- Email: [email protected]
Diosgenin inhibits atherosclerosis via suppressing the MiR-19b-induced downregulation of ATP-binding cassette transporter A1
Accepted Manuscript Diosgenin Inhibits Atherosclerosis via Suppressing the MiR-19b-induced Downregulation of ATP-binding Cassette Transporter A1 Yun-cheng Lv, Jing Yang, Feng Yao, Wei Xie, Yan-yan Tang, Xin-ping Ouyang, Pingping He, Yu-lin Tan, Liang Li, Min Zhang, Dan Liu, Francisco S. Cayabyab, Xi-Long Zheng, Chao-ke Tang PII:
S0021-9150(15)00135-5
DOI:
10.1016/j.atherosclerosis.2015.02.044
Reference:
ATH 13965
To appear in:
Atherosclerosis
Received Date: 21 October 2014 Revised Date:
2 February 2015
Accepted Date: 23 February 2015
Please cite this article as: Lv Y-c, Yang J, Yao F, Xie W, Tang Y-y, Ouyang X-p, He P-p, Tan Yl, Li L, Zhang M, Liu D, Cayabyab FS, Zheng X-L, Tang C-k, Diosgenin Inhibits Atherosclerosis via Suppressing the MiR-19b-induced Downregulation of ATP-binding Cassette Transporter A1, Atherosclerosis (2015), doi: 10.1016/j.atherosclerosis.2015.02.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Diosgenin Inhibits Atherosclerosis via Suppressing the MiR-19b-induced Downregulation of ATP-binding Cassette Transporter A1
Running title: Diosgenin inhibits atherogenesis.
RI PT
Yun-cheng Lva, b, 1, Jing Yangc, 1, Feng Yaoa, d, 1, Wei Xiea, b, Yan-yan Tanga, Xin-ping Ouyanga, Ping-ping Hea, Yu-lin Tana, Liang Lia, Min Zhanga, Dan Liua, Francisco S. Cayabyabe, Xi-Long Zhengf, Chao-ke Tanga*
a
1
TE D
M AN U
SC
Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, Life Science Research Center, Hunan Province Cooperative innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, Hunan 421001, China. b Laboratory of Clinical Anatomy, University of South China, Hengyang, 421001, China. c Department of Metabolism & Endocrinology of the First Affiliated Hospital, University of South China, Hengyang, 421001, China. d Department of Laboratory Animal Science, University of South China, Hengyang 421001, China. e Department of Surgery, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. f Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 4N1.
These authors contributed equally to this work.
EP
To whom correspondence should be addressed: *Chao-ke Tang, PhD, Professor, Institute of Cardiovascular Research, University of South China,
AC C
Hengyang, Hunan 421001, China Email: [email protected] Tel: 86-734-8281853
1
ACCEPTED MANUSCRIPT Rationale: Diosgenin (Dgn), a structural analogue of cholesterol, has been reported to have the hypolipidemic and antiatherogenic properties, but the underlying mechanisms are not fully understood. Given the key roles of macrophages in cholesterol metabolism and atherogenesis, it is critical to investigate macrophage
RI PT
cholesterol efflux and development of atherosclerotic lesion after Dgn treatment. Objective: This study was designed to evaluate the potential effects of Dgn on macrophage cholesterol metabolism and the development of aortic atherosclerosis, and to explore its underlying mechanisms.
SC
Methods and Results: Dgn significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) protein, but didn’t affect liver X receptor α levels in
M AN U
foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by western blotting. The miR-19b levels were markedly down-regulated in Dgn-treated THP-1 macrophages/MPM-derived foam cells. Cholesterol transport assays revealed that treatment with Dgn alone or together with miR-19b inhibitor notably enhanced ABCA1-dependent cholesterol efflux,
TE D
resulting in the reduced levels of total cholesterol, free cholesterol and cholesterol ester as determined by high-performance liquid chromatography. The fecal 3H-sterol originating from cholesterol-laden MPMs was increased in apolipoprotein E knockout
EP
mice treated with Dgn or both Dgn and antagomiR-19b. Treatment with Dgn alone or together with antagomiR-19b elevated plasma high-density lipoprotein levels, but reduced plasma low-density lipoprotein levels. Accordingly, aortic lipid deposition
AC C
and plaque area were reduced, and collagen content and ABCA1 expression were increased in mice treated with Dgn alone or together with antagomiR-19b. However, miR-19b overexpression abrogated the lipid-lowering and atheroprotective effects induced by Dgn.
Conclusion: The present study demonstrates that Dgn enhances ABCA1-dependent cholesterol efflux and inhibits aortic atherosclerosis progression by suppressing macrophage miR-19b expression. Keywords: Diosgenin; ABCA1; macrophage foam cells; cholesterol efflux; reverse cholesterol transport; atherosclerosis. 2
ACCEPTED MANUSCRIPT 1. Introduction Diosgenin (Dgn) is a naturally occurring steroidal sapogenin present in a variety of plants including fenugreek, yam root and soy bean. It has long been used in Ayurvedic and Chinese medicine [1, 2]. Cumulative evidence demonstrates its
RI PT
cardiovascular protective activities and its role in appropriate management of dyslipidemia [3]. Dgn remarkably decreases the level of serum triglyceride (TG) and total cholesterol (TC) in mice, rats, chickens and rabbits fed with a high-cholesterol diet [4-10]. Dgn markedly decreases the extent of fatty liver as well as aortic fatty
SC
streaks in high-cholesterol-fed rabbits [10]. These studies suggest that the lipid-lowering activity and the antiatherogenic property of Dgn are intimately relevant
M AN U
with its suppression of intestinal cholesterol absorption and promotion of hepatic cholesterol secretion [2, 11-13]. It is known that peripherial macrophages play critical roles in cholesterol metabolism and atherosclerosis [14, 15]. However, it is not clear whether and how Dgn regulates macrophage cholesterol metabolism. Cholesterol homeostasis in macrophages is of prime importance in the initiation
TE D
of atherosclerosis, because dysregulation of cholesterol metabolism leads to excessive accumulation of cholesterol in the macrophages and subsequent transformation into foam cells [16]. Our group and others have demonstrated that ATP-binding cassette
EP
transporter A1 (ABCA1)-dependent cholesterol efflux is a crucial event in the prevention of excessive cholesterol accumulation in macrophages and atherosclerosis progression [17-27]. ABCA1 facilitates the efflux of free cholesterol to lipid-poor
AC C
apolipoprotein A-I (apoAI) and contributes to about one third of cholesterol efflux in cholesterol-laden macrophages [28]. The activity of the ABCA1 pathway is elaborately modulated in peripheral macrophages, and an increase in ABCA1 activity in arterial macrophages will prevent foam cell formation in atherosclerotic lesions [29, 30]. Our previous work has verified that macrophage ABCA1 expression is potently inhibited by miR-19b, resulting in an impairment of cholesterol efflux and the formation of macrophage foam cells [31]. In the current study, we first investigated the effects of Dgn on miR-19b level, ABCA1 expression and cholesterol metabolism 3
ACCEPTED MANUSCRIPT in cholesterol-laden macrophages, and then examined the hypolipidemic effects of Dgn on serum lipid profile and reverse cholesterol transport (RCT) in high cholesterol and high fat-fed apolipoprotein E knockout (apoE KO) mice. Finally, we determined the antiatherogenic efficacy of Dgn on aortic lipid deposition and atherosclerotic
RI PT
lesion in apoE KO mice. Our results have provided experimental evidence to support the roles of miR-19b/ABCA1 pathway in Dgn inhibition of macrophage cholesterol accumulation and aortic atherogenesis.
SC
2. Materials and methods 2.1 Cell culture
M AN U
THP-1 cell strain was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells were cultured in RPMI 1640 (Solarbio, China) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco BRL, America) at 37 °C and 5% CO2. Differentiation of THP-1 cells into macrophages was induced using 160 nM phorbol-12-myristate acetate (PMA, Sigma, America) for 24 h. Mouse
TE D
peritoneal macrophages (MPMs) from adult C57BL/6J mice (Jackson Laboratories) were obtained by peritoneal lavage 4 d after the intraperitoneal injection of 2.0 ml thioglycollate broth (4%, w/v). The thioglycollate-elicited MPMs were maintained in
EP
RPMI 1640 medium containing 10% FBS. Finally, macrophages were incubated in RPMI 1640 containing 0.2% (w/v) bovine serum albumin (BSA, Sigma, America) and 50 µg/ml acetylated-low density lipoprotein (acLDL) for 48 h in cell flasks or 12-well
AC C
plates, for full differentiation of THP-1 macrophages/MPMs to foam cells in all experiments analyzing ABCA1 expression and cholesterol metabolism (unless otherwise indicated).
2.2 Dgn treatment and miR-19b/anti-miR-19b transfection Dgn (93% in purity, Sigma, America) was dissolved in ethanol to make a stock solution (100 mM) according to the manufacturer’s instructions. THP-1 macrophage/MPM-derived foam cells were transfected with miR-19b mimic or inhibitor (anti-miR-19b: the suppression of miR-19b) (Ribobio, China) at the 4
ACCEPTED MANUSCRIPT concentrations as indicated in the corresponding figure legends for 24 h using RiboFECTTMCP reagent (Ribobio, China) according to the manufacturer’s protocol. A total volume of 1 ml RPMI 1640 containing 0.2% (w/v) BSA in each well contained 100 µl transfection buffer, 10 µl RiboFECTTMCP reagent and different
RI PT
volumes of miR-19b mimic or inhibitor depending on the concentrations used in the experiments.
2.3 Western blot analysis
SC
Whole cell lysate and tissue proteins were extracted with RIPA buffer (Beyotime, China). The proteins (20 µg per lane) were then loaded on 8% SDS-polyacrylamide
M AN U
electrophoresis gel, electrophoresed for 2 h at 100 V in gel running buffer, and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% (w/v) fat-free dry milk, the membranes were incubated with rabbit antibodies against ABCA1 (Abcam, USA), liver X receptor α (LXRα, Abcam, America), retinoic acid receptor α (RXRα, Sigma, America) or β-actin (Boster, China) overnight at 4 °C, and
TE D
then incubated with horseradish peroxidase-conjugated secondary antibody (Boster, China). Immunoreactive protein bands were detected using the enhanced chemiluminesence immunoblotting detection system (Amersham Biosciences,
EP
America). The relative signal intensities were determined by densitometry using the ImageJ software (national institutes of health, NIH, America). The relative levels of ABCA1, LXRα and RXRα were expressed as the ratio of ABCA1, LXRα or RXRα to
AC C
β-actin densitometric values.
2.4 Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis
Total RNA was obtained from THP-1 macrophage/MPM-derived foam cells using the TRIzol RNA isolation kit (Invitrogen, America). cDNA was generated from total RNA using the Ncode Vilo miRNA cDNA synthesis kit (Invitrogen, America) according to the manufacturer’s instructions. The Ncode SYBR Green miRNA RT-qPCR kit (Invitrogen, America) was used to detect the miRNA expression. The 5
ACCEPTED MANUSCRIPT expression of miR-19b in THP-1 macrophage/MPM-derived foam cells was calculated relative to U6B, a ubiquitously expressed small nuclear RNA. RT-qPCR analyses were performed using the forward primers for miR-19b and U6B as following: hsa-miR-19b, 5'-TGTGCAAATCCATGCAAAACTGA-3'; U6B, 5'-
RI PT
ACGCAAATTCGTGAAGCGTTCCAT-3'. The reverse primer was the Ncode miRNA Universal qPCR Primer (Invitrogen, America). A comparative threshold cycle method was used to compare each condition with the control.
SC
2.5 Cholesterol efflux assay
THP-1 cells and MPMs were seeded in 12-well plates (1×106 cells/well),
M AN U
radiolabeled with 5 µCi/ml 3H-cholesterol (PerkinElmer, America) and loaded with cholesterol (acLDL, 50 µg/ml) for 48 h. Cells were subsequently treated with Dgn, alone or together with miR-19b mimic or inhibitor for an additional 24 h. Then, cells were washed twice with phosphate-buffered saline (PBS, T4174, Sigma, America)) and incubated with RPMI 1640 containing 0.2% (w/v) BSA and 10 µg/ml human
TE D
apoAI. After 6 h, 3H-cholesterol in the medium and cells was measured separately by liquid scintillation counting. The percent efflux was calculated by the following equation: [total media counts/(total cellular counts
EP
31].
total media counts] ×100% [21,
2.6 High-performance liquid chromatography (HPLC) assay
AC C
HPLC assay was conducted as described previously [20, 32]. THP-1
macrophage/ MPM-derived foam cells were treated with Dgn alone or together with miR-19b mimic or inhibitor for 24 h, then incubated with RPMI 1640 containing 0.2% (w/v) BSA and 10 µg/ml human apoAI. After 6 h, cells were detached using 1% EDTA in PBS and collected in a glass tube. The sterol analyses were performed using an HPLC system (model 2790, controlled with Empower Pro software; Waters Corp., Milford, MA, America). Sterols were detected using a photodiode array detector equipped with a 4-lL cell (model 996, Waters Corp, America). Analysis of cholesterol and cholesterol esters was performed after elution with acetonitrile-isopropanol 30:70 6
ACCEPTED MANUSCRIPT (v/v) and detected by absorbance at 210 nm.
2.7 Mice and treatments Six-week old male apoE KO mice were obtained from Laboratory Animal Center
RI PT
of Peking University, China. All mice were maintained on a 12 h light/dark cycle with unlimited access to food and water. After fed with a rodent chow diet containing 4.5% (w/w) fat for a week, apoE KO mice were randomly divided into four groups (15 mice per group): control group; Dgn group; Dgn+agomiR-19b (Dgn+Ago) group; Dgn+
SC
antagomiR-19b (Dgn+Ant) group. All groups were switched to and maintained on Western diet (WD) containing 21% (w/w) fat and 1.25% (w/w) cholesterol, and with
M AN U
or without 1% Dgn (w/w) for 8 wk. The apoE KO mice from Dgn+Ago group and Dgn+Ant group received tail vein injections of 10 nmol/per mouse agomiR-19b or 80 mg/kg antagomiR-19b, respectively, on days 1 to 3 at every other week after starting WD [33]. AgomiR and antagomiR are nucleic acid drugs and optimized version of miRNA mimic or inhibitor, which are specially modified for greater stability and
TE D
binding affinity to their targets than that of miRNA mimic or inhibitor. After fasting for 6 h, mice were euthanized, followed by blood-sampling from the retro-orbital plexus, aortas and hearts for further analyses. All animal experiments were done in
EP
accordance with the Institutional Animal Ethics Committee and the University of
AC C
South China Animal Care guidelines for use of experimental animals.
2.8 In vivo macrophage RCT assay MPMs were radiolabeled with 3H-cholesterol (5 µCi/ml) and loaded with
cholesterol (acLDL, 50 µg/ml) for 48 h. These foam cells were then washed twice, equilibrated in medium with 0.2% (w/v) BSA for 6 h, spun down, and resuspended in 0.5 ml medium. The radio-labeled macrophages were intraperitoneally injected into apoE KO mice (3.6×106 cells at 3.8×106 cpm per mouse) treated with Dgn in the absence or presence of agomiR-19b or antagomiR-19b. Feces were collected continuously from 0 to 48 h and were stored at -20°C until lipid extraction. Blood was collected at 48 h, and plasma was used for liquid scintillation counting and lipid 7
ACCEPTED MANUSCRIPT analyses. After 48 h, mice were anesthetized and euthanized. Their livers were then harvested and stored at -20°C until lipid extraction [34].
2.9 Biliary cholesterol assay
RI PT
After a 6 h fast, mice were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight), followed by laparotomy, distal ligation of the bile duct and gallbladder cannulation. The bile was collected for 10 min, and immediately frozen at -20 °C after homogenization [12]. For the analysis of biliary
SC
cholesterol concentrations, a measured volume (10 µl) of bile was extracted by addition of 3:1 ethanol: ether. A measured aliquot of the organic phase was dried
M AN U
down and then analyzed for cholesterol content by solubilizing the lipids in Triton X-100/water using the Total Cholesterol enzymatic assay kits (Dong’ou, China).
2.10 Plasma lipid analysis
Blood was collected into tubes containing 2 mM EDTA from mice after a 6 h fast,
TE D
and centrifuged for 15 min at 4°C. Plasma samples were separated, aliquoted, and stored frozen prior to analyses. TG, TC and HDL-C were determined by enzymatic methods using commercially available test kits (Rongsheng, China) according to the
EP
manufacturer’s instructions.
2.11 Evaluation of aortic lesions
AC C
Atherosclerotic lesions were quantified by en face analysis of the whole aorta
and by cross-sectional analysis of the proximal aorta. For the en face analysis of the aorta, the Sudan IV-stained aortas were photographed (Nikon Coolpix 990, Japan) for the quantification of atherosclerotic lesions. The total aortic surface area and the lesion area were measured by image analysis (ImageJ software, NIH, America), and the ratio of the lesion area to the total area was calculated. For the cross-sectional analysis of the aorta, the OCT-embedded aortas were sectioned using a cryostat, and 8-µm sections were obtained sequentially beginning at the aortic valve. Twenty sections per mouse obtained every 24 µm from the aortic sinus were stained with 8
ACCEPTED MANUSCRIPT hematoxylin-eosin, Oil red O (Sigma, America) and Masson’s trichrome (MT) (Senbeibio, China), which were used for the quantification of lesion areas and collagen contents. Lesion areas and collagen contents were quantified using NIH
RI PT
ImageJ software. Data were expressed as lesion size ± SEM [17, 31, 35].
2.12 Statistical analysis
Data were expressed as means ± standard deviations (S.D.) of at least three independent experiments. With SPSS 18.0 software, data were analyzed by one way
SC
ANOVA followed by Student-Newman-Keuls test. Values with P < 0.05 were
AC C
EP
TE D
M AN U
accepted as statistical significance comparing with the control groups.
9
ACCEPTED MANUSCRIPT 3. Results 3.1 Dgn increases macrophage ABCA1 expression We first investigated whether Dgn influences the expression of ABCA1 in THP-1 macrophage/MPM-derived foam cells. To do so, THP-1 macrophage/MPM-derived
RI PT
foam cells were treated with Dgn at different concentrations (0, 10, 20, 40, 80 µM) for 24 h or with 40 µM Dgn for different time periods (0, 6, 12, 24, 48 h), followed by western blot analysis. As shown in Fig. 1A–1D, Dgn enhanced the expression of ABCA1 protein in THP-1 macrophage/MPM-derived foam cells in both
SC
concentration- and time-dependent manners. Furthermore, ABCA1 expression was maximally up-regulated by the treatment with 40 µM Dgn for 24 h. However, the
M AN U
expression of LXRα and RXRα, the main posttranscriptional regulators of ABCA1 expression [36], were not altered in Dgn-treated macrophage foam cells (Fig. 1E–1H). These results suggested a potent regulatory role for Dgn in ABCA1 expression in foam cells.
TE D
3.2 Dgn reduces macrophage miR-19b level
Our group has recently shown that miR-19b dramatically represses ABCA1 expression through partial complementary binding to 3’UTR of ABCA1 mRNA [31]. To investigate whether miR-19b is involved in Dgn-induced ABCA1 expression, we
EP
examined miR-19b levels in THP-1 macrophage/MPM-derived foam cells treated with 40 µM Dgn alone or together with 40 nM miR-19b mimic or 80 nM miR-19b
AC C
inhibitor for 24 h. As shown in Fig. 2A–2B, Dgn significantly decreased the levels of miR-19b in foam cells. Dgn-suppressed miR-19b levels were abrogated by miR-19b mimic and enhanced by miR-19b inhibitor, when compared with those treated with Dgn only. We further confirmed the functional role of miR-19b in the effects of Dgn on ABCA1 expression under the condition of the gain or loss of functions of miR-19b in THP-1 macrophage/MPM-derived foam cells (Fig. 2C–2D). We found that the effect of Dgn on ABCA1 expression was blocked by miR-19b mimic and enhanced by miR-19b inhibitor, suggesting that the endogenous miR-19b levels in Dgn-treated cells were decreased so as to facilitate ABCA1 expression. Taken together, these 10
ACCEPTED MANUSCRIPT findings suggested that Dgn regulates ABCA1 expression via inhibiting miR-19b level.
3.3 Dgn enhances macrophage cholesterol efflux
RI PT
Increased expression of ABCA1 is directly associated with an increase in the efflux of macrophage cholesterol. As ABCA1 was upregulated by Dgn, we next examined the effects of Dgn on apoAI-mediated (ABCA1-dependent) cholesterol efflux in THP-1 macrophage/MPM derived foam cells. As shown in Fig. 2E–2F, Dgn
SC
obviously increased cholesterol efflux from foam cells. The apoAI-mediated cholesterol efflux in response to Dgn treatment was impaired when foam cells were
M AN U
co-treated with miR-19b mimic, whereas Dgn-induced efflux was enhanced by the treatment with miR-19b inhibitor. These data indicated that Dgn promotes apoAI-mediated cholesterol efflux via ABCA1-dependent pathway by suppressing miR-19b level.
TE D
3.4 Dgn inhibits macrophage cholesterol accumulation
Cholesterol-laden macrophage foam cells are a central component of atherosclerotic lesions. Here, we performed HPLC to measure cellular cholesterol
EP
levels in THP-1 macrophage/MPM-derived foam cells in response to treatment with 40 µM Dgn alone or in combination with 40 nM miR-19b mimic or 80 nM miR-19b inhibitor. As anticipated, cellular levels of TC, FC and cholesterol ester (CE) in
AC C
Dgn-treated cells were significantly lower than those in the control cells (Table 1 and 2). Comparing with these in Dgn-treated cells, cellular cholesterol levels were increased when foam cells were treated with Dgn and miR-19b mimic, whereas cellular cholesterol levels were further decreased when foam cells were treated with Dgn and miR-19b inhibitor. The percentages of CE/TC were not significantly changed in response to any experimental manipulation. These findings demonstrated that Dgn reduces cellular cholesterol accumulation via suppressing miR-19b levels.
3.5 Dgn improves RCT and plasma lipid profile 11
ACCEPTED MANUSCRIPT ABCA1 is a key player in RCT and is critical in the generation of HDL. To determine the effects of Dgn-induced ABCA1 expression on RCT and plasma lipid profile, 3H-cholesterol tracer amounts in plasma, liver, and feces were quantified to evaluate cholesterol distribution along the RCT pathway with liquid scintillation
RI PT
counting, and the biliary cholesterol levels and the plasma lipid profile were determined by enzymatic methods. As shown in Fig. 3A–3D, the 3H-cholesterol counts in feces after macrophage injection, as expressed as a percentage of total 3
H-counts injected, were significantly increased in Dgn-treated mice compared with
SC
control mice. The counts trended to further increase in mice treated with Dgn together with antagomiR-19b. The fecal radioactivity was slightly increased, but not
M AN U
significantly different, in the Dgn-treated mice with co-treatment with agomiR-19b when compared with control mice. The same alterations were also observed in the biliary cholesterol levels after treatment. The 3H-cholesterol tracer amounts in plasma and liver did not significantly differ among all 4 groups. As expected, the efficiency of macrophage RCT tracked closely with the cholesterol mass in plasma lipoprotein
TE D
distribution and was consistent with HDL levels higher in Dgn-treated mice, the highest in mice treated with Dgn and antagomiR-19b, and was inverse association with the tendencies of LDL. The plasma HDL and LDL levels were not significantly
EP
different in mice treated with Dgn and agomiR-19b when compared with those in control mice (Table 3). Throughout the study, body weight of mice fed different diets was similar. These results clearly demonstrated that Dgn accelerates RCT and
AC C
improves plasma lipid profile likely via the ABCA1 pathway.
3.6 Dgn inhibits aortic atherosclerosis Finally, we further analyzed the atheroprotective effect of Dgn and the potential
mechanism underlying its atheroprotection in apoE KO mice. Consistent with the results from the ex vivo studies, the analysis of atherosclerotic lesions both in en face preparations of the whole aorta and in the aortic root revealed that atherosclerotic lesion areas were significantly reduced in Dgn-treated mice, and further reduced in mice treated with Dgn and antagomiR-19b, when compared with control mice (Fig. 12
ACCEPTED MANUSCRIPT 4A–4B). The assessment of collagen contents in aortic root sections of selected mice in each group showed that Dgn treatment alone or supplemented with antagomiR-19b distinctly increased collagen contents and stabilized aortic atherosclerotic lesions. Lipid deposition in the cross-sections of aortic root was also reduced in Dgn-treated
RI PT
mice or in mice treated with Dgn and antagomiR-19b. Supplement with agomiR-19b abolished the atheroprotective effects of Dgn, resulting in no significant differences in lesion sizes, collagen contents and lipid deposition when compared with those in control mice. Furthermore, we determined ABCA1 expression in tissue homogenate
SC
of aortic roots. Compared with control mice, ABCA1 expression in the aortic roots was significantly up-regulated in Dgn-treated mice or in mice treated with both Dgn
M AN U
and antagomiR-19b, but no significant difference was observed in Dgn-treated mice co-treated with agomiR-19b (Fig. 4C). These results indicated that Dgn inhibits aortic lipid deposition and atherogenesis through miR-19b suppression and subsequent
AC C
EP
TE D
ABCA1 upregulation.
13
ACCEPTED MANUSCRIPT 4. Discussion In this study, we investigated the atheroprotective effect and the molecular mechanisms underlying Dgn effects on cholesterol efflux from macrophage foam cells and atherosclerotic lesions in apoE KO mice. As illustrated in Fig 5, our results
RI PT
showed that Dgn up-regulates macrophage ABCA1 expression via suppressing miR-19b level and induces ABCA1-dependent cholesterol efflux from macrophage foam cells, resulting in the reduction in cholesterol accumulation in peripheral macrophages. Furthermore, Dgn effectively enhances in vivo macrophage RCT and
SC
remarkably attenuates aortic lipid deposition and atherosclerotic lesions in apoE KO mice.
M AN U
Dgn pronouncedly promotes peripheral macrophage ABCA1 expression and ABCA1-dependent cholesterol efflux. Temel et al found that Dgn treatment of wild-type and NPC1L1-knockout mice did not alter intestinal expression of ABCA1, ABCG5 and ABCG8 [12]. Uemura et al verified that Dgn suppressed LXRα transactivation
induced
by
a
synthetic
LXRa
agonist
T0901317
and
TE D
T0901317-induced binding of LXRα to LXRE in HepG2 cells, and inhibited the expression of SREBP-1c and lipogenic genes, leading to a reduction in hepatic lipogenesis and lipid accumulation. However, Uemura et al did not further determine
EP
the levels of ABCA1 in hepatocytes and peripheral macrophages under these conditions [2]. Our study showed that Dgn treatment did not affect macrophage expression of LXRα and RXRα, suggesting another mechanism involved in
AC C
Dgn-induced ABCA1 expression. In addition to classic transcriptional regulation of cholesterol metabolism (e.g. by
LXRα), the members of a class of noncoding RNAs termed microRNAs have recently been identified as potent post-transcriptional regulators of lipid metabolism genes involved in cholesterol homeostasis [37, 38]. ABCA1 has an uncommonly long 3’UTR of >3.3 kb, rendering it particularly susceptible to post-transcriptional regulation by microRNAs [39]. Our previous work has verified that miR-19b potently inhibits macrophage ABCA1 expression by binding to mRNA 3’UTR of ABCA1 [31]. Based on this, we evaluated the role of miR-19b in Dgn-induced ABCA1 expression. 14
ACCEPTED MANUSCRIPT Our data suggest that Dgn-induced ABCA1 expression is at least mainly, if not completely, dependent on miR-19b suppression. However, the pathway through which Dgn regulates miR-19b level needs further investigation. Dgn accelerates macrophage cholesterol efflux via ABCA1 pathway and the
RI PT
process of RCT in vivo. Accumulation of cholesterol-laden macrophages in the intima is a major hallmark in the early stage of atherosclerotic lesions [15, 19]. Cholesterol efflux from macrophages is the first, potentially the most important, step in macrophage RCT. Accordingly, ABCA1-dependent cholesterol efflux is a crucial
SC
event in the prevention of excessive cholesterol accumulation in macrophages of the arterial wall and their transformation into foam cells [16, 25, 40, 41]. In this study, our
M AN U
findings suggest a new mechanism underlying Dgn-accelerated RCT. When the cells were treated with Dgn alone or together with miR-19b inhibitor, cholesterol efflux was accelerated from macrophage foam cells via ABCA1 pathway, resulting in the suppression of cellular cholesterol accumulation. Subsequently, in vivo macrophage RCT was accelerated and blood lipid profile ameliorated. Other mechanisms have
TE D
been reportedly involved in Dgn-accelerated RCT. Dgn competitively inhibits intestinal absorption of dietary cholesterol because of its close chemical structural similarity with cholesterol [8, 9]. Dgn also induces hepatic cholesterol secretion,
EP
which requires the hepatobiliary cholesterol transporters ABCG5 and ABCG8 [11, 42]. Despite accelerated macrophage RCT, Dgn did not significantly decrease plasma TC level. This is likely because enhanced excretion of cholesterol from the liver is
AC C
counteracted by increased peripheral cholesterol transported to plasma and then to the liver.
Dgn inhibits aortic lipid deposition and atherosclerotic development. It has been
believed that the factors which modulate macrophage cholesterol efflux and RCT have major impacts on initiation, progression, and regression of atherosclerosis [16, 29]. Consistently, aortic lipid deposition and atherosclerotic lesion area were dramatically decreased in animals treated with Dgn alone or together with antagomiR-19b. Dgn obviously ameliorated the structure of atherosclerotic plaques by increasing collagen contents and decreasing necrotic core. Our findings further 15
ACCEPTED MANUSCRIPT demonstrated that atheroprotective effect of Dgn is intimately associated with its role in regulating ABCA1 expression in macrophages and lipid accumulation in the artery wall. Previous studies identified that Dgn treatment inhibits lipid peroxidation and production of free radicals in rats fed a high-cholesterol diet [6, 7, 43]. Dgn has a effect
against
atherosclerosis
by
regulating
serum
levels
of
RI PT
protective
proinflammatory mediators such as vascular cell adhesion molecule-1 (VCAM-1), C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) in apoE KO mice [1, 4]. In addition, Manivannan et al demonstrated that Dgn attenuates
SC
vascular calcification in rats with chronic renal failure by preventing the VSMC phenotype changes [44]. All these mechanisms contribute to the atheroprotective
M AN U
action of Dgn.
In the present study, we found that Dgn promotes macrophage ABCA1 expression by suppressing miR-19b level. The hypolipidemic effect of Dgn is dependent on ABCA1-mediated cholesterol efflux from macrophage foam cells and in vivo macrophage RCT. This beneficial effect is reflected by the elevated plasma HDL
TE D
and lowered LDL levels. Aortic lipid deposition and atherosclerotic lesion of apoE KO mice are obviously alleviated by Dgn treatment alone or together with antagomiR-19b. Our ongoing research in collaboration with clinical scientists is to
EP
evaluate the hypolipidemic and antiatherogenic actions of Dgn in the volunteers or even patients. The biochemical tests will be performed to show the alteration of plasma lipids, and the imaging examinations to observe the atherosclerotic lesions in
AC C
common carotid arteries and/or coronary arteries. The data from these investigations will provide direct insight into the hypolipidemic and antiatherogenic actions of Dgn in human, which will be instructive for clinicians. Collectively, our results reveal a novel mechanism by which Dgn ameliorates
macrophage cholesterol metabolism and suppresses the progression of atherosclerosis in vivo. Since Dgn produces potential hypolipidemic activity by enhancing cholesterol removal from macrophages to prevent atherosclerotic cardiovascular diseases, as a natural product, Dgn might be added to the foods and beverages or provide a guide to develop pharmaceutical agents. 16
ACCEPTED MANUSCRIPT Conflict of interest We do have no actual or potential conflict of interest.
Acknowledgments
RI PT
The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (81170278, 81370377, 81300224 and 81100560), the Hunan Provincial Natural Science Foundation of China (14JJ2091) and the Scientific Research Fund of Hunan Provincial Education Department
SC
(12C0339), China, and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, China (2008-244), the
AC C
EP
TE D
M AN U
construct program of the key discipline in Hunan Province, China.
17
ACCEPTED MANUSCRIPT References [1] Song Jia-Xi ML, Kou Jun-Ping, Yu Bo-Yang. Diosgenin reduces leukocytes adhesion and migration linked with inhibition of intercellular adhesion molecule-1 expression and NF-κB p65 activation in endothelial cells. Chinese Journal of Natural Medicines 2012; 10:142-9. [2] Uemura T, Goto T, Kang M-S, Mizoguchi N, Hirai S, Lee J-Y, et al. Diosgenin, the Main Aglycon of Fenugreek, Inhibits LXR{alpha} Activity in HepG2 Cells and Decreases Plasma and Hepatic
RI PT
Triglycerides in Obese Diabetic Mice. J. Nutr. 2011; 141:17-23. [3] Liu K, Zhao W, Gao X, Huang F, Kou J and Liu B. Diosgenin ameliorates palmitate-induced endothelial dysfunction and insulin resistance via blocking IKKbeta and IRS-1 pathways. Atherosclerosis 2012; 223:350-8.
[4] Park H-J, Byeon H-E, Koo H-J and Pyo S. Anti-atherosclerotic effects of diosgenin in ApoE-deficient mice. FASEB journal : official publication of the Federation of American Societies for
SC
Experimental Biology 2011; 25:980-2.
[5] Sangeetha MK, ShriShri Mal N, Atmaja K, Sali VK and Vasanthi HR. PPAR's and Diosgenin a chemico biological insight in NIDDM. Chemico-biological interactions 2013; 206:403-10.
M AN U
[6] Son IS, Kim JH, Sohn HY, Son KH, Kim JS and Kwon CS. Antioxidative and hypolipidemic effects of diosgenin, a steroidal saponin of yam (Dioscorea spp.), on high-cholesterol fed rats. Bioscience, biotechnology, and biochemistry 2007; 71:3063-71.
[7] Gong G, Qin Y, Huang W, Zhou S, Wu X, Yang X, et al. Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis. Chemico-biological interactions 2010; 184:366-75.
[8] Cayen MN and Dvornik D. Effect of diosgenin on lipid metabolism in rats. Journal of lipid research 1979; 20:162-74.
TE D
[9] LAGUNA J, GOMEZ-PUYOU A, PENA A and GUZMAN-GARCIA J. Effect of diosgenin on cholesterol metabolism. J Atheroscler Res 1962; 2:459-70. [10] Pan CH, Tsai CH, Liu FC, Fan MJ, Sheu MJ, Hsieh WT, et al. Influence of different particle processing on hypocholesterolemic and antiatherogenic activities of yam (Dioscorea pseudojaponica) in cholesterol-fed rabbit model. Journal of the science of food and agriculture 2013; 93:1278-83.
EP
[11] Kamisako T and Ogawa H. Regulation of biliary cholesterol secretion is associated with abcg5 and abcg8 expressions in the rats: effects of diosgenin and ethinyl estradiol. Hepatology research : the official journal of the Japan Society of Hepatology 2003; 26:348-52.
AC C
[12] Temel RE, Brown JM, Ma Y, Tang W, Rudel LL, Ioannou YA, et al. Diosgenin stimulation of fecal cholesterol excretion in mice is not NPC1L1 dependent. Journal of lipid research 2009; 50:915-23.
[13] Diaz Zagoya J, Laguna J and Guzman-Garcia J. Studies on the regulation of cholesterol metabolism by the use of the structural analogue, diosgenin. Biochem Pharmacol 1971; 20:3473-80. [14] Moore KJ and Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011; 145:341-55. [15] Moore KJ, Sheedy FJ and Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nature reviews. Immunology 2013; 13:709-21. [16] Pennings M, Meurs I, Ye D, Out R, Hoekstra M, Van Berkel TJ, et al. Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development. FEBS letters 2006; 580:5588-96. [17] Zhao GJ, Tang SL, Lv YC, Ouyang XP, He PP, Yao F, et al. NF-kappaB suppresses the expression
18
ACCEPTED MANUSCRIPT of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice. International journal of cardiology 2014; 171:e93-5. [18] Zhang M, Wu JF, Chen WJ, Tang SL, Mo ZC, Tang YY, et al. MicroRNA-27a/b regulates cellular cholesterol efflux, influx and esterification/hydrolysis in THP-1 macrophages. Atherosclerosis 2014; 234:54-64. [19] Yu XH, Fu YC, Zhang DW, Yin K and Tang CK. Foam cells in atherosclerosis. Clinica chimica acta; international journal of clinical chemistry 2013; 424:245-52.
RI PT
[20] Lu Q, Tang SL, Liu XY, Zhao GJ, Ouyang XP, Lv YC, et al. Tertiary-Butylhydroquinone Upregulates Expression of ATP-Binding Cassette Transporter A1 via Nuclear Factor E2-Related Factor 2/Heme Oxygenase-1 Signaling in THP-1 Macrophage-Derived Foam Cells. Circ J 2013; 77:2399-408. [21] Liu XY, Lu Q, Ouyang XP, Tang SL, Zhao GJ, Lv YC, et al. Apelin-13 increases expression of ATP-binding cassette transporter A1 via activating protein kinase C alpha signaling in THP-1
SC
macrophage-derived foam cells. Atherosclerosis 2013; 226:398-407.
[22] Mo Z, Xiao J, Liu X, Hu Y, Li X, Yi G, et al. AOPPs inhibits cholesterol efflux by down-regulating ABCA1 expression in a JAK/STAT signaling pathway-dependent manner. J Atheroscler Thromb 2011; 18:796-807.
M AN U
[23] Liu X, Xiao J, Mo Z, Yin K, Jiang J, Cui L, et al. Contribution of D4-F to ABCA1 expression and cholesterol efflux in THP-1 macrophage-derived foam cells. Journal of cardiovascular pharmacology 2010; 56:309-19.
[24] Zhao Y, Van Berkel TJ and Van Eck M. Relative roles of various efflux pathways in net cholesterol efflux from macrophage foam cells in atherosclerotic lesions. Curr Opin Lipidol 2010; 21:441-53.
[25] Westerterp M, Bochem AE, Yvan-Charvet L, Murphy AJ, Wang N and Tall AR. ATP-Binding
TE D
Cassette Transporters, Atherosclerosis, and Inflammation. Circ. Res. 2014; 114:157-70. [26] van Capelleveen JC, Brewer HB, Kastelein JJP and Hovingh GK. Novel Therapies Focused on the High-Density Lipoprotein Particle. Circ. Res. 2014; 114:193-204. [27] Rye K-A and Barter PJ. Cardioprotective functions of HDL. J. Lipid Res. 2014; 55:168-79. [28] Rosenson RS, Brewer HB, Jr, Davidson WS, Fayad ZA, Fuster V, Goldstein J, et al. Cholesterol
EP
Efflux and Atheroprotection: Advancing the Concept of Reverse Cholesterol Transport. Circulation 2012; 125:1905-19.
[29] Oram JF and Vaughan AM. ATP-Binding Cassette Cholesterol Transporters and Cardiovascular
AC C
Disease. Circ. Res. 2006; 99:1031-43. [30] Oram JF and Heinecke JW. ATP-Binding Cassette Transporter A1: A Cell Cholesterol Exporter That Protects Against Cardiovascular Disease. Physiol Rev 2005; 85:1343-72. [31] Lv YC, Tang YY, Peng J, Zhao GJ, Yang J, Yao F, et al. MicroRNA-19b promotes macrophage cholesterol accumulation and aortic atherosclerosis by targeting ATP-binding cassette transporter A1. Atherosclerosis 2014; 236:215-26. [32] Tian GP, Chen WJ, He PP, Tang SL, Zhao GJ, Lv YC, et al. MicroRNA-467b targets LPL gene in RAW 264.7 macrophages and attenuates lipid accumulation and proinflammatory cytokine secretion. Biochimie 2012; 94:2749-55. [33] Chen C, Cheng P, Xie H, Zhou HD, Wu XP, Liao EY, et al. MiR-503 regulates osteoclastogenesis via targeting RANK. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2014; 29:338-47. [34] Wang D, Xia M, Yan X, Li D, Wang L, Xu Y, et al. Gut Microbiota Metabolism of Anthocyanin
19
ACCEPTED MANUSCRIPT Promotes Reverse Cholesterol Transport in Mice Via Repressing miRNA-10b. Circ. Res. 2012; 111:967-81. [35] Zhao GJ, Tang SL, Lv YC, Ouyang XP, He PP, Yao F, et al. Antagonism of betulinic acid on LPS-mediated inhibition of ABCA1 and cholesterol efflux through inhibiting nuclear factor-kappaB signaling pathway and miR-33 expression. PloS one 2013; 8:e74782. [36] Schmitz G and Langmann T. Transcriptional regulatory networks in lipid metabolism control ABCA1 expression. Biochimica et biophysica acta 2005; 1735:1-19.
RI PT
[37] Lv YC, Yin K, Fu YC, Zhang DW, Chen WJ and Tang CK. Posttranscriptional Regulation of ATP-Binding Cassette Transporter A1 in Lipid Metabolism. DNA and cell biology 2013; 32:348-58.
[38] Fernandez-Hernand... C, Suarez Y, Rayner K and Moore K. MicroRNAs in lipid metabolism. Curr Opin Lipidol 2011; 22:86-92. Function. Circ. Res. 2014; 114:183-92.
SC
[39] Rayner KJ and Moore KJ. MicroRNA Control of High-Density Lipoprotein Metabolism and [40] De Meyer I, Martinet W and De Meyer G. Therapeutic strategies to deplete macrophages in atherosclerotic plaques. Br J Clin Pharmacol 2012; 74:246-63.
[41] Ouimet M and Marcel YL. Regulation of Lipid Droplet Cholesterol Efflux From Macrophage
M AN U
Foam Cells. Arterioscler Thromb Vasc Biol 2012; 32:575-81.
[42] Kosters A, Kunne C, Looije N, Patel SB, Oude Elferink RP and Groen AK. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice. Journal of lipid research 2006; 47:1959-66. [43] Jayachandran KS, Vasanthi HR and Rajamanickam GV. Antilipoperoxidative and membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction. Molecular and cellular biochemistry 2009; 327:203-10.
[44] Manivannan J, Barathkumar TR, Sivasubramanian J, Arunagiri P, Raja B and Balamurugan E.
TE D
Diosgenin attenuates vascular calcification in chronic renal failure rats. Molecular and cellular
AC C
EP
biochemistry 2013; 378:9-18.
20
ACCEPTED MANUSCRIPT Figure Legends
Fig. 1. The effects of Dgn on the expression of ABCA1 and LXRα in macrophage foam cells. A, B, C, and D: ABCA1 protein levels were determined by western
RI PT
blotting in THP-1 macrophage/MPM-derived foam cells treated with Dgn at different concentrations (0, 10, 20, 40, 80 µM) for 24 h or with 40 µM Dgn at the indicated periods (0, 6, 12, 24, 48 h). E, F, G and H: the protein levels of LXRα and RXRα were determined by western blotting in THP-1 macrophage/MPM-derived foam cells
SC
treated with 40 µM Dgn for 24 h. All results are expressed as mean ± S.D. from three independent experiments in duplicate. * P<0.05 vs. control group. Dgn, diosgenin;
M AN U
LXRα, liver X receptor α; Con, control; Et, ethanol as a vehicle.
Fig. 2. The involvement of miR-19b in the Dgn-mediated increase of ABCA1 expression and cholesterol efflux in macrophage foam cells. A, B, C, and D: miR-19b levels and ABCA1 expression were measured by RT-qPCR or western
TE D
blotting, respectively, in THP-1 macrophage/MPM-derived foam cells treated with 40 µM Dgn alone or together with 80 nM miR-19b inhibitor/40 nM miR-19b mimic for 24 h. E and F: cellular cholesterol efflux was analyzed by liquid scintillation counting
EP
assays as described in Methods. All results are expressed as mean ± S.D. from three independent experiments in duplicate. * P<0.05 vs. control group. Dgn, diosgenin;
AC C
Con, control; Inh, miR-19b inhibitor; Mim, miR-19b mimic.
Fig. 3. The effect of Dgn on the RCT of cholesterol–laden MPMs intraperitoneally injected into apoE KO mice and biliary cholesterol levels. A, B, and C: 3H–cholesterol-labeled and acLDL–loaded MPMs were intraperitoneally injected into apoE KO mice fed with Western diet for 8 wk. After cell injection, the samples of blood, liver and feces were collected for the analysis of in vivo macrophage RCT at 48 h. D: Biliary cholesterol levels in apoE KO mice after a 6 h fast were determined by enzymatic colorimetry. Values are mean ± S.D. (n=15 mice per group). * P<0.05 vs. control group. Dgn, diosgenin; Con, control; Ago, 21
ACCEPTED MANUSCRIPT agomir-19b; Ant, antagomiR-19b.
Fig. 4. The effect of Dgn on aortic lesion area and ABCA1 expression in apoE KO mice. A: Representative images and the quantification of atherosclerotic lesion area in
RI PT
the en face analysis of the whole aorta with Sudan IV staining in apoE KO mice fed with Western diet for 8 wk. Values are mean ± SEM (n=12 mice per group). B: Representative microscopic images and quantification of atherosclerotic plaque area or collagen contents in cross-sections of proximal aorta with the staining of
SC
hematoxylin-eosin, Masson’s trichrome and Oil-red O in apoE KO mice fed with Western diet for 8 wk. Original magnification 40×. Values are mean ± SEM (n=15
M AN U
mice per group). C: ABCA1 levels were determined in the homogenate of aortic arch using western blotting. Values are mean ± S.D. (n=3 mice per group). * P<0.05 vs. control group. Dgn, diosgenin; Con, control; Ago, agomir-19b; Ant, antagomiR-19b; HE, hematoxylin-eosin; MT, Masson’s trichrome.
TE D
Fig. 5. Schematic presentation showing the effects of Dgn on miR-19b level, ABCA1-dependent cholesterol efflux in macrophage foam cell and aortic atherosclerosis development in rodents. Dgn treatment up-regulates macrophage expression
by
suppressing
miR-19b
level,
which
promotes
EP
ABCA1
ABCA1-dependent cholesterol efflux and in vivo macrophage RCT. Consequently, Dgn efficiently ameliorates blood lipid profile and cholesterol homeostasis of
AC C
peripheral macrophage, which potently inhibits aortic lipid deposition and atherosclerosis progression in rodents. Dgn, diosgenin; LD, lipid droplet; FC, free cholesterol; RISC, RNA-induced silencing complex; ↑, promotion; ↓, inhibition.
22
ACCEPTED MANUSCRIPT Table 1. Effects of Dgn on cholesterol contents in THP-1 macrophage-derived foam cells after apoAI–mediated cholesterol efflux FC(mg/g)
CE(mg/g)
CE/TC(%)
Con
347.78±29.14
155.51±15.66
192.27±17.17
55.28
Dgn
316.94±25.48*
144.12±14.83*
172.82±16.40*
54.53
Dgn+Mim
356.33±35.18
157.59±16.24
198.74±18.65
55.77
Dgn+Inh
292.39±23.56*
133.79±13.45*
158.60±14.96*
54.24
RI PT
TC(mg/g)
SC
THP-1 macrophage-derived foam cells were treated with 40 µM Dgn alone or in combination with 40 nM miR-19b mimic or 80 nM miR-19b inhibitor for 24 h. Then,
M AN U
HPLC was performed to determine the levels of cellular total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC). The results were expressed as mean ±
Table 2.
TE D
S.D. of three independent experiments in duplicate. * P<0.05 vs. control group.
Effects of Dgn on cholesterol contents in MPM-derived foam cells after apoAI–mediated cholesterol efflux
Con
CE (mg/g)
CE/TC (%)
301.54±27.63
149.79±14.87
151.75±16.42
50.66
271.05±22.52*
136.78±15.13*
134.27±14.40*
49.54
AC C
Dgn
FC (mg/g)
EP
TC (mg/g)
Dgn+Mim
310.63±29.75
153.32±16.46
157.31±17.73
50.64
Dgn+Inh
250.90±20.38*
127.34±12.93*
123.56±13.69*
49.30
MPM-derived foam cells were treated with 40 µM Dgn alone or in combination with 40 nM miR-19b mimic or 80 nM miR-19b inhibitor for 24 h. Then, HPLC was performed to determine the levels of cellular total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC). The results were expressed as mean ± S.D. of three independent experiments in duplicate. * P<0.05 vs. control group.
1
ACCEPTED MANUSCRIPT Table 3. Body weight and plasma lipid profile in apoE KO mice BW (g)
TG (mmol/l)
TC (mmol/l)
HDL-C (mmol/l)
LDL-C (mmol/l)
30.46±4.04 1.08±0.14
20.56±1.42
2.13±0.21
18.27±2.25
Dgn
29.10±3.73 1.05±0.13
19.39±1.19
2.97±0.26*
15.78±1.65*
Dgn+Ago
30.05±4.17 1.07±0.11
19.70±2.48
2.21±0.19
17.30±2.13
Dgn+Ant
28.89±3.62 1.03±0.10
18.89±1.71
3.21±0.24*
14.94±2.56*
RI PT
Con
SC
Plasma samples from different experimental groups were measured by the enzymatic method as described in Methods. The data were expressed as mean ± S.D. (n=15 mice
AC C
EP
TE D
M AN U
per group). *P < 0.05 vs. control group.
2
ACCEPTED MANUSCRIPT THP-1 derived foam cells
MPM derived foam cells
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 1.
1
ACCEPTED MANUSCRIPT Fig. 2. MPM derived foam cells
AC C
EP
TE D
M AN U
SC
RI PT
THP-1 derived foam cells
2
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 3.
3
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 4.
4
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 5.
5
ACCEPTED MANUSCRIPT Highlights
Dgn increases macrophage ABCA1 expression
③
Dgn enhances macrophage cholesterol efflux
Dgn improves RCT and plasma lipid profile Dgn inhibits aortic atherosclerosis
AC C
EP
TE D
M AN U
⑥
SC
Dgn inhibits macrophage cholesterol accumulation
RI PT
Dgn reduces macrophage miR-19b level
ACCEPTED MANUSCRIPT
AUTHOR DECLARATION
We wish to confirm that there are no known conflicts of interest associated with
RI PT
this publication and there has been no significant financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but
SC
are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.
M AN U
We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.
TE D
We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animal has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the
EP
manuscript.
We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He
AC C
is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected]
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT The full gel images in Figure 1 A: ABCA1
B: ABCA1
B: β-actin
SC
RI PT
A: β-actin
M AN U
C: ABCA1
D: ABCA1
F: LXRα
AC C
EP
E: LXRα
TE D
C: β-actin
D: β-actin
E: β-actin
F: β-actin
G: RXRα H: RXRα
G: β-actin H: β-actin 1
S0021-9150(15)00135-5
DOI:
10.1016/j.atherosclerosis.2015.02.044
Reference:
ATH 13965
To appear in:
Atherosclerosis
Received Date: 21 October 2014 Revised Date:
2 February 2015
Accepted Date: 23 February 2015
Please cite this article as: Lv Y-c, Yang J, Yao F, Xie W, Tang Y-y, Ouyang X-p, He P-p, Tan Yl, Li L, Zhang M, Liu D, Cayabyab FS, Zheng X-L, Tang C-k, Diosgenin Inhibits Atherosclerosis via Suppressing the MiR-19b-induced Downregulation of ATP-binding Cassette Transporter A1, Atherosclerosis (2015), doi: 10.1016/j.atherosclerosis.2015.02.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Diosgenin Inhibits Atherosclerosis via Suppressing the MiR-19b-induced Downregulation of ATP-binding Cassette Transporter A1
Running title: Diosgenin inhibits atherogenesis.
RI PT
Yun-cheng Lva, b, 1, Jing Yangc, 1, Feng Yaoa, d, 1, Wei Xiea, b, Yan-yan Tanga, Xin-ping Ouyanga, Ping-ping Hea, Yu-lin Tana, Liang Lia, Min Zhanga, Dan Liua, Francisco S. Cayabyabe, Xi-Long Zhengf, Chao-ke Tanga*
a
1
TE D
M AN U
SC
Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, Life Science Research Center, Hunan Province Cooperative innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, Hunan 421001, China. b Laboratory of Clinical Anatomy, University of South China, Hengyang, 421001, China. c Department of Metabolism & Endocrinology of the First Affiliated Hospital, University of South China, Hengyang, 421001, China. d Department of Laboratory Animal Science, University of South China, Hengyang 421001, China. e Department of Surgery, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. f Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 4N1.
These authors contributed equally to this work.
EP
To whom correspondence should be addressed: *Chao-ke Tang, PhD, Professor, Institute of Cardiovascular Research, University of South China,
AC C
Hengyang, Hunan 421001, China Email: [email protected] Tel: 86-734-8281853
1
ACCEPTED MANUSCRIPT Rationale: Diosgenin (Dgn), a structural analogue of cholesterol, has been reported to have the hypolipidemic and antiatherogenic properties, but the underlying mechanisms are not fully understood. Given the key roles of macrophages in cholesterol metabolism and atherogenesis, it is critical to investigate macrophage
RI PT
cholesterol efflux and development of atherosclerotic lesion after Dgn treatment. Objective: This study was designed to evaluate the potential effects of Dgn on macrophage cholesterol metabolism and the development of aortic atherosclerosis, and to explore its underlying mechanisms.
SC
Methods and Results: Dgn significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) protein, but didn’t affect liver X receptor α levels in
M AN U
foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by western blotting. The miR-19b levels were markedly down-regulated in Dgn-treated THP-1 macrophages/MPM-derived foam cells. Cholesterol transport assays revealed that treatment with Dgn alone or together with miR-19b inhibitor notably enhanced ABCA1-dependent cholesterol efflux,
TE D
resulting in the reduced levels of total cholesterol, free cholesterol and cholesterol ester as determined by high-performance liquid chromatography. The fecal 3H-sterol originating from cholesterol-laden MPMs was increased in apolipoprotein E knockout
EP
mice treated with Dgn or both Dgn and antagomiR-19b. Treatment with Dgn alone or together with antagomiR-19b elevated plasma high-density lipoprotein levels, but reduced plasma low-density lipoprotein levels. Accordingly, aortic lipid deposition
AC C
and plaque area were reduced, and collagen content and ABCA1 expression were increased in mice treated with Dgn alone or together with antagomiR-19b. However, miR-19b overexpression abrogated the lipid-lowering and atheroprotective effects induced by Dgn.
Conclusion: The present study demonstrates that Dgn enhances ABCA1-dependent cholesterol efflux and inhibits aortic atherosclerosis progression by suppressing macrophage miR-19b expression. Keywords: Diosgenin; ABCA1; macrophage foam cells; cholesterol efflux; reverse cholesterol transport; atherosclerosis. 2
ACCEPTED MANUSCRIPT 1. Introduction Diosgenin (Dgn) is a naturally occurring steroidal sapogenin present in a variety of plants including fenugreek, yam root and soy bean. It has long been used in Ayurvedic and Chinese medicine [1, 2]. Cumulative evidence demonstrates its
RI PT
cardiovascular protective activities and its role in appropriate management of dyslipidemia [3]. Dgn remarkably decreases the level of serum triglyceride (TG) and total cholesterol (TC) in mice, rats, chickens and rabbits fed with a high-cholesterol diet [4-10]. Dgn markedly decreases the extent of fatty liver as well as aortic fatty
SC
streaks in high-cholesterol-fed rabbits [10]. These studies suggest that the lipid-lowering activity and the antiatherogenic property of Dgn are intimately relevant
M AN U
with its suppression of intestinal cholesterol absorption and promotion of hepatic cholesterol secretion [2, 11-13]. It is known that peripherial macrophages play critical roles in cholesterol metabolism and atherosclerosis [14, 15]. However, it is not clear whether and how Dgn regulates macrophage cholesterol metabolism. Cholesterol homeostasis in macrophages is of prime importance in the initiation
TE D
of atherosclerosis, because dysregulation of cholesterol metabolism leads to excessive accumulation of cholesterol in the macrophages and subsequent transformation into foam cells [16]. Our group and others have demonstrated that ATP-binding cassette
EP
transporter A1 (ABCA1)-dependent cholesterol efflux is a crucial event in the prevention of excessive cholesterol accumulation in macrophages and atherosclerosis progression [17-27]. ABCA1 facilitates the efflux of free cholesterol to lipid-poor
AC C
apolipoprotein A-I (apoAI) and contributes to about one third of cholesterol efflux in cholesterol-laden macrophages [28]. The activity of the ABCA1 pathway is elaborately modulated in peripheral macrophages, and an increase in ABCA1 activity in arterial macrophages will prevent foam cell formation in atherosclerotic lesions [29, 30]. Our previous work has verified that macrophage ABCA1 expression is potently inhibited by miR-19b, resulting in an impairment of cholesterol efflux and the formation of macrophage foam cells [31]. In the current study, we first investigated the effects of Dgn on miR-19b level, ABCA1 expression and cholesterol metabolism 3
ACCEPTED MANUSCRIPT in cholesterol-laden macrophages, and then examined the hypolipidemic effects of Dgn on serum lipid profile and reverse cholesterol transport (RCT) in high cholesterol and high fat-fed apolipoprotein E knockout (apoE KO) mice. Finally, we determined the antiatherogenic efficacy of Dgn on aortic lipid deposition and atherosclerotic
RI PT
lesion in apoE KO mice. Our results have provided experimental evidence to support the roles of miR-19b/ABCA1 pathway in Dgn inhibition of macrophage cholesterol accumulation and aortic atherogenesis.
SC
2. Materials and methods 2.1 Cell culture
M AN U
THP-1 cell strain was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells were cultured in RPMI 1640 (Solarbio, China) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco BRL, America) at 37 °C and 5% CO2. Differentiation of THP-1 cells into macrophages was induced using 160 nM phorbol-12-myristate acetate (PMA, Sigma, America) for 24 h. Mouse
TE D
peritoneal macrophages (MPMs) from adult C57BL/6J mice (Jackson Laboratories) were obtained by peritoneal lavage 4 d after the intraperitoneal injection of 2.0 ml thioglycollate broth (4%, w/v). The thioglycollate-elicited MPMs were maintained in
EP
RPMI 1640 medium containing 10% FBS. Finally, macrophages were incubated in RPMI 1640 containing 0.2% (w/v) bovine serum albumin (BSA, Sigma, America) and 50 µg/ml acetylated-low density lipoprotein (acLDL) for 48 h in cell flasks or 12-well
AC C
plates, for full differentiation of THP-1 macrophages/MPMs to foam cells in all experiments analyzing ABCA1 expression and cholesterol metabolism (unless otherwise indicated).
2.2 Dgn treatment and miR-19b/anti-miR-19b transfection Dgn (93% in purity, Sigma, America) was dissolved in ethanol to make a stock solution (100 mM) according to the manufacturer’s instructions. THP-1 macrophage/MPM-derived foam cells were transfected with miR-19b mimic or inhibitor (anti-miR-19b: the suppression of miR-19b) (Ribobio, China) at the 4
ACCEPTED MANUSCRIPT concentrations as indicated in the corresponding figure legends for 24 h using RiboFECTTMCP reagent (Ribobio, China) according to the manufacturer’s protocol. A total volume of 1 ml RPMI 1640 containing 0.2% (w/v) BSA in each well contained 100 µl transfection buffer, 10 µl RiboFECTTMCP reagent and different
RI PT
volumes of miR-19b mimic or inhibitor depending on the concentrations used in the experiments.
2.3 Western blot analysis
SC
Whole cell lysate and tissue proteins were extracted with RIPA buffer (Beyotime, China). The proteins (20 µg per lane) were then loaded on 8% SDS-polyacrylamide
M AN U
electrophoresis gel, electrophoresed for 2 h at 100 V in gel running buffer, and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% (w/v) fat-free dry milk, the membranes were incubated with rabbit antibodies against ABCA1 (Abcam, USA), liver X receptor α (LXRα, Abcam, America), retinoic acid receptor α (RXRα, Sigma, America) or β-actin (Boster, China) overnight at 4 °C, and
TE D
then incubated with horseradish peroxidase-conjugated secondary antibody (Boster, China). Immunoreactive protein bands were detected using the enhanced chemiluminesence immunoblotting detection system (Amersham Biosciences,
EP
America). The relative signal intensities were determined by densitometry using the ImageJ software (national institutes of health, NIH, America). The relative levels of ABCA1, LXRα and RXRα were expressed as the ratio of ABCA1, LXRα or RXRα to
AC C
β-actin densitometric values.
2.4 Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis
Total RNA was obtained from THP-1 macrophage/MPM-derived foam cells using the TRIzol RNA isolation kit (Invitrogen, America). cDNA was generated from total RNA using the Ncode Vilo miRNA cDNA synthesis kit (Invitrogen, America) according to the manufacturer’s instructions. The Ncode SYBR Green miRNA RT-qPCR kit (Invitrogen, America) was used to detect the miRNA expression. The 5
ACCEPTED MANUSCRIPT expression of miR-19b in THP-1 macrophage/MPM-derived foam cells was calculated relative to U6B, a ubiquitously expressed small nuclear RNA. RT-qPCR analyses were performed using the forward primers for miR-19b and U6B as following: hsa-miR-19b, 5'-TGTGCAAATCCATGCAAAACTGA-3'; U6B, 5'-
RI PT
ACGCAAATTCGTGAAGCGTTCCAT-3'. The reverse primer was the Ncode miRNA Universal qPCR Primer (Invitrogen, America). A comparative threshold cycle method was used to compare each condition with the control.
SC
2.5 Cholesterol efflux assay
THP-1 cells and MPMs were seeded in 12-well plates (1×106 cells/well),
M AN U
radiolabeled with 5 µCi/ml 3H-cholesterol (PerkinElmer, America) and loaded with cholesterol (acLDL, 50 µg/ml) for 48 h. Cells were subsequently treated with Dgn, alone or together with miR-19b mimic or inhibitor for an additional 24 h. Then, cells were washed twice with phosphate-buffered saline (PBS, T4174, Sigma, America)) and incubated with RPMI 1640 containing 0.2% (w/v) BSA and 10 µg/ml human
TE D
apoAI. After 6 h, 3H-cholesterol in the medium and cells was measured separately by liquid scintillation counting. The percent efflux was calculated by the following equation: [total media counts/(total cellular counts
EP
31].
total media counts] ×100% [21,
2.6 High-performance liquid chromatography (HPLC) assay
AC C
HPLC assay was conducted as described previously [20, 32]. THP-1
macrophage/ MPM-derived foam cells were treated with Dgn alone or together with miR-19b mimic or inhibitor for 24 h, then incubated with RPMI 1640 containing 0.2% (w/v) BSA and 10 µg/ml human apoAI. After 6 h, cells were detached using 1% EDTA in PBS and collected in a glass tube. The sterol analyses were performed using an HPLC system (model 2790, controlled with Empower Pro software; Waters Corp., Milford, MA, America). Sterols were detected using a photodiode array detector equipped with a 4-lL cell (model 996, Waters Corp, America). Analysis of cholesterol and cholesterol esters was performed after elution with acetonitrile-isopropanol 30:70 6
ACCEPTED MANUSCRIPT (v/v) and detected by absorbance at 210 nm.
2.7 Mice and treatments Six-week old male apoE KO mice were obtained from Laboratory Animal Center
RI PT
of Peking University, China. All mice were maintained on a 12 h light/dark cycle with unlimited access to food and water. After fed with a rodent chow diet containing 4.5% (w/w) fat for a week, apoE KO mice were randomly divided into four groups (15 mice per group): control group; Dgn group; Dgn+agomiR-19b (Dgn+Ago) group; Dgn+
SC
antagomiR-19b (Dgn+Ant) group. All groups were switched to and maintained on Western diet (WD) containing 21% (w/w) fat and 1.25% (w/w) cholesterol, and with
M AN U
or without 1% Dgn (w/w) for 8 wk. The apoE KO mice from Dgn+Ago group and Dgn+Ant group received tail vein injections of 10 nmol/per mouse agomiR-19b or 80 mg/kg antagomiR-19b, respectively, on days 1 to 3 at every other week after starting WD [33]. AgomiR and antagomiR are nucleic acid drugs and optimized version of miRNA mimic or inhibitor, which are specially modified for greater stability and
TE D
binding affinity to their targets than that of miRNA mimic or inhibitor. After fasting for 6 h, mice were euthanized, followed by blood-sampling from the retro-orbital plexus, aortas and hearts for further analyses. All animal experiments were done in
EP
accordance with the Institutional Animal Ethics Committee and the University of
AC C
South China Animal Care guidelines for use of experimental animals.
2.8 In vivo macrophage RCT assay MPMs were radiolabeled with 3H-cholesterol (5 µCi/ml) and loaded with
cholesterol (acLDL, 50 µg/ml) for 48 h. These foam cells were then washed twice, equilibrated in medium with 0.2% (w/v) BSA for 6 h, spun down, and resuspended in 0.5 ml medium. The radio-labeled macrophages were intraperitoneally injected into apoE KO mice (3.6×106 cells at 3.8×106 cpm per mouse) treated with Dgn in the absence or presence of agomiR-19b or antagomiR-19b. Feces were collected continuously from 0 to 48 h and were stored at -20°C until lipid extraction. Blood was collected at 48 h, and plasma was used for liquid scintillation counting and lipid 7
ACCEPTED MANUSCRIPT analyses. After 48 h, mice were anesthetized and euthanized. Their livers were then harvested and stored at -20°C until lipid extraction [34].
2.9 Biliary cholesterol assay
RI PT
After a 6 h fast, mice were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight), followed by laparotomy, distal ligation of the bile duct and gallbladder cannulation. The bile was collected for 10 min, and immediately frozen at -20 °C after homogenization [12]. For the analysis of biliary
SC
cholesterol concentrations, a measured volume (10 µl) of bile was extracted by addition of 3:1 ethanol: ether. A measured aliquot of the organic phase was dried
M AN U
down and then analyzed for cholesterol content by solubilizing the lipids in Triton X-100/water using the Total Cholesterol enzymatic assay kits (Dong’ou, China).
2.10 Plasma lipid analysis
Blood was collected into tubes containing 2 mM EDTA from mice after a 6 h fast,
TE D
and centrifuged for 15 min at 4°C. Plasma samples were separated, aliquoted, and stored frozen prior to analyses. TG, TC and HDL-C were determined by enzymatic methods using commercially available test kits (Rongsheng, China) according to the
EP
manufacturer’s instructions.
2.11 Evaluation of aortic lesions
AC C
Atherosclerotic lesions were quantified by en face analysis of the whole aorta
and by cross-sectional analysis of the proximal aorta. For the en face analysis of the aorta, the Sudan IV-stained aortas were photographed (Nikon Coolpix 990, Japan) for the quantification of atherosclerotic lesions. The total aortic surface area and the lesion area were measured by image analysis (ImageJ software, NIH, America), and the ratio of the lesion area to the total area was calculated. For the cross-sectional analysis of the aorta, the OCT-embedded aortas were sectioned using a cryostat, and 8-µm sections were obtained sequentially beginning at the aortic valve. Twenty sections per mouse obtained every 24 µm from the aortic sinus were stained with 8
ACCEPTED MANUSCRIPT hematoxylin-eosin, Oil red O (Sigma, America) and Masson’s trichrome (MT) (Senbeibio, China), which were used for the quantification of lesion areas and collagen contents. Lesion areas and collagen contents were quantified using NIH
RI PT
ImageJ software. Data were expressed as lesion size ± SEM [17, 31, 35].
2.12 Statistical analysis
Data were expressed as means ± standard deviations (S.D.) of at least three independent experiments. With SPSS 18.0 software, data were analyzed by one way
SC
ANOVA followed by Student-Newman-Keuls test. Values with P < 0.05 were
AC C
EP
TE D
M AN U
accepted as statistical significance comparing with the control groups.
9
ACCEPTED MANUSCRIPT 3. Results 3.1 Dgn increases macrophage ABCA1 expression We first investigated whether Dgn influences the expression of ABCA1 in THP-1 macrophage/MPM-derived foam cells. To do so, THP-1 macrophage/MPM-derived
RI PT
foam cells were treated with Dgn at different concentrations (0, 10, 20, 40, 80 µM) for 24 h or with 40 µM Dgn for different time periods (0, 6, 12, 24, 48 h), followed by western blot analysis. As shown in Fig. 1A–1D, Dgn enhanced the expression of ABCA1 protein in THP-1 macrophage/MPM-derived foam cells in both
SC
concentration- and time-dependent manners. Furthermore, ABCA1 expression was maximally up-regulated by the treatment with 40 µM Dgn for 24 h. However, the
M AN U
expression of LXRα and RXRα, the main posttranscriptional regulators of ABCA1 expression [36], were not altered in Dgn-treated macrophage foam cells (Fig. 1E–1H). These results suggested a potent regulatory role for Dgn in ABCA1 expression in foam cells.
TE D
3.2 Dgn reduces macrophage miR-19b level
Our group has recently shown that miR-19b dramatically represses ABCA1 expression through partial complementary binding to 3’UTR of ABCA1 mRNA [31]. To investigate whether miR-19b is involved in Dgn-induced ABCA1 expression, we
EP
examined miR-19b levels in THP-1 macrophage/MPM-derived foam cells treated with 40 µM Dgn alone or together with 40 nM miR-19b mimic or 80 nM miR-19b
AC C
inhibitor for 24 h. As shown in Fig. 2A–2B, Dgn significantly decreased the levels of miR-19b in foam cells. Dgn-suppressed miR-19b levels were abrogated by miR-19b mimic and enhanced by miR-19b inhibitor, when compared with those treated with Dgn only. We further confirmed the functional role of miR-19b in the effects of Dgn on ABCA1 expression under the condition of the gain or loss of functions of miR-19b in THP-1 macrophage/MPM-derived foam cells (Fig. 2C–2D). We found that the effect of Dgn on ABCA1 expression was blocked by miR-19b mimic and enhanced by miR-19b inhibitor, suggesting that the endogenous miR-19b levels in Dgn-treated cells were decreased so as to facilitate ABCA1 expression. Taken together, these 10
ACCEPTED MANUSCRIPT findings suggested that Dgn regulates ABCA1 expression via inhibiting miR-19b level.
3.3 Dgn enhances macrophage cholesterol efflux
RI PT
Increased expression of ABCA1 is directly associated with an increase in the efflux of macrophage cholesterol. As ABCA1 was upregulated by Dgn, we next examined the effects of Dgn on apoAI-mediated (ABCA1-dependent) cholesterol efflux in THP-1 macrophage/MPM derived foam cells. As shown in Fig. 2E–2F, Dgn
SC
obviously increased cholesterol efflux from foam cells. The apoAI-mediated cholesterol efflux in response to Dgn treatment was impaired when foam cells were
M AN U
co-treated with miR-19b mimic, whereas Dgn-induced efflux was enhanced by the treatment with miR-19b inhibitor. These data indicated that Dgn promotes apoAI-mediated cholesterol efflux via ABCA1-dependent pathway by suppressing miR-19b level.
TE D
3.4 Dgn inhibits macrophage cholesterol accumulation
Cholesterol-laden macrophage foam cells are a central component of atherosclerotic lesions. Here, we performed HPLC to measure cellular cholesterol
EP
levels in THP-1 macrophage/MPM-derived foam cells in response to treatment with 40 µM Dgn alone or in combination with 40 nM miR-19b mimic or 80 nM miR-19b inhibitor. As anticipated, cellular levels of TC, FC and cholesterol ester (CE) in
AC C
Dgn-treated cells were significantly lower than those in the control cells (Table 1 and 2). Comparing with these in Dgn-treated cells, cellular cholesterol levels were increased when foam cells were treated with Dgn and miR-19b mimic, whereas cellular cholesterol levels were further decreased when foam cells were treated with Dgn and miR-19b inhibitor. The percentages of CE/TC were not significantly changed in response to any experimental manipulation. These findings demonstrated that Dgn reduces cellular cholesterol accumulation via suppressing miR-19b levels.
3.5 Dgn improves RCT and plasma lipid profile 11
ACCEPTED MANUSCRIPT ABCA1 is a key player in RCT and is critical in the generation of HDL. To determine the effects of Dgn-induced ABCA1 expression on RCT and plasma lipid profile, 3H-cholesterol tracer amounts in plasma, liver, and feces were quantified to evaluate cholesterol distribution along the RCT pathway with liquid scintillation
RI PT
counting, and the biliary cholesterol levels and the plasma lipid profile were determined by enzymatic methods. As shown in Fig. 3A–3D, the 3H-cholesterol counts in feces after macrophage injection, as expressed as a percentage of total 3
H-counts injected, were significantly increased in Dgn-treated mice compared with
SC
control mice. The counts trended to further increase in mice treated with Dgn together with antagomiR-19b. The fecal radioactivity was slightly increased, but not
M AN U
significantly different, in the Dgn-treated mice with co-treatment with agomiR-19b when compared with control mice. The same alterations were also observed in the biliary cholesterol levels after treatment. The 3H-cholesterol tracer amounts in plasma and liver did not significantly differ among all 4 groups. As expected, the efficiency of macrophage RCT tracked closely with the cholesterol mass in plasma lipoprotein
TE D
distribution and was consistent with HDL levels higher in Dgn-treated mice, the highest in mice treated with Dgn and antagomiR-19b, and was inverse association with the tendencies of LDL. The plasma HDL and LDL levels were not significantly
EP
different in mice treated with Dgn and agomiR-19b when compared with those in control mice (Table 3). Throughout the study, body weight of mice fed different diets was similar. These results clearly demonstrated that Dgn accelerates RCT and
AC C
improves plasma lipid profile likely via the ABCA1 pathway.
3.6 Dgn inhibits aortic atherosclerosis Finally, we further analyzed the atheroprotective effect of Dgn and the potential
mechanism underlying its atheroprotection in apoE KO mice. Consistent with the results from the ex vivo studies, the analysis of atherosclerotic lesions both in en face preparations of the whole aorta and in the aortic root revealed that atherosclerotic lesion areas were significantly reduced in Dgn-treated mice, and further reduced in mice treated with Dgn and antagomiR-19b, when compared with control mice (Fig. 12
ACCEPTED MANUSCRIPT 4A–4B). The assessment of collagen contents in aortic root sections of selected mice in each group showed that Dgn treatment alone or supplemented with antagomiR-19b distinctly increased collagen contents and stabilized aortic atherosclerotic lesions. Lipid deposition in the cross-sections of aortic root was also reduced in Dgn-treated
RI PT
mice or in mice treated with Dgn and antagomiR-19b. Supplement with agomiR-19b abolished the atheroprotective effects of Dgn, resulting in no significant differences in lesion sizes, collagen contents and lipid deposition when compared with those in control mice. Furthermore, we determined ABCA1 expression in tissue homogenate
SC
of aortic roots. Compared with control mice, ABCA1 expression in the aortic roots was significantly up-regulated in Dgn-treated mice or in mice treated with both Dgn
M AN U
and antagomiR-19b, but no significant difference was observed in Dgn-treated mice co-treated with agomiR-19b (Fig. 4C). These results indicated that Dgn inhibits aortic lipid deposition and atherogenesis through miR-19b suppression and subsequent
AC C
EP
TE D
ABCA1 upregulation.
13
ACCEPTED MANUSCRIPT 4. Discussion In this study, we investigated the atheroprotective effect and the molecular mechanisms underlying Dgn effects on cholesterol efflux from macrophage foam cells and atherosclerotic lesions in apoE KO mice. As illustrated in Fig 5, our results
RI PT
showed that Dgn up-regulates macrophage ABCA1 expression via suppressing miR-19b level and induces ABCA1-dependent cholesterol efflux from macrophage foam cells, resulting in the reduction in cholesterol accumulation in peripheral macrophages. Furthermore, Dgn effectively enhances in vivo macrophage RCT and
SC
remarkably attenuates aortic lipid deposition and atherosclerotic lesions in apoE KO mice.
M AN U
Dgn pronouncedly promotes peripheral macrophage ABCA1 expression and ABCA1-dependent cholesterol efflux. Temel et al found that Dgn treatment of wild-type and NPC1L1-knockout mice did not alter intestinal expression of ABCA1, ABCG5 and ABCG8 [12]. Uemura et al verified that Dgn suppressed LXRα transactivation
induced
by
a
synthetic
LXRa
agonist
T0901317
and
TE D
T0901317-induced binding of LXRα to LXRE in HepG2 cells, and inhibited the expression of SREBP-1c and lipogenic genes, leading to a reduction in hepatic lipogenesis and lipid accumulation. However, Uemura et al did not further determine
EP
the levels of ABCA1 in hepatocytes and peripheral macrophages under these conditions [2]. Our study showed that Dgn treatment did not affect macrophage expression of LXRα and RXRα, suggesting another mechanism involved in
AC C
Dgn-induced ABCA1 expression. In addition to classic transcriptional regulation of cholesterol metabolism (e.g. by
LXRα), the members of a class of noncoding RNAs termed microRNAs have recently been identified as potent post-transcriptional regulators of lipid metabolism genes involved in cholesterol homeostasis [37, 38]. ABCA1 has an uncommonly long 3’UTR of >3.3 kb, rendering it particularly susceptible to post-transcriptional regulation by microRNAs [39]. Our previous work has verified that miR-19b potently inhibits macrophage ABCA1 expression by binding to mRNA 3’UTR of ABCA1 [31]. Based on this, we evaluated the role of miR-19b in Dgn-induced ABCA1 expression. 14
ACCEPTED MANUSCRIPT Our data suggest that Dgn-induced ABCA1 expression is at least mainly, if not completely, dependent on miR-19b suppression. However, the pathway through which Dgn regulates miR-19b level needs further investigation. Dgn accelerates macrophage cholesterol efflux via ABCA1 pathway and the
RI PT
process of RCT in vivo. Accumulation of cholesterol-laden macrophages in the intima is a major hallmark in the early stage of atherosclerotic lesions [15, 19]. Cholesterol efflux from macrophages is the first, potentially the most important, step in macrophage RCT. Accordingly, ABCA1-dependent cholesterol efflux is a crucial
SC
event in the prevention of excessive cholesterol accumulation in macrophages of the arterial wall and their transformation into foam cells [16, 25, 40, 41]. In this study, our
M AN U
findings suggest a new mechanism underlying Dgn-accelerated RCT. When the cells were treated with Dgn alone or together with miR-19b inhibitor, cholesterol efflux was accelerated from macrophage foam cells via ABCA1 pathway, resulting in the suppression of cellular cholesterol accumulation. Subsequently, in vivo macrophage RCT was accelerated and blood lipid profile ameliorated. Other mechanisms have
TE D
been reportedly involved in Dgn-accelerated RCT. Dgn competitively inhibits intestinal absorption of dietary cholesterol because of its close chemical structural similarity with cholesterol [8, 9]. Dgn also induces hepatic cholesterol secretion,
EP
which requires the hepatobiliary cholesterol transporters ABCG5 and ABCG8 [11, 42]. Despite accelerated macrophage RCT, Dgn did not significantly decrease plasma TC level. This is likely because enhanced excretion of cholesterol from the liver is
AC C
counteracted by increased peripheral cholesterol transported to plasma and then to the liver.
Dgn inhibits aortic lipid deposition and atherosclerotic development. It has been
believed that the factors which modulate macrophage cholesterol efflux and RCT have major impacts on initiation, progression, and regression of atherosclerosis [16, 29]. Consistently, aortic lipid deposition and atherosclerotic lesion area were dramatically decreased in animals treated with Dgn alone or together with antagomiR-19b. Dgn obviously ameliorated the structure of atherosclerotic plaques by increasing collagen contents and decreasing necrotic core. Our findings further 15
ACCEPTED MANUSCRIPT demonstrated that atheroprotective effect of Dgn is intimately associated with its role in regulating ABCA1 expression in macrophages and lipid accumulation in the artery wall. Previous studies identified that Dgn treatment inhibits lipid peroxidation and production of free radicals in rats fed a high-cholesterol diet [6, 7, 43]. Dgn has a effect
against
atherosclerosis
by
regulating
serum
levels
of
RI PT
protective
proinflammatory mediators such as vascular cell adhesion molecule-1 (VCAM-1), C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) in apoE KO mice [1, 4]. In addition, Manivannan et al demonstrated that Dgn attenuates
SC
vascular calcification in rats with chronic renal failure by preventing the VSMC phenotype changes [44]. All these mechanisms contribute to the atheroprotective
M AN U
action of Dgn.
In the present study, we found that Dgn promotes macrophage ABCA1 expression by suppressing miR-19b level. The hypolipidemic effect of Dgn is dependent on ABCA1-mediated cholesterol efflux from macrophage foam cells and in vivo macrophage RCT. This beneficial effect is reflected by the elevated plasma HDL
TE D
and lowered LDL levels. Aortic lipid deposition and atherosclerotic lesion of apoE KO mice are obviously alleviated by Dgn treatment alone or together with antagomiR-19b. Our ongoing research in collaboration with clinical scientists is to
EP
evaluate the hypolipidemic and antiatherogenic actions of Dgn in the volunteers or even patients. The biochemical tests will be performed to show the alteration of plasma lipids, and the imaging examinations to observe the atherosclerotic lesions in
AC C
common carotid arteries and/or coronary arteries. The data from these investigations will provide direct insight into the hypolipidemic and antiatherogenic actions of Dgn in human, which will be instructive for clinicians. Collectively, our results reveal a novel mechanism by which Dgn ameliorates
macrophage cholesterol metabolism and suppresses the progression of atherosclerosis in vivo. Since Dgn produces potential hypolipidemic activity by enhancing cholesterol removal from macrophages to prevent atherosclerotic cardiovascular diseases, as a natural product, Dgn might be added to the foods and beverages or provide a guide to develop pharmaceutical agents. 16
ACCEPTED MANUSCRIPT Conflict of interest We do have no actual or potential conflict of interest.
Acknowledgments
RI PT
The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (81170278, 81370377, 81300224 and 81100560), the Hunan Provincial Natural Science Foundation of China (14JJ2091) and the Scientific Research Fund of Hunan Provincial Education Department
SC
(12C0339), China, and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, China (2008-244), the
AC C
EP
TE D
M AN U
construct program of the key discipline in Hunan Province, China.
17
ACCEPTED MANUSCRIPT References [1] Song Jia-Xi ML, Kou Jun-Ping, Yu Bo-Yang. Diosgenin reduces leukocytes adhesion and migration linked with inhibition of intercellular adhesion molecule-1 expression and NF-κB p65 activation in endothelial cells. Chinese Journal of Natural Medicines 2012; 10:142-9. [2] Uemura T, Goto T, Kang M-S, Mizoguchi N, Hirai S, Lee J-Y, et al. Diosgenin, the Main Aglycon of Fenugreek, Inhibits LXR{alpha} Activity in HepG2 Cells and Decreases Plasma and Hepatic
RI PT
Triglycerides in Obese Diabetic Mice. J. Nutr. 2011; 141:17-23. [3] Liu K, Zhao W, Gao X, Huang F, Kou J and Liu B. Diosgenin ameliorates palmitate-induced endothelial dysfunction and insulin resistance via blocking IKKbeta and IRS-1 pathways. Atherosclerosis 2012; 223:350-8.
[4] Park H-J, Byeon H-E, Koo H-J and Pyo S. Anti-atherosclerotic effects of diosgenin in ApoE-deficient mice. FASEB journal : official publication of the Federation of American Societies for
SC
Experimental Biology 2011; 25:980-2.
[5] Sangeetha MK, ShriShri Mal N, Atmaja K, Sali VK and Vasanthi HR. PPAR's and Diosgenin a chemico biological insight in NIDDM. Chemico-biological interactions 2013; 206:403-10.
M AN U
[6] Son IS, Kim JH, Sohn HY, Son KH, Kim JS and Kwon CS. Antioxidative and hypolipidemic effects of diosgenin, a steroidal saponin of yam (Dioscorea spp.), on high-cholesterol fed rats. Bioscience, biotechnology, and biochemistry 2007; 71:3063-71.
[7] Gong G, Qin Y, Huang W, Zhou S, Wu X, Yang X, et al. Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis. Chemico-biological interactions 2010; 184:366-75.
[8] Cayen MN and Dvornik D. Effect of diosgenin on lipid metabolism in rats. Journal of lipid research 1979; 20:162-74.
TE D
[9] LAGUNA J, GOMEZ-PUYOU A, PENA A and GUZMAN-GARCIA J. Effect of diosgenin on cholesterol metabolism. J Atheroscler Res 1962; 2:459-70. [10] Pan CH, Tsai CH, Liu FC, Fan MJ, Sheu MJ, Hsieh WT, et al. Influence of different particle processing on hypocholesterolemic and antiatherogenic activities of yam (Dioscorea pseudojaponica) in cholesterol-fed rabbit model. Journal of the science of food and agriculture 2013; 93:1278-83.
EP
[11] Kamisako T and Ogawa H. Regulation of biliary cholesterol secretion is associated with abcg5 and abcg8 expressions in the rats: effects of diosgenin and ethinyl estradiol. Hepatology research : the official journal of the Japan Society of Hepatology 2003; 26:348-52.
AC C
[12] Temel RE, Brown JM, Ma Y, Tang W, Rudel LL, Ioannou YA, et al. Diosgenin stimulation of fecal cholesterol excretion in mice is not NPC1L1 dependent. Journal of lipid research 2009; 50:915-23.
[13] Diaz Zagoya J, Laguna J and Guzman-Garcia J. Studies on the regulation of cholesterol metabolism by the use of the structural analogue, diosgenin. Biochem Pharmacol 1971; 20:3473-80. [14] Moore KJ and Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011; 145:341-55. [15] Moore KJ, Sheedy FJ and Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nature reviews. Immunology 2013; 13:709-21. [16] Pennings M, Meurs I, Ye D, Out R, Hoekstra M, Van Berkel TJ, et al. Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development. FEBS letters 2006; 580:5588-96. [17] Zhao GJ, Tang SL, Lv YC, Ouyang XP, He PP, Yao F, et al. NF-kappaB suppresses the expression
18
ACCEPTED MANUSCRIPT of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice. International journal of cardiology 2014; 171:e93-5. [18] Zhang M, Wu JF, Chen WJ, Tang SL, Mo ZC, Tang YY, et al. MicroRNA-27a/b regulates cellular cholesterol efflux, influx and esterification/hydrolysis in THP-1 macrophages. Atherosclerosis 2014; 234:54-64. [19] Yu XH, Fu YC, Zhang DW, Yin K and Tang CK. Foam cells in atherosclerosis. Clinica chimica acta; international journal of clinical chemistry 2013; 424:245-52.
RI PT
[20] Lu Q, Tang SL, Liu XY, Zhao GJ, Ouyang XP, Lv YC, et al. Tertiary-Butylhydroquinone Upregulates Expression of ATP-Binding Cassette Transporter A1 via Nuclear Factor E2-Related Factor 2/Heme Oxygenase-1 Signaling in THP-1 Macrophage-Derived Foam Cells. Circ J 2013; 77:2399-408. [21] Liu XY, Lu Q, Ouyang XP, Tang SL, Zhao GJ, Lv YC, et al. Apelin-13 increases expression of ATP-binding cassette transporter A1 via activating protein kinase C alpha signaling in THP-1
SC
macrophage-derived foam cells. Atherosclerosis 2013; 226:398-407.
[22] Mo Z, Xiao J, Liu X, Hu Y, Li X, Yi G, et al. AOPPs inhibits cholesterol efflux by down-regulating ABCA1 expression in a JAK/STAT signaling pathway-dependent manner. J Atheroscler Thromb 2011; 18:796-807.
M AN U
[23] Liu X, Xiao J, Mo Z, Yin K, Jiang J, Cui L, et al. Contribution of D4-F to ABCA1 expression and cholesterol efflux in THP-1 macrophage-derived foam cells. Journal of cardiovascular pharmacology 2010; 56:309-19.
[24] Zhao Y, Van Berkel TJ and Van Eck M. Relative roles of various efflux pathways in net cholesterol efflux from macrophage foam cells in atherosclerotic lesions. Curr Opin Lipidol 2010; 21:441-53.
[25] Westerterp M, Bochem AE, Yvan-Charvet L, Murphy AJ, Wang N and Tall AR. ATP-Binding
TE D
Cassette Transporters, Atherosclerosis, and Inflammation. Circ. Res. 2014; 114:157-70. [26] van Capelleveen JC, Brewer HB, Kastelein JJP and Hovingh GK. Novel Therapies Focused on the High-Density Lipoprotein Particle. Circ. Res. 2014; 114:193-204. [27] Rye K-A and Barter PJ. Cardioprotective functions of HDL. J. Lipid Res. 2014; 55:168-79. [28] Rosenson RS, Brewer HB, Jr, Davidson WS, Fayad ZA, Fuster V, Goldstein J, et al. Cholesterol
EP
Efflux and Atheroprotection: Advancing the Concept of Reverse Cholesterol Transport. Circulation 2012; 125:1905-19.
[29] Oram JF and Vaughan AM. ATP-Binding Cassette Cholesterol Transporters and Cardiovascular
AC C
Disease. Circ. Res. 2006; 99:1031-43. [30] Oram JF and Heinecke JW. ATP-Binding Cassette Transporter A1: A Cell Cholesterol Exporter That Protects Against Cardiovascular Disease. Physiol Rev 2005; 85:1343-72. [31] Lv YC, Tang YY, Peng J, Zhao GJ, Yang J, Yao F, et al. MicroRNA-19b promotes macrophage cholesterol accumulation and aortic atherosclerosis by targeting ATP-binding cassette transporter A1. Atherosclerosis 2014; 236:215-26. [32] Tian GP, Chen WJ, He PP, Tang SL, Zhao GJ, Lv YC, et al. MicroRNA-467b targets LPL gene in RAW 264.7 macrophages and attenuates lipid accumulation and proinflammatory cytokine secretion. Biochimie 2012; 94:2749-55. [33] Chen C, Cheng P, Xie H, Zhou HD, Wu XP, Liao EY, et al. MiR-503 regulates osteoclastogenesis via targeting RANK. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2014; 29:338-47. [34] Wang D, Xia M, Yan X, Li D, Wang L, Xu Y, et al. Gut Microbiota Metabolism of Anthocyanin
19
ACCEPTED MANUSCRIPT Promotes Reverse Cholesterol Transport in Mice Via Repressing miRNA-10b. Circ. Res. 2012; 111:967-81. [35] Zhao GJ, Tang SL, Lv YC, Ouyang XP, He PP, Yao F, et al. Antagonism of betulinic acid on LPS-mediated inhibition of ABCA1 and cholesterol efflux through inhibiting nuclear factor-kappaB signaling pathway and miR-33 expression. PloS one 2013; 8:e74782. [36] Schmitz G and Langmann T. Transcriptional regulatory networks in lipid metabolism control ABCA1 expression. Biochimica et biophysica acta 2005; 1735:1-19.
RI PT
[37] Lv YC, Yin K, Fu YC, Zhang DW, Chen WJ and Tang CK. Posttranscriptional Regulation of ATP-Binding Cassette Transporter A1 in Lipid Metabolism. DNA and cell biology 2013; 32:348-58.
[38] Fernandez-Hernand... C, Suarez Y, Rayner K and Moore K. MicroRNAs in lipid metabolism. Curr Opin Lipidol 2011; 22:86-92. Function. Circ. Res. 2014; 114:183-92.
SC
[39] Rayner KJ and Moore KJ. MicroRNA Control of High-Density Lipoprotein Metabolism and [40] De Meyer I, Martinet W and De Meyer G. Therapeutic strategies to deplete macrophages in atherosclerotic plaques. Br J Clin Pharmacol 2012; 74:246-63.
[41] Ouimet M and Marcel YL. Regulation of Lipid Droplet Cholesterol Efflux From Macrophage
M AN U
Foam Cells. Arterioscler Thromb Vasc Biol 2012; 32:575-81.
[42] Kosters A, Kunne C, Looije N, Patel SB, Oude Elferink RP and Groen AK. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice. Journal of lipid research 2006; 47:1959-66. [43] Jayachandran KS, Vasanthi HR and Rajamanickam GV. Antilipoperoxidative and membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction. Molecular and cellular biochemistry 2009; 327:203-10.
[44] Manivannan J, Barathkumar TR, Sivasubramanian J, Arunagiri P, Raja B and Balamurugan E.
TE D
Diosgenin attenuates vascular calcification in chronic renal failure rats. Molecular and cellular
AC C
EP
biochemistry 2013; 378:9-18.
20
ACCEPTED MANUSCRIPT Figure Legends
Fig. 1. The effects of Dgn on the expression of ABCA1 and LXRα in macrophage foam cells. A, B, C, and D: ABCA1 protein levels were determined by western
RI PT
blotting in THP-1 macrophage/MPM-derived foam cells treated with Dgn at different concentrations (0, 10, 20, 40, 80 µM) for 24 h or with 40 µM Dgn at the indicated periods (0, 6, 12, 24, 48 h). E, F, G and H: the protein levels of LXRα and RXRα were determined by western blotting in THP-1 macrophage/MPM-derived foam cells
SC
treated with 40 µM Dgn for 24 h. All results are expressed as mean ± S.D. from three independent experiments in duplicate. * P<0.05 vs. control group. Dgn, diosgenin;
M AN U
LXRα, liver X receptor α; Con, control; Et, ethanol as a vehicle.
Fig. 2. The involvement of miR-19b in the Dgn-mediated increase of ABCA1 expression and cholesterol efflux in macrophage foam cells. A, B, C, and D: miR-19b levels and ABCA1 expression were measured by RT-qPCR or western
TE D
blotting, respectively, in THP-1 macrophage/MPM-derived foam cells treated with 40 µM Dgn alone or together with 80 nM miR-19b inhibitor/40 nM miR-19b mimic for 24 h. E and F: cellular cholesterol efflux was analyzed by liquid scintillation counting
EP
assays as described in Methods. All results are expressed as mean ± S.D. from three independent experiments in duplicate. * P<0.05 vs. control group. Dgn, diosgenin;
AC C
Con, control; Inh, miR-19b inhibitor; Mim, miR-19b mimic.
Fig. 3. The effect of Dgn on the RCT of cholesterol–laden MPMs intraperitoneally injected into apoE KO mice and biliary cholesterol levels. A, B, and C: 3H–cholesterol-labeled and acLDL–loaded MPMs were intraperitoneally injected into apoE KO mice fed with Western diet for 8 wk. After cell injection, the samples of blood, liver and feces were collected for the analysis of in vivo macrophage RCT at 48 h. D: Biliary cholesterol levels in apoE KO mice after a 6 h fast were determined by enzymatic colorimetry. Values are mean ± S.D. (n=15 mice per group). * P<0.05 vs. control group. Dgn, diosgenin; Con, control; Ago, 21
ACCEPTED MANUSCRIPT agomir-19b; Ant, antagomiR-19b.
Fig. 4. The effect of Dgn on aortic lesion area and ABCA1 expression in apoE KO mice. A: Representative images and the quantification of atherosclerotic lesion area in
RI PT
the en face analysis of the whole aorta with Sudan IV staining in apoE KO mice fed with Western diet for 8 wk. Values are mean ± SEM (n=12 mice per group). B: Representative microscopic images and quantification of atherosclerotic plaque area or collagen contents in cross-sections of proximal aorta with the staining of
SC
hematoxylin-eosin, Masson’s trichrome and Oil-red O in apoE KO mice fed with Western diet for 8 wk. Original magnification 40×. Values are mean ± SEM (n=15
M AN U
mice per group). C: ABCA1 levels were determined in the homogenate of aortic arch using western blotting. Values are mean ± S.D. (n=3 mice per group). * P<0.05 vs. control group. Dgn, diosgenin; Con, control; Ago, agomir-19b; Ant, antagomiR-19b; HE, hematoxylin-eosin; MT, Masson’s trichrome.
TE D
Fig. 5. Schematic presentation showing the effects of Dgn on miR-19b level, ABCA1-dependent cholesterol efflux in macrophage foam cell and aortic atherosclerosis development in rodents. Dgn treatment up-regulates macrophage expression
by
suppressing
miR-19b
level,
which
promotes
EP
ABCA1
ABCA1-dependent cholesterol efflux and in vivo macrophage RCT. Consequently, Dgn efficiently ameliorates blood lipid profile and cholesterol homeostasis of
AC C
peripheral macrophage, which potently inhibits aortic lipid deposition and atherosclerosis progression in rodents. Dgn, diosgenin; LD, lipid droplet; FC, free cholesterol; RISC, RNA-induced silencing complex; ↑, promotion; ↓, inhibition.
22
ACCEPTED MANUSCRIPT Table 1. Effects of Dgn on cholesterol contents in THP-1 macrophage-derived foam cells after apoAI–mediated cholesterol efflux FC(mg/g)
CE(mg/g)
CE/TC(%)
Con
347.78±29.14
155.51±15.66
192.27±17.17
55.28
Dgn
316.94±25.48*
144.12±14.83*
172.82±16.40*
54.53
Dgn+Mim
356.33±35.18
157.59±16.24
198.74±18.65
55.77
Dgn+Inh
292.39±23.56*
133.79±13.45*
158.60±14.96*
54.24
RI PT
TC(mg/g)
SC
THP-1 macrophage-derived foam cells were treated with 40 µM Dgn alone or in combination with 40 nM miR-19b mimic or 80 nM miR-19b inhibitor for 24 h. Then,
M AN U
HPLC was performed to determine the levels of cellular total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC). The results were expressed as mean ±
Table 2.
TE D
S.D. of three independent experiments in duplicate. * P<0.05 vs. control group.
Effects of Dgn on cholesterol contents in MPM-derived foam cells after apoAI–mediated cholesterol efflux
Con
CE (mg/g)
CE/TC (%)
301.54±27.63
149.79±14.87
151.75±16.42
50.66
271.05±22.52*
136.78±15.13*
134.27±14.40*
49.54
AC C
Dgn
FC (mg/g)
EP
TC (mg/g)
Dgn+Mim
310.63±29.75
153.32±16.46
157.31±17.73
50.64
Dgn+Inh
250.90±20.38*
127.34±12.93*
123.56±13.69*
49.30
MPM-derived foam cells were treated with 40 µM Dgn alone or in combination with 40 nM miR-19b mimic or 80 nM miR-19b inhibitor for 24 h. Then, HPLC was performed to determine the levels of cellular total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC). The results were expressed as mean ± S.D. of three independent experiments in duplicate. * P<0.05 vs. control group.
1
ACCEPTED MANUSCRIPT Table 3. Body weight and plasma lipid profile in apoE KO mice BW (g)
TG (mmol/l)
TC (mmol/l)
HDL-C (mmol/l)
LDL-C (mmol/l)
30.46±4.04 1.08±0.14
20.56±1.42
2.13±0.21
18.27±2.25
Dgn
29.10±3.73 1.05±0.13
19.39±1.19
2.97±0.26*
15.78±1.65*
Dgn+Ago
30.05±4.17 1.07±0.11
19.70±2.48
2.21±0.19
17.30±2.13
Dgn+Ant
28.89±3.62 1.03±0.10
18.89±1.71
3.21±0.24*
14.94±2.56*
RI PT
Con
SC
Plasma samples from different experimental groups were measured by the enzymatic method as described in Methods. The data were expressed as mean ± S.D. (n=15 mice
AC C
EP
TE D
M AN U
per group). *P < 0.05 vs. control group.
2
ACCEPTED MANUSCRIPT THP-1 derived foam cells
MPM derived foam cells
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 1.
1
ACCEPTED MANUSCRIPT Fig. 2. MPM derived foam cells
AC C
EP
TE D
M AN U
SC
RI PT
THP-1 derived foam cells
2
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 3.
3
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 4.
4
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 5.
5
ACCEPTED MANUSCRIPT Highlights
Dgn increases macrophage ABCA1 expression
③
Dgn enhances macrophage cholesterol efflux
Dgn improves RCT and plasma lipid profile Dgn inhibits aortic atherosclerosis
AC C
EP
TE D
M AN U
⑥
SC
Dgn inhibits macrophage cholesterol accumulation
RI PT
Dgn reduces macrophage miR-19b level
ACCEPTED MANUSCRIPT
AUTHOR DECLARATION
We wish to confirm that there are no known conflicts of interest associated with
RI PT
this publication and there has been no significant financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but
SC
are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.
M AN U
We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.
TE D
We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animal has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the
EP
manuscript.
We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He
AC C
is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected]
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT The full gel images in Figure 1 A: ABCA1
B: ABCA1
B: β-actin
SC
RI PT
A: β-actin
M AN U
C: ABCA1
D: ABCA1
F: LXRα
AC C
EP
E: LXRα
TE D
C: β-actin
D: β-actin
E: β-actin
F: β-actin
G: RXRα H: RXRα
G: β-actin H: β-actin 1