liver X receptor-alpha (LXR-α); ATP binding cassette transporter A1 (ABCA1), ABCG1.
Introduction
A critical event in the progression of atherosclerosis is the differentiation of monocytes into macrophages that accumulate lipoprotein-derived cholesterol and develop into foam cells to help plaque formation in arteries (1). Reverse cholesterol transport (RCT) is a process to transport excess cellular cholesterol from peripheral tissues to the liver for excretion, and thus prevents atherosclerosis (2). Major constituents of RCT include acceptors, such as high-density lipoprotein (HDL) and apolipoprotein A-I (ApoA-I), and enzymes, such as lecithin: cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) (3). A critical part of RCT is cholesterol efflux (3), in which lipid-poor ApoA-I, the major component of HDL, interacts with ATP-binding cassette A1 (ABCA1) to export free cholesterol (FC) and phospholipids (PL) from macrophages and serves as the first step in RCT (4-7). Additionally, accumulated cholesterol can be removed from macrophages by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1) (8, 9), caveolins (10) and sterol 27-hydroxylase (11), and collected by HDL and ApoA-I (3). Esterified cholesterol in the HDL is then delivered to the liver for excretion.
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Alpinetin (7-hydroxy-5-methoxyflavanone, molecular formula C16H14O4, molecular weight 270.28), is a natural flavonoid abundantly found in the ginger family, such as turmeric, cardamom, and radix curcumae (12-14). Oral administration of a turmeric extract has been shown to inhibit low-density lipoprotein (LDL) oxidation and exhibit hypocholesterolemic effects in rabbits with experimental atherosclerosis (15). Cardamom extract can protect platelets from aggregation and lipid peroxidation (16). It seems that the members of ginger family have anti-atherosclerosis effects. In the present study, we sought to investigate the effect of alpinetin on cholesterol efflux, an important process for preventing atherosclerosis.
Methods
Cell Culture Human monocytic THP-1 cells were bought from Shanghai Cells Bank (Shanghai, China). THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Invitrogen) in a humidified atmosphere of 5% CO2 at 37 °C. Differentiation of THP-1 monocytes into macrophages was induced by culturing the cells in the presence of 100 nM of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) for 72 h. Fresh human peripheral blood mononuclear cells were isolated from healthy volunteers with Ficoll density centrifugation according to the approval of the ethics committee of Zhengzhou University. Healthy volunteers gave their written informed consent. Monocytes were This article is protected by copyright. All rights reserved.
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prepared by negative selection using human monocyte enrichment set-DM (BD Biosciences, San Jose, CA). After 2h of incubation (37 °C, 5% CO2) for adherence, the medium was replaced with RPMI 1640 containing 20% autologous human serum (1). Adhered moncytes were cultured at 37 °C in 5% CO2 for 7 days to induce differentiation into macrophages with change of medium every 3 days (1). THP-1 macrophages or HMDMs were exposed to 50 μg/ml of ox-LDL for 24 h and then incubated with varying concentrations of alpinetin (the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; 50, 100, and 150 μg/ml) for 24 h.
Cholesterol efflux assay Percentage of cholesterol efflux was analyzed by liquid scintillation counting. Cells were labeled with 1.0 μCi/ml [3H] cholesterol. 10 μg/ml of ApoA-I or 50 μg/mL of HDL was also added to media. The percentage of cholesterol efflux was calculated by the following equation: [total media count/ (total cellular count + total media count)] × 100%.
Cell transfection Human PPAR-γ- and LXR-α-specific small interfering RNA (siRNA) and scrambled control RNA were obtained from Ambion Inc. (Austin, TX, USA). The transfection of siRNA was conducted with lipofectamine 2000 (Invitrogen). Briefly, THP-1 cells were stimulated with 100 nM PMA for 72 h and then exposed to 50 μg/ml of ox-LDL for another 24 h. Cells were incubated with 25 or 50 nM of control, PPAR-γ- or LXR-α-specific siRNA for 24 h and then indicated concentrations of alpinetin were added. Cells were collected 24 h after the treatment This article is protected by copyright. All rights reserved.
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with alpinetin. The oligonucleotide sequences used to construct siRNA in the present study were: 5’-GGAUGCAAGGGUUUCUUCCtt-3’ and 5’-GGAAGAAACCCUUGCAUCCtt-3’ for
PPAR-γ
siRNA;
and
5’-GGAGUGUGUCCUGUCAGAAtt-3’
and
5’-UUCUGACAGGACACACUCCtc-3’ for LXR-α siRNA.
Reverse transcription polymerase chain reaction Total RNA from cells was extracted with TRIzol reagent (Invitrogen) in according to the manufacturer’s instructions. RNA was reverse transcribed into complementary DNA (cDNA) by a Superscript First-Strand Synthesis System for RT-PCR kit (Invitrogen). The primer sequences used in this study were shown in Table 1. These primers were used to amplify cDNA fragments by PCR using Taq DNA polymerase (Invitrogen). The PCR cycles were conducted in a thermal cycler (Sigma). The PCR products were subjected to eletrophoresis in a 1% agarose gel.
Western blot analysis Cells were harvested and protein extracts were prepared with the ReadyPrep Protein Extraction kit (Total protein, Bio-Rad, Hercules, CA, USA). Protein samples were subjected to Western blot analyses (10% SDS-PAGE; 20 μg/lane) with anti-PPAR-γ, LXR-α, ABCA1, ABCG1, SR-B1, and β-actin (Santa Cruz, Santa Cruz, CA, USA) antibodies. The proteins were visualized by a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA, USA).
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Oil Red O staining After treatment, THP-1 cells were fixed with 10% neutral buffered formalin at room temperature for 1 h. Cells were washed with PBS and stained with 0.5% Oil Red O (in 60% isopropanol) for 1 h. Stained cells were washed with distilled water and observed under a microscope. The stained lipid droplets were extracted with isopropanol for quantification by measuring its absorbance at 490 nm.
Statistical analysis Quantitative data were represented as mean ± standard deviation (SD). Statistical significance of the data was analyzed by analysis of variance (ANOVA). P < 0.05 was considered significant. For nonquantitative data, results represent 3 independent experiments.
Results
Alpinetin enhances cholesterol efflux from ox-LDL-treated THP-1 macrophages and HMDMs. Dose-dependent enhancement of cholesterol efflux to ApoA-I was observed in response to alpinetin in THP-1 macrophages and HMDMs (Figure 1A). At 50, 100, and 150 μg/ml of alpinetin, the cholesterol efflux from THP-1 macrophages showed increases by 1.4-, 1.9-, and 2.6- fold of control group (with the treatment of ApoA-I only; Figure 1A), respectively. Similarly, alpinetin enhanced cholesterol efflux from HMDMs by 1.3-, 1.6-, and 2.3- fold of control at the concentration of 50, 100, 150 μg/ml (Figure 1A), respectively. This article is protected by copyright. All rights reserved.
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We also explored the effects of alpinetin on HDL-mediated cholesterol efflux (Figure 1B). Only 150 μg/ml of alpinetin, but not 50 or 100 μg/ml of alpinetin, enhanced HDL-induced cholesterol efflux in THP-1 macrophages and HMDMs (Figure 1A)
PPAR-γ
is
involved
in
alpinetin-induced
increase
of
cholesterol
efflux
from
ox-LDL-stimulated THP-1 macrophages and HMDMs. To determine how alpinetin increases cholesterol efflux, the expression of PPAR-γ, a key molecule involved in cholesterol efflux, was analyzed in ox-LDL loaded THP-1 macrophages and HMDMs. Treatment with alpinetin dose-dependently enhanced the mRNA levels in THP-1 macrophages and HMDMs, compared with control (Figure 2A). Consistent with mRNA data, the protein levels of PPAR-γ were also raised by alpinetin in a dose-dependent manner in both THP-1 macrophages and HMDMs (Figure 2B). PPAR-γ siRNA was used to analyze whether alpinetin-promoted cholesterol efflux is dependent on PPAR-γ. 25 and 50 nM of PPAR-γ siRNA suppressed PPAR-γ protein expression by up to 60% and 92%, respectively (Figure 2C). Transient transfection of THP-1 macrophages with PPAR-γ siRNA substantially abolished alpinetin-mediated induction of cholesterol efflux to ApoA-I and HDL. However, scrambled control siRNA had no effects on alpinetin-promoted cholesterol efflux to ApoA-I and HDL (Figure 2C). These results indicate that alpinetin enhances cholesterol efflux at least partially through PPAR-γ signaling pathway.
LXR-α confers alpinetin-induced increase of cholesterol efflux from ox-LDL-loaded THP-1 macrophages and HMDMs. This article is protected by copyright. All rights reserved.
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To analyze whether LXR-α expression is affected by alpinetin in ox-LDL-loaded THP-1 macrophages, RT-PCR and Western blot analysis were performed. As shown in Figure 3A and B, the expressions of LXR-α mRNA and protein were increased in a dose-dependent manner in ox-LDL–loaded THP-1 macrophages and HMDMs when cells were treated with alpinetin. We further examined the effect of LXR-α siRNA on alpinetin-induced cholesterol efflux. 25 and 50 nM of LXR-α siRNA decreased LXR-α protein expression by about 78% and 93%, respectively (Figure 3C). Cellular cholesterol efflux to ApoA-I and HDL was significantly reduced in THP-1 macrophages treated by the combination of LXR-α siRNA and alpinetin, compared with cells treated by alpinetin alone (Figure 3D). Thus, our results suggest that LXR-α is implicated in alpinetin-promoted cholesterol efflux.
Alpinetin decreases ox-LDL-induced lipid accumulation in THP-1 cells. Alpinetin inhibited ox-LDL-induced lipid accumulation by 16%, 40%, and 60% at the concentrations of 50, 100, and 150 μg/ml, respectively (Figure 4A). Alpinetin-mediated suppression of lipid accumulation in ox-LDL-treated THP-1 cells was almost completely reversed by 50 μg/ml of PPAR-γ siRNA (Figure 4B) or LXR-α siRNA (Figure 4C).
The expression of ABCA1 and ABCG1 is upregulated by alpinetin in ox-LDL-loaded THP-1 macrophages and HMDMs. The expression of ABCA1, ABCG1 and SR-B1 was also measured in THP-1 macrophages and HMDMs by RT-PCR and Western blot analysis (Figure 5). Alpinetin enhanced ABCA1 and ABCG1 mRNA (Figure 5A) and protein (Figure 5B) levels in a concentration-dependent This article is protected by copyright. All rights reserved.
9
manner in THP-1 macrophages and HMDMs, but not SR-B1.
The expression of LXR-α, PPAR-γ, ABCA1, and ABCG1 in PPAR-γ siRNA-transfected or LXR-α-transfect THP-1 cells. The expression of LXR-α, PPAR-γ, ABCA1 and ABCG1 proteins was also measured in alpinetin-stimulated THP-1 macrophages, which were transfected with PPAR-γ siRNA or LXR-α siRNA (Figure 6). Specific targeting of PPAR-γ decreased the expression of LXR-α, ABCA1 and ABCG1 at protein levels (Figure 6A). Similarly, specific silencing of LXR-α attenuated the expression of PPAR-γ, ABCA1 and ABCG1 (Figure 6B).
Discussion
Alpinetin promoted ApoA-I- and HDL-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in ox-LDL-treated THP-1 macrophages and HMDMs. Targeting of PPAR-γ or LXR-α, reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Our results indicate that alpinetin enhances cholesterol efflux and decreases ox-LDL-mediated lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.
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10
PPAR-γ, a transcription factor, is abundantly expressed in the foam cells of atherosclerotic lesions (17, 18). PPAR-γ has been shown to coordinate a complex physiologic response to ox-LDL in macrophages, which involves cholesterol uptake, processing and removal through ABCA1 (19). Alpinetin is a novel plant flavonoid derived from Alpinia katsumadai Hayata (20). It has been shown that alpinetin inhibits lipopolysaccharide (LPS)-induced inflammatory mediator response through activation of PPAR-γ in THP-1 macrophages (21). We hypothesized that alpinetin might have effects on cholesterol efflux in human macrophages. To test this hypothesis, we analyzed the effects of alpinetin on ApoA-I-induced cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. Interestingly, we found that alpinetin dose-dependently elevated cholesterol efflux in both THP-1 macrophages. Moreover, similar results were observed in ox-LDL-treated HMDMs. Both results indicate that alpinetin has positive effects on cholesterol efflux.
We found that alpinetin raised the expression of PPAR-γ at both mRNA and protein levels. Specific knock-down of PPAR-γ significantly reversed alpinetin-mediated positive effects on cholesterol efflux, indicating that to some extent, PPAR-γ is necessary for alpinetin-induced increase of cholesterol efflux. However, PPAR-γ inhibition does not completely suppress alpinetin-mediate effects, suggesting that additional pathways are involved. We also found that alpinetin suppressed ox-LDL-induced lipid accumulation, which was reversed by specific targeting of PPAR-γ. Our results suggest that PPAR-γ is a critical factor for alpinetin-mediated biology effects on THP-1 cells. Actually, many Chinese traditional medicines regulate cholesterol efflux or lipid accumulation through PPAR-γ signaling This article is protected by copyright. All rights reserved.
11
pathways, such as quercetin (22), resveratrol (23) and arctigenin (24). On the other hand, alpinetin induces the activation of PPAR-γ in THP-1 macrophages to inhibit LPS-induced inflammation (21). It seems that increasing the expression and promoting the activation of PPAR-γ are new strategies for alpinetin to regulate various cell behaviors under different cell contexts.
The nuclear receptors LXR-α is a direct target gene of PPAR-γ/retinoid X receptor (RXR) heterodimer (25). Previous work has documented that macrophage lipid loading induces ligand activation of PPAR-γ, which leads to primary induction of LXR-α, coupled with the upregulation of LXR-α target genes ABCA1 (19, 26). Here, we found that alpinetin enhanced the expression of LXR-α at both mRNA and protein levels in THP-1 macrophages and HMDMs. Silencing of LXR-α substantially inhibited alpinetin-mediated increase in cholesterol efflux and recovered alpinetin-mediated inhibition of ox-LDL-induced lipid accumulation
in
THP-1 macrophages,
indicating
the importance
of
LXR-α
in
alpinetin-mediated effect on THP-1 cells. ABCA1, a member of the ATP binding cassette (ABC) family, is a key contributor to modulate the cholesterol efflux and lipid uptake (27, 28). In addition to ABCA1, ABCG1 plays an important role in maintaining cholesterol homeostasis (29). ABCA1 regulates ApoA-I-medaited cholesterol efflux, but ABCG1 regulates HDL-induced cholesterol efflux (30, 31). SR-B1 increases bidirectional cholesterol exchange between cells and lipoprotein including HDL (32). In the present study, we found that alpinetin elevated ABCA1 and ABCG1 mRNA and protein expression, but not SR-B1 in both THP-1 macrophages and HMDMs, indicating that alpinetin enhances cholesterol efflux This article is protected by copyright. All rights reserved.
12
and lipid accumulation through ABCA1 and ABCG1. Consistent with the previous studies (19, 25, 26), specific silencing of PPAR-γ decreased the expression of LXR-α, ABCA1 and ABCG1. Knockdown of LXR-α reduced the expression of PPAR-γ, ABCA1 and ABCG1. These results suggested that PPAR-γ and LXR-α, might be co-regulators, which modulate the expression of ABCA1 and ABCG1. How LXR-α silencing affects the expression of PPAR-γ is not clear yet. One possible mechanism might be through NF-κB signaling pathway. LXR-α agonist treatment reduces IκB-α degradation and NF-κB p65 activation in a mouse model of reperfusion
(SAO)
shock
(33).
On
the
other
hand,
the
activation
of
ERK
1/2-NF-κB-Nox4-dependent signaling pathway results in the reduction of PPAR-γ expression under hypoxic condition in human pulmonary artery smooth muscle cells (34). PPAR-γ expression is inhibited by the crosstalk between the canonical NF-κB and Notch signaling pathways, which promotes pancreatic cancer progression in mice (35). It seems that LXR-α can reduce the activation of NF-κB, which negatively regulates the expression of PPAR-γ. Alternatively, LXR-α silencing might induce the activation of NF-κB, which in turn attenuates the expression of PPAR-γ.
In sum, our observations reveal that alpinetin enhances cholesterol efflux and suppresses lipid accumulation through PPAR-γ/ LXR-α/ ABCA1/ABCG1 pathway in THP-1 macrophages and HMDMs.
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13
References
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Figure 1 Effects of alpinetin on cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. THP-1 macrophages or HMDMs were exposed to 50 μg/ml of ox-LDL for 24 h and then incubated with varying concentrations of alpinetin (50, 100, and 150 μg/ml) and 10 μg/ml of ApoA-I (A) or 50 μg/ml of HDL (B) for 24 h. Data represent mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 2 PPAR-γ is involved in alpinetin-promoted cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. (A) PPAR-γ mRNA expression in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (B) PPAR-γ protein expression in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (C) PPAR-γ protein expression in ox-LDL-loaded THP-1 macrophages transfected with PPAR-γ siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. (A-C) representative blots are from three independent experiments with similar results. THP-1 macrophages were exposed to 50 μg/ml of ox-LDL for 24 h and transfected with PPAR-γ siRNA or control siRNA. 24 h after transfection, cells were incubated with 150 μg/ml of alpinetin and 10 μg/ml of ApoA-I (D) or 50 μg/ml of HDL (E) for another 24 h. Data represent mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 3 LXR-α confers alpinetin-increased cholesterol efflux in ox-LDL-loaded THP-1 macrophages and HMDMs. (A) LXR-α mRNA levels in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (B) The expression of LXR-α protein in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (C) LXR-α protein expression in ox-LDL-loaded THP-1 macrophages transfected with LXR-α siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. (A-C) blots are from three independent experiments with similar results. THP-1 macrophages were exposed to 50 μg/ml of ox-LDL for 24 h and transfected with LXR-α siRNA or control siRNA. 24 h after transfection, cells were incubated with 150 μg/ml of alpinetin and 10 μg/ml of ApoA-I (D) or 50 μg/ml of HDL (E) for another 24 h. Data represent mean ± SD (n = 6). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 4 Alpinetin inhibits ox-LDL-induced lipid accumulation in THP-1 macrophages. Cells were stained with Oil Red O for the presence of neutral lipids in ox-LDL-stimulated THP-1 macrophages (A), or ox-LDL-stimulated THP-1 macrophages transfected with PPAR-γ siRNA (B) or LXR-α siRNA (C). The absorbance of cell monolayers was spectrophotometrically determined at 490 nm after Oil Red O staining. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus indicated controls.
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Figure 5 Alpinetin enhances ABCA1 and ABCG1 mRNA and protein expression in ox-LDL-loaded THP-1 macrophages and HMDMs. (A) ABCA1, ABCG1 and SR-B1 mRNA expression in THP-1 macrophages and HMDMs. β-actin was used as an internal control. (B) ABCA1, ABCG1 and SR-B1 protein levels in THP-1 macrophages and HMDMs. β-actin was used as an internal control. Blots represent three independent experiments with similar results`. Representative blots are from three independent experiments with similar results. Data represent mean ± SD (n = 3). **P < 0.01 versus indicated controls.
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Figure 6 PPAR-γ and LXR-α confer alpinetin-increased expression of ABCA1 and ABCG1 in THP-1 macrophages and HMDMs. (A) the protein levels of LXR-α, ABCA1 and ABCG1 in ox-LDL-loaded THP-1 macrophages transfected with PPAR-γ siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. (B) The protein levels of PPAR-γ, ABCA1 and ABCG1 in ox-LDL-loaded THP-1 macrophages transfected with LXR-α siRNA and treated with 150 μg/ml alpinetin. β-actin was used as an internal control. Data represent mean ± SD (n = 3). **P < 0.01 versus LXR-α (A) or PPAR-γ controls (B); ^^P < 0.01 versus ABCA1 controls (A) and (B); ##P < 0.01 versus ABCG1 controls (A) and (B).
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Table 1 RT-PCR primer sequences. Gene
Primer sequences
PPAR-γ
Forward: 5’-CACAAGAACAGATCCAGTGGTTGCAG-3’; Reverse: 5’-AATAATAAGGTGGAGATGCAGGCTCC-3’
LXR-α
Forward:5′-AAGCCCTGCATGCCTACGT-3′; Reverse:5′-TGCAGACGCAGTGCAAACA-3′
ABCA1
Forward: 5′-CCCTGTGGAATGTACCTATGTG-3′; Reverse:5′-GAGGTGTCCCAAAGATGCAA-3′
ABCG1
Forward: 5′-CGGAGCCCAAGTCGGTGTG-3′; Reverse:5′-TTTCAGATGTCCATTCAGCAGGTC-3′
SR-B1
Forward: 5’-ATGATCGTGATGGTGCCGTC-3’ Reverse: 5’-GGAAGGAGATCCCTATCCCC-3’
β-actin
Forward: 5’-GTGGGGCGCCCCAGGCACCA-3’; Reverse: 5’-CTCCTTAATGTCACGCACGATTTC-3’
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