Ethanolic extracts of Brazilian red propolis increase ABCA1 expression and promote cholesterol efflux from THP-1 macrophages

Phytomedicine 19 (2012) 383–388

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Ethanolic extracts of Brazilian red propolis increase ABCA1 expression and promote cholesterol efflux from THP-1 macrophages Akio Iio a,∗ , Kenji Ohguchi a,b , Hiroe Maruyama c , Shigemi Tazawa c , Yoko Araki c , Kenji Ichihara c , Yoshinori Nozawa a,d , Masafumi Ito a a

Gifu International Institute of Biotechnology, 1-1 Naka-Fudogaoka, Kakamigahara, Gifu 504-0838, Japan Department of Human Nutrition, Sugiyama Jogakuen University, 17-3 Hoshigaoka-Motomachi, Nagoya, Aichi 464-8662, Japan c Nagaragawa Research Center, Api Co. Ltd., 692-3 Yamasaki, Nagara, Gifu 502-0071, Japan d Department of Food and Health, Tokai Gakuin University, 5-68 Naka-Kirinocho, Kakamigahara, Gifu 504-8511, Japan b

a r t i c l e Keywords: Red propolis LXR␣ PPAR␥ ABCA1-expression Cholesterol efflux

i n f o

a b s t r a c t The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by regulating the cellular efflux of cholesterol. Since ABCA1 plays a pivotal role in cholesterol homeostasis and HDL metabolism, identification of a novel substance that is capable of increasing its expression would be beneficial for the prevention and therapy of atherosclerosis. In the present study, we studied the effects of ethanolic extracts of Brazilian red propolis (EERP) on ABCA1 expression and cholesterol efflux in THP-1 macrophages. EERP enhanced PPAR␥ and liver X receptor (LXR) transcriptional activity at 5–15 ␮g/ml, which was associated with upregulation of PPAR␥ and LXR␣ expression. It was also found that EERP increase the activity of the ABCA1 promoter, which is positively regulated by LXR. Consistent with these findings, treatment with EERP increased both mRNA and protein expression of ABCA1. Finally, EERP upregulated ApoA-I-mediated cholesterol efflux. Our results showed that EERP promote ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression via induction of PPAR␥/LXR. © 2011 Elsevier GmbH. All rights reserved.

Introduction Propolis, the resinous substance collected by honey bees from various plants, has been popularly used in folk medicine, and a variety of biological functions have been identified including antioxidant (Pascual et al. 1994), -microbial (Grange and Davey 1990), -inflammatory (Khayyal et al. 1993), -carcinogenic (Grunberger et al. 1988), and -cariogenic properties (Park et al. 1998). Red propolis, a novel type of propolis, was recently found in northeastern Brazil (Trusheva et al. 2006), and its botanical origin was described as Dalbergia ecastophyllum (L) Taub. (Leguminosae) (Daugsch et al. 2008). Fourteen compounds have been identified from red propolis, including simple phenolics, triterpenoids, isoflavonoids, prenylated benzophenones, and naphthoquinone epoxide (Trusheva et al. 2006). The chemical constituents of Brazilian red propolis (BRP) are similar to those of propolis from Cuba (CURP; CuestaRubio et al. 2007), but apparently different from those from China (CHRP; Izuta et al. 2009). Isoliquiritigenin, liquiritigenin, naringenin, isoflavones, isoflavans and pterocarpans were detected in BRP and CURP, but not in CHRP, whereas caffeic acid, caffeic acid phenethyl ester, chrysin, galangin and pinocembrin were detected

∗ Corresponding author. Tel.: +81 58 371 4646; fax: +81 58 371 4412. E-mail address: [email protected] (A. Iio). 0944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2011.10.007

only in CHRP (Izuta et al. 2009; Iio et al. 2010; Piccinelli et al. 2011). Polyisoprenylated benzophenones were detected only in BRP. Atherosclerosis is a chronic inflammatory disease characterized by the early and prolonged presence of macrophages within the innermost layer of the artery wall (Lusis 2000; Glass and Witztum 2001). Circulating monocytes that trapped low-density lipoproteins (LDL) are recruited to the vascular intima. After entering the intima, monocytes differentiate into macrophages, ingest oxidized LDL, and subsequently transform into lipid-laden foam cells leading to intimal fatty-streak lesions (Berliner and Heinecke 1996). Accumulation of the lipid-laden foam cells mainly caused by uncontrolled uptake of modified LDL or impaired cholesterol efflux is a key feature of the initiation and development of atherosclerosis. In a later stage, the continued foam cell accumulation and the subsequent apoptosis of plaque cells lead to the formation of a necrotic core, which is more prone to rupture. Finally, these lesions form fatal thrombosis resulting in acute coronary syndromes (Viles-Gonzalez et al. 2004). Therefore, reduction of intracellular cholesterol accumulation in arterial macrophages may be a promising strategy to prevent and treat atherosclerosis. It has been shown that high density lipoprotein (HDL) cholesterol and its most abundant protein constituent, apolipoprotein A-I (ApoA-I), can promote the efflux of cholesterol from foam cells of the artery wall and mediate the transport of cholesterol to the liver for catabolism (Fielding and Fielding 1995; Panagotopulos

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et al. 2002) and further decrease atherosclerotic lesion progression (Choudhury et al. 2004). Schmitz et al. (1999) showed that ABCA1, one of the ATP-binding cassette (ABC) transporters present in macrophages, mediates the efflux of intracellular cholesterol forming HDL. It was also shown that ABCA1 is a definite liver X receptor (LXR) target gene both in vitro and in vivo (Costet et al. 2000; Venkateswaran et al. 2000; Joseph et al. 2002). Although the synthetic LXR agonist, GW3965, increases the plasma HDL level and inhibits the development of atherosclerosis (Joseph et al. 2002), relatively few compounds have been reported that can upregulate ABCA1 expression. Thus, identification of a novel substance capable of increasing ABCA1 expression may lead to a therapeutic benefit for patients with cardiovascular diseases. We have recently reported that the ethanolic extracts of Brazilian red propolis (EERP) promote adipocyte differentiation, and exhibit anti-diabetic activity by upregulating PPAR␥ and adiponectin expression and inhibiting the TNF-␣ activity (Iio et al. 2010). It has been also shown that propolis reduces the serum level of LDL–cholesterol and increases that of HDL–cholesterol in fasting rats (Fuliang et al. 2005). It is therefore possible that EERP have multiple roles in cholesterol metabolism. In the present study, we investigated the effect of EERP on ABCA1 expression and cholesterol efflux, and characterized the underlying molecular mechanisms. Materials and methods Reagents Bovine serum albumin (BSA) and skim milk were purchased from Nacalai Tesque (Kyoto, Japan). RPMI1640 medium, Cell counting kit-8, and Pikkagene Dual-SeaPancy luminescence kit were purchased from Wako (Tokyo, Japan). Charcoal-stripped fetal bovine serum (FBS) was obtained from Biological Industries (Kibbutz Beit-Ha’Emek, Israel). PrimeScript RT reagent kit, SYBR Premix Ex Taq II, and RNase-free DNase I were from Takara (Otsu, Japan). Apolipoprotein A-I, phorbol 12-myristate 13-acetate (TPA) were purchased from Sigma (St Louis, USA). The firefly luciferase reporter vector pGL4.27 was obtained from Promega (Madison, USA). AntiABCA1 antibody was from Novus Biologicals (Littleton, USA). DC protein assay kit was obtained from Bio-Rad (Hercules, USA). Horseradish peroxidase-conjugated donkey anti-rabbit/mouse IgG and ECL Plus Western blotting detection reagents were from GE (Fairfield, USA). PolyScreen PVDF membrane was from PerkinElmer (Winter Street Waltham, USA). Oligonucleotide primers were from Rikaken (Nagoya, Japan). 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl) amino)-23,24-bisnor-5-cholen-3s-ol (NBD cholesterol) and TRIzol were purchased from Invitrogen (Carlsbad, USA). T0901317 (T09), Formononetin, and Rosiglitazone maleate (Rosi) were purchased from Alexis (Lausen, Switzerland). FuGENE6 and fatty acid-free BSA were obtained from Roche (Penzberg, Germany). ProteoJET membrane protein extraction kit was purchased from Fermentas (Vilnius, Lithuania). Anti-Flotillin2 antibody was from BD Biosciences (Franklin Lakes, USA). The PPREx3-tk reporter gene and PPAR␥ expression vector (pCMX-hPPAR␥1; Hotta et al. 2010) were kind gifts from Dr. Inoue (Nara Women’s University, Nara, Japan). Ethanol extracts of Brazilian red propolis (EERP) were prepared at Api Co. Ltd. according to the methods previously described (Iio et al. 2010). Cell culture, treatment, and cytotoxic assay THP-1 cells were obtained from ATCC (Manassas, USA) and grown in RPMI1640 supplemented with 10% (v/v) heat-inactivated FBS at 37 ◦ C in an atmosphere containing 5% CO2 . To induce macrophage differentiation, THP-1 cells were stimulated for 4 days

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Fig. 1. Effects of EERP on cell viability of THP-1 macrophages. THP-1 macrophages were treated with various concentrations of EERP (0–40 ␮g/ml) for 24 h, and then cell viability was determined.

with TPA (80 nM). Cell viability was determined calorimetrically using the Cell counting kit-8 according to the manufacturer’s protocol.

Measurement of LXR and PPAR transcriptional activity, and ABCA1 promoter activity The LXR reporter gene, 3xLXRE-luc, was constructed by inserting 3 copies of the LXRE responsive element (LXRE; DR4) derived from the human ABCA1 promoter into the BglII site of the enhancerless minimal artificial promoter/luciferase expression vector, pGL4.27, using the following primer set; 5 -GATCGGCTTTGACCGATAGTAACCTCTGCGCTCG-3 and 5 GATCCGAGCGCAGAGGTTACTATCGGTCAAAGCC-3 . To measure the LXR transcriptional activity and ABCA1 promoter activity, the 3xLXRE-luc and pGL3-ABCA1 promoter reporter vector (WT1; Iwamoto et al. 2007) were co-transfected with Renilla luciferase expression vector (TK-RL), respectively. To measure the PPAR␥ transcriptional activity of EERP, the PPREx3-tk reporter gene and PPAR␥ expression vector (pCMX-hPPAR␥ 1) were co-transfected with TK-RL as previously described (Iio et al. 2010). Transfection into post-differentiated THP-1 macrophages was carried out using FuGENE6. EERP or formononetin were added to the culture medium 24 h after transfection. Four hours or twenty-four hours later, cells were lysed, and luciferase activities were measured using the Pikkagene Dual-SeaPancy luminescence kit according to the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.

Real-time RT-PCR Total RNA extracted from cells using TRIzol was treated with RNase-free DNase I, and then reverse transcribed using PrimeScript RT reagent kit. Quantitative real-time PCR was performed using SYBR Premix Ex Taq II on a real-time thermal cycler Dice (TP800, Takara). GAPDH was used as an internal control. Primer sets were as follows: GAPDH, 5 CCACATCGCTCAGACACCAT-3 and 5 -GCAACAATATCCACTTTACCAGAGTTAA-3 ; ABCA1, 5 -GCCTGCTAGTGGTCATCCTG-3 and 5 CCACGCTGGGATCACTGTA-3 ; LXR␣, 5 -CAGGGCTCCAGAAAGAGATG-3 and 5 -ACAGCTCCACCGCAGAGT-3 ; PPAR␥, 5 GACAGGAAAGACAACAGACAAATC-3 and 5 -GGGGTGATGTGTTTGAACTTG-3 . Crossing points for each transcript were determined using the second derivative maximum method, and quantification was performed using the comparative Ct (Ct) method according to the manufacturer’s protocol.

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Fig. 2. Effects of EERP on LXR transcriptional activity and ABCA1 promoter activity. THP-1 macrophages were transfected with 3xLXRE-luc (450 ng) and TK-RL (50 ng) vectors (A), or the pGL3-ABCA1 promoter reporter (450 ng) and TK-RL (50 ng) vectors (B). Twenty-four hours later, cells were treated with T09 (1 ␮M), LXRs agonist for positive control, or EERP (0–15 ␮g/ml) for additional four hours (A), or 24 h (B). LXR transcriptional activity and ABCA1 promoter activity were measured by dual-luciferase assay. Firefly luciferase activity was normalized to Renilla luciferase activity (*p < 0.05, **p < 0.01).

Western blot analysis For preparation of the cell membrane fraction, THP-1 macrophages were washed twice with PBS and resuspended in cell permiabilization buffer containing protease inhibitor cocktail and extracted with membrane protein extraction buffer according to the manufacturer’s protocol. Protein content was measured with a DC protein assay kit. Ten micrograms of the cell membrane fraction were separated by SDS–PAGE on 5–12% polyacrylamide gel and then electroblotted onto a PVDF membrane. After blocking for 2 h by 5% skim milk in TBST (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20), the membrane was incubated for 2 h with anti-ABCA1 or anti-Flotillin2 antibody. After washing four times with TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibody, and then washed four times with TBST. Protein bands were detected with the ECL kit and chemiluminescence detector LAS-4000 (Fujifilm, Tokyo, Japan).

Fig. 3. Effects of Formononetin on LXR transcriptional activity and ABCA1 promoter activity. THP-1 macrophages were transfected with 3xLXRE-luc (450 ng) and TKRL (50 ng) vectors (A), or the pGL3-ABCA1 promoter reporter (450 ng) and TK-RL (50 ng) vectors (B). Twenty-four hours later, cells were treated with T09 (1 ␮M) or formononetin (0–10 ␮M) for additional four hours (A), or 24 h (B). LXR transcriptional activity and ABCA1 promoter activity were measured by dual-luciferase assay. Firefly luciferase activity was normalized to Renilla luciferase activity (***p < 0.001).

Statistical analysis Results were expressed as mean ± S.D. of three independent experiments. Data were analyzed using Student’s t-test. A value of p < 0.05 was considered significant. Results and discussion Viability of THP-1 macrophages To assess the effects of EERP on viability of THP-1 macrophages, we performed the WST-8 assay, which measures the mitochondrial activity of cells. When THP-1 macrophages were treated with various concentrations of EERP (0–40 ␮g/ml), cell viability was not decreased (Fig. 1). Thus, EERP do not cause significant cytotoxicity at concentrations of ≤40 ␮g/ml. LXR transcriptional activity and ABCA1 promoter activity in THP-1 macrophages

Cholesterol efflux assay macrophages were equilibrated with Differentiated NBD–cholesterol (1 ␮g/ml) for 12 h (Lee et al. 2010). NBD–cholesterol-labeled cells were washed with PBS and incubated in RPMI1640 medium containing 0.2% (w/v) fatty acid-free BSA and 10 ␮g/ml ApoA-1 for 6 h. The fluorescence-labeled cholesterol released from cells into the medium was measured with MT-600F fluorescence microplate reader (Corona Electric, Hitachinaka, Japan). Cholesterol efflux was expressed as a percentage of fluorescence in the medium relative to the total amounts of fluorescence detected in cells and the medium.

LXRs play an important role in cholesterol homeostasis by serving as regulatory sensors of cholesterol levels in cells. We performed a luciferase reporter gene assay to test whether EERP could enhance transcriptional activity of LXRs. As shown in Fig. 2A, EERP dose-dependently (5–15 ␮g/ml) increased expression of the LXR luciferase reporter gene 4 h after treatment. In addition, EERP enhanced the ABCA1 promoter activity 24 h after treatment (Fig. 2B). Since it has been shown that LXR binds to the LXRE in the ABCA1 promoter, thereby activating its transcription (Costet et al. 2000), these results suggested that EERP may up-regulate ABCA1 transcription through enhancing LXR transcriptional activity.

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Fig. 5. Effects of EERP on ABCA1 mRNA and protein expression in THP-1 macrophages. (A) THP-1 macrophages were cultured in the presence of EERP (0–15 ␮g/ml) for 24 h, and then quantitative real-time RT-PCR was performed for ABCA1 (*p < 0.05, ***p < 0.001). (B) THP-1 macrophages were cultured in the presence of EERP (0–15 ␮g/ml) for 48 h. Then, the membrane fraction was harvested and subjected to Western blotting analysis to detect ABCA1 and integral membrane protein, Flotillin2.

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Fig. 4. Effects of EERP on PPAR␥ transcriptional activity and expression of PPAR␥ and LXR␣. (A) THP-1 macrophages were transfected with PPREx3-tk (450 ng) and TK-RL (50 ng) vectors along with the PPAR␥ expression vector (pCMX-hPPAR␥1). Twentyfour hours later, cells were treated with Rosiglitazone (1 ␮M), PPAR␥ agonist for positive control, or EERP (0–15 ␮g/ml) for additional 24 h. The PPAR␥ transcriptional activity was measured by dual-luciferase assay. Firefly luciferase activity was normalized to Renilla luciferase activity (*p < 0.05, ***p < 0.001). (B, C) THP-1 macrophages were cultured in the presence or absence of EERP (0–15 ␮g/ml) for 24 h, and then quantitative real-time RT-PCR was performed for PPAR␥ (B) and LXR␣ (C). Expression of each gene was normalized to GAPDH (*p < 0.05).

Formononetin, one of isoflavones, is a major component of Brazilian red propolis (Daugsch et al. 2008). We therefore performed a luciferase reporter gene assay to test whether formononetin could enhance LXR transcriptional and ABCA1 promoter activities. As shown in Fig. 3, formononetin enhanced LXR transcriptional and ABCA1 promoter activities. These results suggest that formononetin may represent one of the major ABCA1upregulating activities in Brazilian red propolis. PPAR transcriptional activity and PPAR/LXR˛ expression in THP-1 macrophages We have previously shown that EERP promote adipocyte differentiation through PPAR␥ activation (Iio et al. 2010). Many studies have indicated that PPAR␥ regulates expression of ABCA1

through induction of LXRs (Chinetti et al. 2001; Chawla et al. 2001; Nakaya et al. 2007). Moreover, PPAR␥ functions by forming obligate heterodimers with LXRs (Yue et al. 2005). These previous findings suggest that PPAR␥ and LXR␣ might upregulate ABCA1 transcription in a cooperative manner. In order to examine the effects of EERP on PPAR␥ transcriptional activity in THP-1 macrophages, we performed a luciferase reporter gene assay using PPREx3-tk and PPAR␥ expression vectors. As shown in Fig. 4A, EERP dose-dependently increased PPAR␥ transcriptional activity in macrophages as observed in adipocytes (Iio et al. 2010). In addition, mRNA levels of both PPAR␥ and LXR␣ were upregulated by EERP treatment (Fig. 4B and C). Taken all data together, it was suggested that EERP increase expression of both PPAR␥ and LXR␣, and thereby upregulate ABCA1 transcription through acting on its promoter presumably in a cooperative manner.

ABCA1 mRNA and protein expression in THP-1 macrophages To investigate whether EERP could increase mRNA and protein levels of ABCA1, we performed quantitative real-time RT-PCR and Western blot analysis. Consistent with the effect on ABCA1 promoter activity, EERP increased both mRNA (Fig. 5A) and protein (Fig. 5B) expression of ABCA1 in a dose-dependent manner.

Cholesterol efflux in THP-1 macrophages Because ABCA1 expression was upregulated by EERP, we next determined the effect of EERP on the cholesterol efflux from THP-1 macrophages by measuring fluorescent-labeled cholesterol content (Fig. 6). The cholesterol efflux to ApoA-I was found to be significantly increased in a dose-dependent manner in response to EERP treatment.

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Relative cholesterol efflux rate

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Appendix A.

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ATP: adenosine triphosphate, PPAR␥: peroxisome proliferator-activated receptor ␥, TNF-␣: tumor necrosis factor-␣, PVDF: polyvinylidene difluoride, RT-PCR; reverse transcription-polymerase chain reaction, GAPDH; glyceraldehyde3-phosphate dehydrogenase, SDS–PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis, PBS: phosphatebuffered saline.

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Fig. 6. Effects of EERP on cholesterol efflux in THP-1 macrophages. THP-1 macrophages were treated with EERP (0–15 ␮g/ml) and NBD–cholesterol (1 ␮g/ml) to detect cholesterol efflux as described in Materials and methods (*p < 0.05, **p < 0.01).

EERP induction

induction

PPAR

LXR transactivation transactivation

ABCA1 Enhanced cholesterol efflux

HDL Fig. 7. A scheme showing the molecular mechanisms underlying upregulation of ABCA1-dependent cholesterol efflux in macrophages. EERP induce PPAR␥ and LXR expression. PPAR␥ may induce LXR expression, Thus, EERP activate ABCA1 transcription directly through LXR or indirectly through PPAR␥ induction and LXR activation, thereby enhancing ApoA-I-mediated cholesterol efflux in macrophages.

Conclusion In the present study, we demonstrated that EERP significantly enhanced ApoA-I-mediated cholesterol efflux in THP-1 macrophages, which was accompanied by a marked induction of ABCA1 gene that is critical for cholesterol metabolism. The effect of EERP on ABCA1-dependent cholesterol efflux was explained in part by its potency of induction of PPAR␥ and LXR␣ expression (Fig. 7). These results suggest that EERP have a potential as a diet supplement for prevention and treatment of cardiovascular disease such as atherosclerosis. Acknowledgements This work was supported in part by Grant for Biological Research from Gifu prefecture, Japan, and Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Dr. Inoue at Nara Women’s University, Nara, Japan, for providing us with PPREx3-tk reporter gene and PPAR␥ expression vector.

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