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Panax notoginseng saponins decrease cholesterol ester via up-regulating ATP-binding cassette transporter A1 in foam cells
Journal of Ethnopharmacology 132 (2010) 297–302
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Panax notoginseng saponins decrease cholesterol ester via up-regulating ATP-binding cassette transporter A1 in foam cells Yi Jia a , Zhuo-Ying Li a , Hai-Gang Zhang a , Hai-Bo Li b , Ya Liu a , Xiao-Hui Li a,∗ a Institute of Materia Medica and Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, People’s Republic of China b Department of Medicinal Chemistry, College of Pharmacy, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, People’s Republic of China
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Article history: Received 9 June 2010 Received in revised form 12 August 2010 Accepted 13 August 2010 Available online 19 August 2010 Keywords: Panax notoginseng saponins ATP-binding cassette transporter A1 Foam cell Macrophage Cholesterol ester
a b s t r a c t Aim: Accumulating evidence has indicated that Panax notoginseng saponins (PNS), the major ingredients in Panax notoginseng (Burk.) F.H. Chen which could be found widely in Asia, can attenuate atherogenesis in vivo. This study was designed to examine the relationship of PNS with cholesterol ester in foam cells sourced from macrophages and the effect of PNS on the expression of ATP-binding cassette transporter A1 (ABCA1). Materials and methods: Foam cells sourced from macrophages were cultured with PNS. The content of cholesterol ester in foam cells was analyzed and expressions of ABCA1 and liver X receptor ␣ (LXR␣) in foam cells were measured by real-time PCR and western blotting methods. Results: The results showed that PNS could significantly decrease the level of cholesterol ester in foam cells at middle and high dosages. The real-time PCR and western blotting assays indicated that the expression of ABCA1 was up-regulated by PNS in a dose-dependent manner. Analysis based on these results showed that the cholesterol ester level was negatively correlated with ABCA1 expression. Conclusions: As a result, we conclude that by up-regulating the expression of ABCA1, PNS could lower the cholesterol ester level, which resulted in the attenuation of the foam cell formation. This bioactivity might be associated with the special chemical structures of PNS that are similar to the natural agonist of LXR␣. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Atherosclerosis (AS) is a fundamental pathological change that leads to serious cardiovascular diseases, such as stroke, coronary artery disease and hypertension. Although the pathogenesis of AS is still unclear, it is generally accepted that macrophage foam cells are the characteristic feature of atherosclerotic plaques (Webb and Moore, 2007). During foam cell formation, modified lipoproteins enter cells by receptor-mediated uptake and excess neutral lipids are stored as lipid droplets, creating the typical foamy appearance. The formation of foam cells is considered to be the initiation of AS and can influence the progression and destination of pathological changes. Oxidized forms of low density lipoproteins (LDL) are the modified lipoproteins usually considered to be causative during foam cell formation (Hiroyuki, 2003; Liu et al., 2008), illustrating
Abbreviations: AS, atherosclerosis; PNS, Panax notoginseng saponins; ABCA1, ATP-binding cassette transporter A1; LXR␣, liver X receptor ␣. ∗ Corresponding author. Tel.: +86 023 68752318; fax: +86 023 68753397. E-mail address: [email protected] (X.-H. Li). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.08.033
a model for macrophage foam cell formation with predominantly lipid droplets. ATP-binding cassette transporter A1 (ABCA1) is the critical channel of cholesterol efflux in macrophages and other tissues (Santamarina-Fojo et al., 2000; Schmitz and Langmann, 2001). ABCA1 can reduce the redundant lipids in cells, which protects against the formation of foam cells (Wang et al., 2007). The mutation of ABCA1 in humans causes a defect in cellular cholesterol efflux and deposition of lipid in tissues, also called Tangier disease (Mott et al., 2000). Notoginseng, the root of Panax notoginseng (Burk.) F.H. Chen, has been used as a health product and natural remedy in traditional medicine for cardiovascular diseases for more than 1000 years in Asia, including China, Japan, Korea, et al. Panax notoginseng saponins (PNS) are the major effective ingredients extracted from Panax notoginseng. Various studies on PNS showed that they exhibited significant anti-atherogenic effects, including the abilities to limit the proliferation of vascular smooth muscle cells (Wang and Hu, 2006), reduce hyperlipidemia (Cicero et al., 2003), protect against artery injury (Chen et al., 2004), regulate blood lipid profiles and control anti-inflammatory capability (Zhang et al., 2008). As the typical traditional Chinese medicines with cardiovascular
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beneficial actions, it is necessary to evaluate their pharmacological profiles in vitro. Several previous studies have elucidated the effects of PNS on the formation of foam cells, however, little empirical evidence has been presented on the relationship between PNS and ABCA1, that plays an essential role in cellular cholesterol efflux and helps to prevent macrophages from becoming foam cells (Brunham et al., 2009). In the current study, we examined cholesterol ester content in foam cells and the expression of ABCA1 to investigate the potentially mechanism underlying the anti-atherogenic activities of PNS. 2. Materials and methods 2.1. Cell culture Macrophages (NR8383) were cultured in six well cell culture clusters (Costar, Corning Incorporated, NY, USA) and adjusted to 105 /mL. The cells were cultured in phenol red-free RPMI 1640 medium (GIBCO, Invitrogen Corporation, Grand Island, NY, USA), containing 10% (v/v) fetal calf serum (GIBCO), 100 U/mL of penicillin and 100 g/mL of streptomycin, and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 for 24 h. After incubation with oxidized low density lipoprotein (ox-LDL, 100 g/mL, purchased from Institute of Basic Medicine, Peking Union Medical College) for 24 h, cells were treated with ox-LDL free medium supplemented with different concentrations of PNS (0 (as model group), 20, 40, 80 mg/L) (Yunnan Phytopharmaceutical Co. LTD., Yunnan, China) and CD36 blocking antibody (sc-5522p, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) for another 24 h. Control cells were incubated over the same time period in cell culture media without PNS or ox-LDL. The potential toxic effect on cell viability by the treatments of ox-LDL and PNS was examined by the MTT method in 96 well cell culture clusters (Costar). The number of replicates per experiment is 5 (n = 5). After administration, all cells were rinsed with heparin in order to remove lipoproteins attached to the cell surface. 2.2. Analysis of cellular cholesterol ester content Lipid-laden cells (lipid droplet positive cells) were stained with oil red O (Yang et al., 2004), and observed under an optical microscope (Olympus BX51, Olympus, Tokyo, Japan). Foam cells, which concentration was about 105 /mL, were collected after treatment with trypsin (0.25%, 5 min). Then, cells were washed three times with PBS. Each sample was sonicated for 3 min using the microtip of a sonicator. Samples (usually 100 L) were transferred into tubes and extracted with a chloroform/methanol (2:1; v/v) mixture. The chloroform phase was separated, dried and re-suspended in assay buffers supplied with the commercially available test kits for total cholesterol (TC) and free cholesterol (FC) content measurement (Rongshen Inc., Shanghai, China). The sediments were collected for the measurement of protein content. All samples were stored at −70 ◦ C for further experimentation. The method of TC test kits is CHOP-PAP. Samples were treated with cholesterol esterase first, in order to change cholesterol ester (CE) into cholesterol. Then oxidized the total cholesterol in samples by cholesterol oxidase. The oxidation reaction released H2 O2 , and the concentration of H2 O2 is in direct proportion to the concentration of cholesterol. The concentration of H2 O2 was measured by a peroxidase and 4-amino antipyrine method, then the OD values in 500 nm of the products were tested. The method of FC test kits is the same as TC test kits except the first step of reaction with cholesterol esterase. CE contents were then calculated (CE = TC − FC). The protein amount was determined using protein assay kit (Bio-Rad Lab., Hercules, CA, USA).
2.3. RNA extraction and real-time PCR Total RNA was isolated from cells using Tripure reagents (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. RNA samples were dissolved in nuclease-free water and stored at −70 ◦ C for real-time PCR after DNA was removed by DNase I (Takara, Shiga, Japan) treatment for 30 min at 37 ◦ C. Total RNA (1 g) was reversely transcribed using AMV reverse transcriptase (Promega, Madison, WI, USA) at 42 ◦ C for 1 h. PCR primers used were designed by Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) based on the cDNA sequences of ABCA1 and LXR␣. (forward: 5 -GCT CCT GCT GAA ATA CCG ACA A-3 and reverse: 5 -AGG GAC GAT TCC ACA TCT TTC TTG-3 , with an amplified product of 198 bp for ABCA1; forward: 5 -GAT GTT CCC ACG GAT GCT AAT G-3 and reverse: 5 -CAG GAA TGT TTG CCC TCC TCA-3 , with an amplified product of 232 bp for LXR␣) Rat -actin (forward: 5 -TGT TGT CCC TGT ATG CCT CT-3 ; reverse: 5 -CTC TTT AAT GTC ACG CAC GAT-3 , with an amplified product of 225 bp) was used as a control. Real-time PCR reactions were performed using iCycler iQ (Bio-Rad) and Quanti Tect SYBR Green PCR Master Mix (MJ Research, Waltham, MA, USA) under the following conditions: 5 min at 95 ◦ C, 40 cycles at 95 ◦ C for 10 s, 58 ◦ C for 15 s, 72 ◦ C for 20 s, and 82.5 ◦ C for 5 s. After amplification, a melting curve analysis was performed by collecting fluorescence data while increasing the temperature from 72 to 99 ◦ C over 135 s. The Ct (cycle threshold) values were normalized to the expression levels of -actin. 2.4. Protein extraction and western blotting Foam cells (collected by scraping) were pooled and collected by centrifugation. Cells were washed with ice-cold PBS, lysed with the protein extraction reagent (Pierce Labs, Rockford, IL, USA), and heated to 100 ◦ C for 5 min after adding loading buffer. The samples were stored at −70 ◦ C until further analysis. Protein concentration was measured using a modified BCA protein assay (Pierce Lab). An equal amount of total protein (10 g/lane) from each sample was used for immunoblot analysis. The proteins were resolved using SDS-PAGE (15%). Proteins were transferred to polyvinyl difluoride membranes (Millipore) using a semidry transfer system (Bio-Rad Lab.). The membranes were blocked with 5% nonfat milk (BioRad Lab.) for 1 h at room temperature. Then, the membranes were incubated with mouse monoclonal anti-ABCA1 (1:500 dilution) (ab-18180, Abcam Inc., Abcam, MA, USA) and goat polyclonal antibody against LXR␣ (sc-1202, Santa Cruz, 1:500) at 4 ◦ C overnight. The membranes were then washed three times with PBST (phosphate buffered saline with 0.05% Tween 20), and incubated with species-specific horseradish peroxidase (HRP)-coupled secondary antibodies (1:2000 dilution) for 1 h at room temperature. The membrane was re-probed with HRP-conjugated monoclonal mouse anti--actin (1:1000 dilution) (KC-5A08, Kangchen Inc., Kangchen, Shanghai, China) to ensure equal loading. The immune complex was visualized with a chemiluminescence method (Pierce Lab.) using a BIO-RAD image system (Bio-Rad, XRS). 2.5. Statistical analysis All data were expressed as the mean ± SD. Statistical analysis of all data was accomplished using SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). Comparisons between groups were made using one-way ANOVA analysis with post hoc least significant difference (LSD) test. We performed Pearson correlation analysis to examine the relationship between cholesterol ester content and ABCA1. Values of p < 0.05 were considered to be statistically significant.
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Fig. 1. Cellular cholesterol ester aggregation was attenuated by PNS in foam cells (n = 5). a, control group; b, model group; c, PNS 20 mg/ml group; d, PNS 40 mg/ml group; e, PNS 80mg/ml group. *p < 0.05 versus model group.
3. Results
3.3. ABCA1 mRNA and protein expression
3.1. Cell viability measurement
The results of real-time PCR showed that ABCA1 mRNA level was up-regulated in the model group versus the control group. Compared with the model group, dose-dependent changes of ABCA1 mRNA were demonstrated. In comparison to the model group, treatment with PNS at doses of 40 and 80 mg/L significantly increased the expression of ABCA1 mRNA by 39.79% and 47.72%, respectively (Fig. 2A). Simultaneously, treatment with PNS (40 and 80 mg/L) could also increase the ABCA1 protein level remarkably (Fig. 2C and D).
Cell viability was measured by the MTT method after 24 h culture with ox-LDL and PNS. The data showed that there was no significant difference between the control and administered groups (Fig. 1A).
3.2. Analysis of cholesterol ester content For direct-viewing of the effects of PNS on cellular cholesterol content, foam cells stained by red oil O were observed by light microscopy. Only a few lipid aggregates could be found in the control group, while lipid aggregation was abundant in the cellular plasma in the model group. Compared with that in the control group, the size of cells was significantly larger in the model group. Treatment with PNS (40 and 80 mg/L) caused a visible decrease of lipid levels in cells compared to the model group (Fig. 1B). Compared with that in model group, the administration of PNS (40 mg/L) decreased the CE content by 30.47%, and PNS (80 mg/L) decreased the CE content by 40.23% (Fig. 1C).
3.4. LXR˛ mRNA and protein expression Compared with that in the control group, the LXR␣ mRNA level was increased in the model group. Administration of PNS (40 and 80 mg/L) could significantly up-regulate the level of LXR␣ mRNA by 41.46% and 55.00%, respectively, versus the model group (Fig. 2B). ox-LDL treatment caused an increase in the expression of LXR␣ protein in the model group compared with that in the control group. Treatment with PNS (40 and 80 mg/L) could significantly increase LXR␣ protein expression compared to the model group (Fig. 2C and D).
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Fig. 2. Regulation of ABCA1 and LXR␣ in foam cells by PNS (real-time PCR and western blot methods) (n = 5). *p < 0.05 versus model group.
3.5. Correlation analysis of CE content and ABCA1 To confirm whether the cellular aggregation of cholesterol ester is retarded by PNS via up-regulating the expression of ABCA1, the linear correlation of cholesterol ester content and ABCA1 was examined. As shown in Fig. 3, the expressions of ABCA1 mRNA and protein were negatively correlated with cellular cholesterol ester content (ABCA1 mRNA: R = −0.948, p < 0.01; ABCA1 protein: R = −0.932, p < 0.01). 4. Discussion Panax notoginseng saponins (PNS) are the effective ingredients extracted from Panax notoginseng (Burk.) F.H. Chen, which has been used to prevent cardiovascular diseases and enhance wound
Fig. 3. Linear correlation of the expression of ABCA1 and the cholesterol ester content.
healing for more than 1000 years in Asia. PNS are the mixture of more than 20 dammarane-type saponins, including ginsenosides Rg1, Rg2, Rb1, Rb2, Rb3 and Rc, and notoginsenosides R1, R2, R3, R4 and R6. Among the ingredients, Panax notoginsenoside Rb1 (about 30%), and ginsenosides Rg1 (about 30%), Rd and Re are considered to be the principal active constituents. All of the active constituents are derivatives of 20(S)-protopanaxadiol, 20(S)protopanaxpanaxatriol or dammarane-type triterpene. Although PNS injection (commercial product: Xuesaitong) has been used as an anti-thrombosis treatment during the rehabilitation of stroke patients in Chinese clinical practice, the traditional utilization of PNS is mainly focused on cardiovascular protection via oral administration. Studies on PNS have demonstrated their anti-atherogenic effects, while the exact mechanism underlying their functions is still unclear (Cicero et al., 2003; Chen et al., 2004). Recent study of us has demonstrated that PNS could attenuate atherosclerosis in rats via their regulation of the blood lipid profile and their anti-inflammatory roles (Zhang et al., 2008). In addition to the aforementioned results, in vitro study is also one requisite for the complete elucidation of PNS pharmacological actions. There are several well-recognized in vitro models in the study of atherosclerosis (AS), including formation of macrophages foam cells, impairment of endotheliocyte and proliferation of vascular smooth cell. Among them, formation of macrophage foam cells is the most classic one because of its importance in the initiation, processing and termination of AS. Since the content of cholesterol ester (CE) can function as a measure of lipid quantity in foam cells (Pomerantz and Hajjar, 1990), we first sought to examine the effects of PNS on cholesterol ester content in macrophage foam cells. To determine the potential cytotoxicity of PNS, a cell viability test via MTT method was performed first to examine the possible membrane integrity change caused by saponins due to their physicochemical properties. The result revealed that the cell viability was not affected by treatments with ox-LDL or PNS. In addition, adding PNS into cell culture media after lipoprotein incubation eliminated other factors such as uptake of lipid that could have affected CE content in foam cells.
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Therefore, it was clear that cells utilized stored lipids when macrophages were lipid-loaded by incubating with ox-LDL and that lipid content was reduced by different doses of PNS administration. Treatments with PNS at doses of 40 and 80 mg/L caused a 30% and 40% (p < 0.05) decrease in cholesterol ester content, respectively. This indicated that PNS could considerably decrease cellular cholesterol ester levels, through which the formation of foam cells was inhibited. The results highlight the effect of PNS on the formation of foam cells, which is helpful for understanding its pharmacological actions against AS. In addition, this result also suggests that the decreased cellular cholesterol ester content after incubation with PNS is the result of increased intracellular lipid catabolism and/or efflux. As macrophage foam cells lack pathways for cholesterol degradation, excess lipids can only be removed from cells via reverse cholesterol transport pathways. Next, we investigated the relationship between PNS and the critical gate-keeper in the process of cholesterol efflux, ABCA1. It is well known that the removal of excess cholesterol from cells by HDL or its apolipoproteins is important for maintaining cellular cholesterol homeostasis. Excess cholesterol is removed from macrophage foam cells as oxy-sterols by four pathways, passive diffusion, CD36, ABCA1 and apolipoprotein E (Huang et al., 2001). Among them, the most important one is ABCA1 pathway (Yancey et al., 2003). ABCA1 is a member of a large family of ATP-binding cassette transporters that have common structural motifs and use ATP as an energy source to transport a variety of substrates, including ions, lipids, and cytotoxins (Dean et al., 2001). Among patients with Tangier disease, a disease resulting from the homozygote mutation of the ABCA1 gene, cardiovascular disease occurs at a rate about sixfold higher than among healthy adults. This is analogous to findings in ABCA1−/− mice (Singaraja et al., 2001). Additional study showed that over-expression of ABCA1 could increase the efflux of cellular cholesterol and attenuate the formation of AS lesions (Joyce et al., 2002). These studies provided valid evidence of the positive effects of ABCA1 on AS formation. The expression of ABCA1 was regulated by physiological factors such as specified cholesterol, cAMP and interferon. Whether cellular cholesterol ester content was decreased by PNS via regulating the expression of ABCA1 had not yet been discovered. In order to exclude the influence of CD36 in this study, antiCD36 antibody was added. Anti-CD36 not only prevents the cellular cholesterol efflux via CD36, but also inhibits the uptake of cholesterol after ox-LDL treatment. The test results showed that, compared with that in the control group, the expression of ABCA1 in the model group was increased significantly. The up-regulation of ABCA1 expression, both mRNA and protein, caused increased efflux of cellular cholesterol ester in foam cells, which could be considered as a defensive response of the lipid-loaded cell. However, the presence of abundant cholesterol ester also indicated that the defensive response in the model group was not powerful and effective enough to remove cholesterol ester from foam cells. We examined the expression of ABCA1 in PNS administered foam cells and observed the dose-dependent changes of ABCA1 expression compared with that in the model group. No noticeable differences of ABCA1 expression and cholesterol ester content between PNS groups and the model group, however, could be found, until the PNS dose was increased to 40 mg/L. We further examined the expression of LXRa mRNA and protein in foam cells to investigate the possible mechanism underlying the changes in ABCA1 expression. LXR␣ is a transcription factor which could stimulate the expression of ABCA1 and promote cholesterol efflux (Steffensen and Gustafsson, 2004; Rode et al., 2008). Our results showed that the expression of LXR␣ was also up-regulated in PNS treated groups. This suggested that PNS could stimulate the expression of ABCA1 via up-regulating the
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Fig. 4. The chemical structures of 24-hydroxycholesterol and derivatives of 20(S)protopanaxadiol.
expression of LXR␣ and subsequently increased the efflux of cholesterol. Last, correlation analysis of CE content and ABCA1 was performed. The test confirmed that the expressions of ABCA1 mRNA and protein were negatively correlated with cellular cholesterol ester content. Therefore, we could come to the conclusion that cellular cholesterol ester content could be decreased by PNS via up-regulating the expression of ABCA1 from LXRa stimulation. Although the precise mechanisms involved remain to be determined from this study, we summarized an important cue that the effect of PNS on the accumulation of cellular cholesterol was correlated with efflux. Furthermore, to explore the possible mechanism of LXR␣ activation by PNS, we analyzed the chemical structure of the principal active constituents of PNS. Compared with the natural agonists of LXR␣, 22-hydroxycholesterol, 24-hydroxycholesterol and 24(s), 25-epoxycholesterol, the major active ingredients of PNS possess a similar structure and an 8-C side-chain at the 17-position (Fig. 4). Although saponins contain complex carbohydrates groups, the carbohydrates groups would be hydrolyzed first during the bio-metabolism of saponins. For example, in compound K (the metabolite of Rb1, one of the main components of PNS), the parent structure is retained and all of the carbohydrates groups are hydrolyzed completely. Therefore, the structural similarities of PNS with the natural agonists might contribute to their pharmacological activities such as the activation of LXR␣ and the up-regulation of ABCA1 expression. Previous studies on phytosterols have found that they could remarkably reduce LDL and TC levels in humans and increase HDL levels because of structural similarities with cholesterol (Mannarino et al., 2009). Our results suggested that some saponins might have the same or similar action as phytosterols. Further study is necessary to find the most active monomer of PNS for the discovery of new drugs with anti-atherosclerotic or anti-hypercholesterolemic properties. In conclusion, PNS could attenuate the formation of foam cells via up-regulating the expression of ABCA1. This bioactivity might be associated with their special chemical structures that are similar to the natural agonists of liver X receptor ␣, just like gynosaponin TR1 (Huang et al., 2005).
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Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 30470465 and 30371768). References Brunham, L.R., Singaraja, R.R., Duong, M., Timmins, J.M., Fievet, C., Bissada, N., Kang, M.H., Samra, A., Fruchart, J.C., McManus, B., Staels, B., Parks, J.S., Hayden, M.R., 2009. Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis. Arteriosclerosis Thrombosis and Vascular Biology 29, 548–554. Cicero, A.F., Vitale, G., Savino, G., Arletti, R., 2003. Panax notoginseng effects on fibrinogen and lipid plasma level in rats fed on a high-fat diet. Phytotherapy Research 17, 174–178. Chen, S.W., Li, X.H., Ye, K.H., Jiang, Z.F., Ren, X.D., 2004. Total saponins of Panax notoginseng protected rabbit iliac artery against balloon endothelial denudation injury. Acta Pharmacologica Sinica 25, 1151–1156. Dean, M., Hamon, Y., Chimini, G., 2001. The human ATP-binding cassette (ABC) transporter superfamily. Journal of Lipid Research 42, 1007–1017. Huang, Z.H., Lin, C.Y., Oram, J.F., Mazzone, T., 2001. Sterol efflux mediated by endogenous macrophage apoE expression is independent of ABCA1. Arteriosclerosis Thrombosis and Vascular Biology 21, 2019–2025. Hiroyuki, I., 2003. Oxidized low-density lipoproteins: What is understood and what remains to be clarified. Biological Pharmaceutical Bulletin 26, 1–9. Huang, T.H., Razmovski-Naumovski, V., Salam, N.K., Duke, R.K., Tran, V.H., Duke, C.C., Roufogalis, B.D., 2005. A novel LXR-alpha activator identified from the natural product Gynostemma pentaphyllum. Biochemical Pharmacology 70, 1298–1308. Joyce, C.W., Amar, M.J., Lambert, G., Vaisman, B.L., Paigen, B., Najib-Fruchart, J., Hoyt Jr., R.F., Neufeld, E.D., Remaley, A.T., Fredrickson, D.S., Brewer Jr., H.B., Santamarina-Fojo, S., 2002. The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 mice and apoEknockout mice. Proceedings of the National Academy of Sciences of the United States of America 99, 407–412. Liu, L.K., Lee, H.J., Shih, Y.W., Chyau, C.C., Wang, C.J., 2008. Mulberry anthocyanin extracts inhibit LDL oxidation and macrophage-derived foam cell formation induced by oxidative LDL. Journal of Food Science 73, H113–H121. Mott, S., Yu, L., Marcil, M., Boucher, B., Rondeau, C., Genest, J., 2000. Decreased cellular cholesterol efflux is a common cause of familial hypoalphalipoproteinemia: role of the ABCA1 gene mutations. Atherosclerosis 152, 457–468. Mannarino, E., Pirro, M., Cortese, C., Lupattelli, G., Siepi, D., Mezzetti, A., Bertolini, S., Parillo, M., Fellin, R., Pujia, A., Averna, M., Nicolle, C., Notarbartolo, A., 2009. Effects of a phytosterol-enriched dairy product on lipids, sterols and 8-isoprostane in hypercholesterolemic patients: a multicenter Italian study. Nutrition Metabolism Cardiovascular Diseases 19, 84–90.
Pomerantz, K.B., Hajjar, D.P., 1990. High-density-lipoprotein-induced cholesterol efflux from arterial smooth muscle cell derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis. Biochemistry 29, 1892–1899. Rode, S., Rubic, T., Lorenz, R.L., 2008. alpha-Tocopherol disturbs macrophage LXR alpha regulation of ABCA1/G1 and cholesterol handling. Biochemical and Biophysical Research Communications 369, 868–872. Santamarina-Fojo, S., Peterson, K., Knapper, C., Qiu, Y., Freeman, L., Cheng, J.F., Osorio, J., Remaley, A., Yang, X.P., Haudenschild, C., Prades, C., Chimini, G., Blackmon, E., Francois, T., Duverger, N., Rubin, E.M., Rosier, M., Denèfle, P., Fredrickson, D.S., Brewer, H.B., 2000. Complete genomic sequence of the human ABCA1: analysis of the human and mouse ATP-binding cassette A promoter. Proceedings of the National Academy of Sciences of the United States of America 97, 7987–7992. Schmitz, G., Langmann, T., 2001. Structure, function, and regulation of the ABC1 gene product. Current Opinion in Lipidology 12, 129–140. Singaraja, R.R., Bocher, V., James, E.R., Clee, S.M., Zhang, L.H., Leavitt, B.R., Tan, B., Brooks-Wilson, A., Kwok, A., Bissada, N., Yang, Y.Z., Liu, G., Tafuri, S.R., Fievet, C., Wellington, C.L., Staels, B., Hayden, M.R., 2001. Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1. Journal of Biological Chemistry 276, 33969–33979. Steffensen, K.P., Gustafsson, J.A., 2004. Putative metabolic effects of the liver X receptor (LXR). Diabetes 53 (Suppl. 1), S36–S42. Wang, J., Hu, J.H., 2006. Inhibitory effect of Panax notoginseng on the VSMC proliferation induced by hyperlipidemia serum. Zhongguo Zhong Yao Za Zhi 31, 588–590. Wang, Y.F., Chen, Z.J., Liao, Y.H., Mei, C.L., Peng, H.Y., Wang, M., Guo, H.P., Lu, H., 2007. Angiotensin II increases the cholesterol content of foam cells via downregulating the expression of ATP-binding cassette transporter A1. Biochemical and Biophysical Research Communications 353, 650–654. Webb, N.R., Moore, K.J., 2007. Macrophage-derived foam cells in atherosclerosis: lessons from murine models and implications for therapy. Current Drug Targets 8, 1249–1263. Yancey, P.G., Bortnick, A.E., Kellner-Weibel, G., de la Llera-Moya, M., Phillips, M.C., Rothblat, G.H., 2003. Importance of different pathways of cellular cholesterol efflux. Arteriosclerosis Thrombosis and Vascular Biology 23, 712–719. Yang, L., Yang, J.B., Chen, J., Yu, G.Y., Zhou, P., Lei, L., Wang, Z.Z., Cy Chang, C., Yang, X.Y., Chang, T.Y., Li, B.L., 2004. Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone. Cell Research 14, 315–323. Zhang, Y.G., Zhang, H.G., Zhang, G.Y., Fan, J.S., Li, X.H., Liu, Y.H., Li, S.H., Lian, X.M., Tang, Z., 2008. Panax notoginseng saponins attenuate atherosclerosis in rats by regulating the blood lipid profile and an anti-inflammatory action. Clinical and Experimental Pharmacology and Physiology 35, 1238–1244.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Panax notoginseng saponins decrease cholesterol ester via up-regulating ATP-binding cassette transporter A1 in foam cells Yi Jia a , Zhuo-Ying Li a , Hai-Gang Zhang a , Hai-Bo Li b , Ya Liu a , Xiao-Hui Li a,∗ a Institute of Materia Medica and Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, People’s Republic of China b Department of Medicinal Chemistry, College of Pharmacy, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, People’s Republic of China
a r t i c l e
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Article history: Received 9 June 2010 Received in revised form 12 August 2010 Accepted 13 August 2010 Available online 19 August 2010 Keywords: Panax notoginseng saponins ATP-binding cassette transporter A1 Foam cell Macrophage Cholesterol ester
a b s t r a c t Aim: Accumulating evidence has indicated that Panax notoginseng saponins (PNS), the major ingredients in Panax notoginseng (Burk.) F.H. Chen which could be found widely in Asia, can attenuate atherogenesis in vivo. This study was designed to examine the relationship of PNS with cholesterol ester in foam cells sourced from macrophages and the effect of PNS on the expression of ATP-binding cassette transporter A1 (ABCA1). Materials and methods: Foam cells sourced from macrophages were cultured with PNS. The content of cholesterol ester in foam cells was analyzed and expressions of ABCA1 and liver X receptor ␣ (LXR␣) in foam cells were measured by real-time PCR and western blotting methods. Results: The results showed that PNS could significantly decrease the level of cholesterol ester in foam cells at middle and high dosages. The real-time PCR and western blotting assays indicated that the expression of ABCA1 was up-regulated by PNS in a dose-dependent manner. Analysis based on these results showed that the cholesterol ester level was negatively correlated with ABCA1 expression. Conclusions: As a result, we conclude that by up-regulating the expression of ABCA1, PNS could lower the cholesterol ester level, which resulted in the attenuation of the foam cell formation. This bioactivity might be associated with the special chemical structures of PNS that are similar to the natural agonist of LXR␣. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Atherosclerosis (AS) is a fundamental pathological change that leads to serious cardiovascular diseases, such as stroke, coronary artery disease and hypertension. Although the pathogenesis of AS is still unclear, it is generally accepted that macrophage foam cells are the characteristic feature of atherosclerotic plaques (Webb and Moore, 2007). During foam cell formation, modified lipoproteins enter cells by receptor-mediated uptake and excess neutral lipids are stored as lipid droplets, creating the typical foamy appearance. The formation of foam cells is considered to be the initiation of AS and can influence the progression and destination of pathological changes. Oxidized forms of low density lipoproteins (LDL) are the modified lipoproteins usually considered to be causative during foam cell formation (Hiroyuki, 2003; Liu et al., 2008), illustrating
Abbreviations: AS, atherosclerosis; PNS, Panax notoginseng saponins; ABCA1, ATP-binding cassette transporter A1; LXR␣, liver X receptor ␣. ∗ Corresponding author. Tel.: +86 023 68752318; fax: +86 023 68753397. E-mail address: [email protected] (X.-H. Li). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.08.033
a model for macrophage foam cell formation with predominantly lipid droplets. ATP-binding cassette transporter A1 (ABCA1) is the critical channel of cholesterol efflux in macrophages and other tissues (Santamarina-Fojo et al., 2000; Schmitz and Langmann, 2001). ABCA1 can reduce the redundant lipids in cells, which protects against the formation of foam cells (Wang et al., 2007). The mutation of ABCA1 in humans causes a defect in cellular cholesterol efflux and deposition of lipid in tissues, also called Tangier disease (Mott et al., 2000). Notoginseng, the root of Panax notoginseng (Burk.) F.H. Chen, has been used as a health product and natural remedy in traditional medicine for cardiovascular diseases for more than 1000 years in Asia, including China, Japan, Korea, et al. Panax notoginseng saponins (PNS) are the major effective ingredients extracted from Panax notoginseng. Various studies on PNS showed that they exhibited significant anti-atherogenic effects, including the abilities to limit the proliferation of vascular smooth muscle cells (Wang and Hu, 2006), reduce hyperlipidemia (Cicero et al., 2003), protect against artery injury (Chen et al., 2004), regulate blood lipid profiles and control anti-inflammatory capability (Zhang et al., 2008). As the typical traditional Chinese medicines with cardiovascular
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beneficial actions, it is necessary to evaluate their pharmacological profiles in vitro. Several previous studies have elucidated the effects of PNS on the formation of foam cells, however, little empirical evidence has been presented on the relationship between PNS and ABCA1, that plays an essential role in cellular cholesterol efflux and helps to prevent macrophages from becoming foam cells (Brunham et al., 2009). In the current study, we examined cholesterol ester content in foam cells and the expression of ABCA1 to investigate the potentially mechanism underlying the anti-atherogenic activities of PNS. 2. Materials and methods 2.1. Cell culture Macrophages (NR8383) were cultured in six well cell culture clusters (Costar, Corning Incorporated, NY, USA) and adjusted to 105 /mL. The cells were cultured in phenol red-free RPMI 1640 medium (GIBCO, Invitrogen Corporation, Grand Island, NY, USA), containing 10% (v/v) fetal calf serum (GIBCO), 100 U/mL of penicillin and 100 g/mL of streptomycin, and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 for 24 h. After incubation with oxidized low density lipoprotein (ox-LDL, 100 g/mL, purchased from Institute of Basic Medicine, Peking Union Medical College) for 24 h, cells were treated with ox-LDL free medium supplemented with different concentrations of PNS (0 (as model group), 20, 40, 80 mg/L) (Yunnan Phytopharmaceutical Co. LTD., Yunnan, China) and CD36 blocking antibody (sc-5522p, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:1000) for another 24 h. Control cells were incubated over the same time period in cell culture media without PNS or ox-LDL. The potential toxic effect on cell viability by the treatments of ox-LDL and PNS was examined by the MTT method in 96 well cell culture clusters (Costar). The number of replicates per experiment is 5 (n = 5). After administration, all cells were rinsed with heparin in order to remove lipoproteins attached to the cell surface. 2.2. Analysis of cellular cholesterol ester content Lipid-laden cells (lipid droplet positive cells) were stained with oil red O (Yang et al., 2004), and observed under an optical microscope (Olympus BX51, Olympus, Tokyo, Japan). Foam cells, which concentration was about 105 /mL, were collected after treatment with trypsin (0.25%, 5 min). Then, cells were washed three times with PBS. Each sample was sonicated for 3 min using the microtip of a sonicator. Samples (usually 100 L) were transferred into tubes and extracted with a chloroform/methanol (2:1; v/v) mixture. The chloroform phase was separated, dried and re-suspended in assay buffers supplied with the commercially available test kits for total cholesterol (TC) and free cholesterol (FC) content measurement (Rongshen Inc., Shanghai, China). The sediments were collected for the measurement of protein content. All samples were stored at −70 ◦ C for further experimentation. The method of TC test kits is CHOP-PAP. Samples were treated with cholesterol esterase first, in order to change cholesterol ester (CE) into cholesterol. Then oxidized the total cholesterol in samples by cholesterol oxidase. The oxidation reaction released H2 O2 , and the concentration of H2 O2 is in direct proportion to the concentration of cholesterol. The concentration of H2 O2 was measured by a peroxidase and 4-amino antipyrine method, then the OD values in 500 nm of the products were tested. The method of FC test kits is the same as TC test kits except the first step of reaction with cholesterol esterase. CE contents were then calculated (CE = TC − FC). The protein amount was determined using protein assay kit (Bio-Rad Lab., Hercules, CA, USA).
2.3. RNA extraction and real-time PCR Total RNA was isolated from cells using Tripure reagents (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. RNA samples were dissolved in nuclease-free water and stored at −70 ◦ C for real-time PCR after DNA was removed by DNase I (Takara, Shiga, Japan) treatment for 30 min at 37 ◦ C. Total RNA (1 g) was reversely transcribed using AMV reverse transcriptase (Promega, Madison, WI, USA) at 42 ◦ C for 1 h. PCR primers used were designed by Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) based on the cDNA sequences of ABCA1 and LXR␣. (forward: 5 -GCT CCT GCT GAA ATA CCG ACA A-3 and reverse: 5 -AGG GAC GAT TCC ACA TCT TTC TTG-3 , with an amplified product of 198 bp for ABCA1; forward: 5 -GAT GTT CCC ACG GAT GCT AAT G-3 and reverse: 5 -CAG GAA TGT TTG CCC TCC TCA-3 , with an amplified product of 232 bp for LXR␣) Rat -actin (forward: 5 -TGT TGT CCC TGT ATG CCT CT-3 ; reverse: 5 -CTC TTT AAT GTC ACG CAC GAT-3 , with an amplified product of 225 bp) was used as a control. Real-time PCR reactions were performed using iCycler iQ (Bio-Rad) and Quanti Tect SYBR Green PCR Master Mix (MJ Research, Waltham, MA, USA) under the following conditions: 5 min at 95 ◦ C, 40 cycles at 95 ◦ C for 10 s, 58 ◦ C for 15 s, 72 ◦ C for 20 s, and 82.5 ◦ C for 5 s. After amplification, a melting curve analysis was performed by collecting fluorescence data while increasing the temperature from 72 to 99 ◦ C over 135 s. The Ct (cycle threshold) values were normalized to the expression levels of -actin. 2.4. Protein extraction and western blotting Foam cells (collected by scraping) were pooled and collected by centrifugation. Cells were washed with ice-cold PBS, lysed with the protein extraction reagent (Pierce Labs, Rockford, IL, USA), and heated to 100 ◦ C for 5 min after adding loading buffer. The samples were stored at −70 ◦ C until further analysis. Protein concentration was measured using a modified BCA protein assay (Pierce Lab). An equal amount of total protein (10 g/lane) from each sample was used for immunoblot analysis. The proteins were resolved using SDS-PAGE (15%). Proteins were transferred to polyvinyl difluoride membranes (Millipore) using a semidry transfer system (Bio-Rad Lab.). The membranes were blocked with 5% nonfat milk (BioRad Lab.) for 1 h at room temperature. Then, the membranes were incubated with mouse monoclonal anti-ABCA1 (1:500 dilution) (ab-18180, Abcam Inc., Abcam, MA, USA) and goat polyclonal antibody against LXR␣ (sc-1202, Santa Cruz, 1:500) at 4 ◦ C overnight. The membranes were then washed three times with PBST (phosphate buffered saline with 0.05% Tween 20), and incubated with species-specific horseradish peroxidase (HRP)-coupled secondary antibodies (1:2000 dilution) for 1 h at room temperature. The membrane was re-probed with HRP-conjugated monoclonal mouse anti--actin (1:1000 dilution) (KC-5A08, Kangchen Inc., Kangchen, Shanghai, China) to ensure equal loading. The immune complex was visualized with a chemiluminescence method (Pierce Lab.) using a BIO-RAD image system (Bio-Rad, XRS). 2.5. Statistical analysis All data were expressed as the mean ± SD. Statistical analysis of all data was accomplished using SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). Comparisons between groups were made using one-way ANOVA analysis with post hoc least significant difference (LSD) test. We performed Pearson correlation analysis to examine the relationship between cholesterol ester content and ABCA1. Values of p < 0.05 were considered to be statistically significant.
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Fig. 1. Cellular cholesterol ester aggregation was attenuated by PNS in foam cells (n = 5). a, control group; b, model group; c, PNS 20 mg/ml group; d, PNS 40 mg/ml group; e, PNS 80mg/ml group. *p < 0.05 versus model group.
3. Results
3.3. ABCA1 mRNA and protein expression
3.1. Cell viability measurement
The results of real-time PCR showed that ABCA1 mRNA level was up-regulated in the model group versus the control group. Compared with the model group, dose-dependent changes of ABCA1 mRNA were demonstrated. In comparison to the model group, treatment with PNS at doses of 40 and 80 mg/L significantly increased the expression of ABCA1 mRNA by 39.79% and 47.72%, respectively (Fig. 2A). Simultaneously, treatment with PNS (40 and 80 mg/L) could also increase the ABCA1 protein level remarkably (Fig. 2C and D).
Cell viability was measured by the MTT method after 24 h culture with ox-LDL and PNS. The data showed that there was no significant difference between the control and administered groups (Fig. 1A).
3.2. Analysis of cholesterol ester content For direct-viewing of the effects of PNS on cellular cholesterol content, foam cells stained by red oil O were observed by light microscopy. Only a few lipid aggregates could be found in the control group, while lipid aggregation was abundant in the cellular plasma in the model group. Compared with that in the control group, the size of cells was significantly larger in the model group. Treatment with PNS (40 and 80 mg/L) caused a visible decrease of lipid levels in cells compared to the model group (Fig. 1B). Compared with that in model group, the administration of PNS (40 mg/L) decreased the CE content by 30.47%, and PNS (80 mg/L) decreased the CE content by 40.23% (Fig. 1C).
3.4. LXR˛ mRNA and protein expression Compared with that in the control group, the LXR␣ mRNA level was increased in the model group. Administration of PNS (40 and 80 mg/L) could significantly up-regulate the level of LXR␣ mRNA by 41.46% and 55.00%, respectively, versus the model group (Fig. 2B). ox-LDL treatment caused an increase in the expression of LXR␣ protein in the model group compared with that in the control group. Treatment with PNS (40 and 80 mg/L) could significantly increase LXR␣ protein expression compared to the model group (Fig. 2C and D).
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Fig. 2. Regulation of ABCA1 and LXR␣ in foam cells by PNS (real-time PCR and western blot methods) (n = 5). *p < 0.05 versus model group.
3.5. Correlation analysis of CE content and ABCA1 To confirm whether the cellular aggregation of cholesterol ester is retarded by PNS via up-regulating the expression of ABCA1, the linear correlation of cholesterol ester content and ABCA1 was examined. As shown in Fig. 3, the expressions of ABCA1 mRNA and protein were negatively correlated with cellular cholesterol ester content (ABCA1 mRNA: R = −0.948, p < 0.01; ABCA1 protein: R = −0.932, p < 0.01). 4. Discussion Panax notoginseng saponins (PNS) are the effective ingredients extracted from Panax notoginseng (Burk.) F.H. Chen, which has been used to prevent cardiovascular diseases and enhance wound
Fig. 3. Linear correlation of the expression of ABCA1 and the cholesterol ester content.
healing for more than 1000 years in Asia. PNS are the mixture of more than 20 dammarane-type saponins, including ginsenosides Rg1, Rg2, Rb1, Rb2, Rb3 and Rc, and notoginsenosides R1, R2, R3, R4 and R6. Among the ingredients, Panax notoginsenoside Rb1 (about 30%), and ginsenosides Rg1 (about 30%), Rd and Re are considered to be the principal active constituents. All of the active constituents are derivatives of 20(S)-protopanaxadiol, 20(S)protopanaxpanaxatriol or dammarane-type triterpene. Although PNS injection (commercial product: Xuesaitong) has been used as an anti-thrombosis treatment during the rehabilitation of stroke patients in Chinese clinical practice, the traditional utilization of PNS is mainly focused on cardiovascular protection via oral administration. Studies on PNS have demonstrated their anti-atherogenic effects, while the exact mechanism underlying their functions is still unclear (Cicero et al., 2003; Chen et al., 2004). Recent study of us has demonstrated that PNS could attenuate atherosclerosis in rats via their regulation of the blood lipid profile and their anti-inflammatory roles (Zhang et al., 2008). In addition to the aforementioned results, in vitro study is also one requisite for the complete elucidation of PNS pharmacological actions. There are several well-recognized in vitro models in the study of atherosclerosis (AS), including formation of macrophages foam cells, impairment of endotheliocyte and proliferation of vascular smooth cell. Among them, formation of macrophage foam cells is the most classic one because of its importance in the initiation, processing and termination of AS. Since the content of cholesterol ester (CE) can function as a measure of lipid quantity in foam cells (Pomerantz and Hajjar, 1990), we first sought to examine the effects of PNS on cholesterol ester content in macrophage foam cells. To determine the potential cytotoxicity of PNS, a cell viability test via MTT method was performed first to examine the possible membrane integrity change caused by saponins due to their physicochemical properties. The result revealed that the cell viability was not affected by treatments with ox-LDL or PNS. In addition, adding PNS into cell culture media after lipoprotein incubation eliminated other factors such as uptake of lipid that could have affected CE content in foam cells.
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Therefore, it was clear that cells utilized stored lipids when macrophages were lipid-loaded by incubating with ox-LDL and that lipid content was reduced by different doses of PNS administration. Treatments with PNS at doses of 40 and 80 mg/L caused a 30% and 40% (p < 0.05) decrease in cholesterol ester content, respectively. This indicated that PNS could considerably decrease cellular cholesterol ester levels, through which the formation of foam cells was inhibited. The results highlight the effect of PNS on the formation of foam cells, which is helpful for understanding its pharmacological actions against AS. In addition, this result also suggests that the decreased cellular cholesterol ester content after incubation with PNS is the result of increased intracellular lipid catabolism and/or efflux. As macrophage foam cells lack pathways for cholesterol degradation, excess lipids can only be removed from cells via reverse cholesterol transport pathways. Next, we investigated the relationship between PNS and the critical gate-keeper in the process of cholesterol efflux, ABCA1. It is well known that the removal of excess cholesterol from cells by HDL or its apolipoproteins is important for maintaining cellular cholesterol homeostasis. Excess cholesterol is removed from macrophage foam cells as oxy-sterols by four pathways, passive diffusion, CD36, ABCA1 and apolipoprotein E (Huang et al., 2001). Among them, the most important one is ABCA1 pathway (Yancey et al., 2003). ABCA1 is a member of a large family of ATP-binding cassette transporters that have common structural motifs and use ATP as an energy source to transport a variety of substrates, including ions, lipids, and cytotoxins (Dean et al., 2001). Among patients with Tangier disease, a disease resulting from the homozygote mutation of the ABCA1 gene, cardiovascular disease occurs at a rate about sixfold higher than among healthy adults. This is analogous to findings in ABCA1−/− mice (Singaraja et al., 2001). Additional study showed that over-expression of ABCA1 could increase the efflux of cellular cholesterol and attenuate the formation of AS lesions (Joyce et al., 2002). These studies provided valid evidence of the positive effects of ABCA1 on AS formation. The expression of ABCA1 was regulated by physiological factors such as specified cholesterol, cAMP and interferon. Whether cellular cholesterol ester content was decreased by PNS via regulating the expression of ABCA1 had not yet been discovered. In order to exclude the influence of CD36 in this study, antiCD36 antibody was added. Anti-CD36 not only prevents the cellular cholesterol efflux via CD36, but also inhibits the uptake of cholesterol after ox-LDL treatment. The test results showed that, compared with that in the control group, the expression of ABCA1 in the model group was increased significantly. The up-regulation of ABCA1 expression, both mRNA and protein, caused increased efflux of cellular cholesterol ester in foam cells, which could be considered as a defensive response of the lipid-loaded cell. However, the presence of abundant cholesterol ester also indicated that the defensive response in the model group was not powerful and effective enough to remove cholesterol ester from foam cells. We examined the expression of ABCA1 in PNS administered foam cells and observed the dose-dependent changes of ABCA1 expression compared with that in the model group. No noticeable differences of ABCA1 expression and cholesterol ester content between PNS groups and the model group, however, could be found, until the PNS dose was increased to 40 mg/L. We further examined the expression of LXRa mRNA and protein in foam cells to investigate the possible mechanism underlying the changes in ABCA1 expression. LXR␣ is a transcription factor which could stimulate the expression of ABCA1 and promote cholesterol efflux (Steffensen and Gustafsson, 2004; Rode et al., 2008). Our results showed that the expression of LXR␣ was also up-regulated in PNS treated groups. This suggested that PNS could stimulate the expression of ABCA1 via up-regulating the
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Fig. 4. The chemical structures of 24-hydroxycholesterol and derivatives of 20(S)protopanaxadiol.
expression of LXR␣ and subsequently increased the efflux of cholesterol. Last, correlation analysis of CE content and ABCA1 was performed. The test confirmed that the expressions of ABCA1 mRNA and protein were negatively correlated with cellular cholesterol ester content. Therefore, we could come to the conclusion that cellular cholesterol ester content could be decreased by PNS via up-regulating the expression of ABCA1 from LXRa stimulation. Although the precise mechanisms involved remain to be determined from this study, we summarized an important cue that the effect of PNS on the accumulation of cellular cholesterol was correlated with efflux. Furthermore, to explore the possible mechanism of LXR␣ activation by PNS, we analyzed the chemical structure of the principal active constituents of PNS. Compared with the natural agonists of LXR␣, 22-hydroxycholesterol, 24-hydroxycholesterol and 24(s), 25-epoxycholesterol, the major active ingredients of PNS possess a similar structure and an 8-C side-chain at the 17-position (Fig. 4). Although saponins contain complex carbohydrates groups, the carbohydrates groups would be hydrolyzed first during the bio-metabolism of saponins. For example, in compound K (the metabolite of Rb1, one of the main components of PNS), the parent structure is retained and all of the carbohydrates groups are hydrolyzed completely. Therefore, the structural similarities of PNS with the natural agonists might contribute to their pharmacological activities such as the activation of LXR␣ and the up-regulation of ABCA1 expression. Previous studies on phytosterols have found that they could remarkably reduce LDL and TC levels in humans and increase HDL levels because of structural similarities with cholesterol (Mannarino et al., 2009). Our results suggested that some saponins might have the same or similar action as phytosterols. Further study is necessary to find the most active monomer of PNS for the discovery of new drugs with anti-atherosclerotic or anti-hypercholesterolemic properties. In conclusion, PNS could attenuate the formation of foam cells via up-regulating the expression of ABCA1. This bioactivity might be associated with their special chemical structures that are similar to the natural agonists of liver X receptor ␣, just like gynosaponin TR1 (Huang et al., 2005).
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Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 30470465 and 30371768). References Brunham, L.R., Singaraja, R.R., Duong, M., Timmins, J.M., Fievet, C., Bissada, N., Kang, M.H., Samra, A., Fruchart, J.C., McManus, B., Staels, B., Parks, J.S., Hayden, M.R., 2009. Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis. Arteriosclerosis Thrombosis and Vascular Biology 29, 548–554. Cicero, A.F., Vitale, G., Savino, G., Arletti, R., 2003. Panax notoginseng effects on fibrinogen and lipid plasma level in rats fed on a high-fat diet. Phytotherapy Research 17, 174–178. Chen, S.W., Li, X.H., Ye, K.H., Jiang, Z.F., Ren, X.D., 2004. Total saponins of Panax notoginseng protected rabbit iliac artery against balloon endothelial denudation injury. Acta Pharmacologica Sinica 25, 1151–1156. Dean, M., Hamon, Y., Chimini, G., 2001. The human ATP-binding cassette (ABC) transporter superfamily. Journal of Lipid Research 42, 1007–1017. Huang, Z.H., Lin, C.Y., Oram, J.F., Mazzone, T., 2001. Sterol efflux mediated by endogenous macrophage apoE expression is independent of ABCA1. Arteriosclerosis Thrombosis and Vascular Biology 21, 2019–2025. Hiroyuki, I., 2003. Oxidized low-density lipoproteins: What is understood and what remains to be clarified. Biological Pharmaceutical Bulletin 26, 1–9. Huang, T.H., Razmovski-Naumovski, V., Salam, N.K., Duke, R.K., Tran, V.H., Duke, C.C., Roufogalis, B.D., 2005. A novel LXR-alpha activator identified from the natural product Gynostemma pentaphyllum. Biochemical Pharmacology 70, 1298–1308. Joyce, C.W., Amar, M.J., Lambert, G., Vaisman, B.L., Paigen, B., Najib-Fruchart, J., Hoyt Jr., R.F., Neufeld, E.D., Remaley, A.T., Fredrickson, D.S., Brewer Jr., H.B., Santamarina-Fojo, S., 2002. The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 mice and apoEknockout mice. Proceedings of the National Academy of Sciences of the United States of America 99, 407–412. Liu, L.K., Lee, H.J., Shih, Y.W., Chyau, C.C., Wang, C.J., 2008. Mulberry anthocyanin extracts inhibit LDL oxidation and macrophage-derived foam cell formation induced by oxidative LDL. Journal of Food Science 73, H113–H121. Mott, S., Yu, L., Marcil, M., Boucher, B., Rondeau, C., Genest, J., 2000. Decreased cellular cholesterol efflux is a common cause of familial hypoalphalipoproteinemia: role of the ABCA1 gene mutations. Atherosclerosis 152, 457–468. Mannarino, E., Pirro, M., Cortese, C., Lupattelli, G., Siepi, D., Mezzetti, A., Bertolini, S., Parillo, M., Fellin, R., Pujia, A., Averna, M., Nicolle, C., Notarbartolo, A., 2009. Effects of a phytosterol-enriched dairy product on lipids, sterols and 8-isoprostane in hypercholesterolemic patients: a multicenter Italian study. Nutrition Metabolism Cardiovascular Diseases 19, 84–90.
Pomerantz, K.B., Hajjar, D.P., 1990. High-density-lipoprotein-induced cholesterol efflux from arterial smooth muscle cell derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis. Biochemistry 29, 1892–1899. Rode, S., Rubic, T., Lorenz, R.L., 2008. alpha-Tocopherol disturbs macrophage LXR alpha regulation of ABCA1/G1 and cholesterol handling. Biochemical and Biophysical Research Communications 369, 868–872. Santamarina-Fojo, S., Peterson, K., Knapper, C., Qiu, Y., Freeman, L., Cheng, J.F., Osorio, J., Remaley, A., Yang, X.P., Haudenschild, C., Prades, C., Chimini, G., Blackmon, E., Francois, T., Duverger, N., Rubin, E.M., Rosier, M., Denèfle, P., Fredrickson, D.S., Brewer, H.B., 2000. Complete genomic sequence of the human ABCA1: analysis of the human and mouse ATP-binding cassette A promoter. Proceedings of the National Academy of Sciences of the United States of America 97, 7987–7992. Schmitz, G., Langmann, T., 2001. Structure, function, and regulation of the ABC1 gene product. Current Opinion in Lipidology 12, 129–140. Singaraja, R.R., Bocher, V., James, E.R., Clee, S.M., Zhang, L.H., Leavitt, B.R., Tan, B., Brooks-Wilson, A., Kwok, A., Bissada, N., Yang, Y.Z., Liu, G., Tafuri, S.R., Fievet, C., Wellington, C.L., Staels, B., Hayden, M.R., 2001. Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1. Journal of Biological Chemistry 276, 33969–33979. Steffensen, K.P., Gustafsson, J.A., 2004. Putative metabolic effects of the liver X receptor (LXR). Diabetes 53 (Suppl. 1), S36–S42. Wang, J., Hu, J.H., 2006. Inhibitory effect of Panax notoginseng on the VSMC proliferation induced by hyperlipidemia serum. Zhongguo Zhong Yao Za Zhi 31, 588–590. Wang, Y.F., Chen, Z.J., Liao, Y.H., Mei, C.L., Peng, H.Y., Wang, M., Guo, H.P., Lu, H., 2007. Angiotensin II increases the cholesterol content of foam cells via downregulating the expression of ATP-binding cassette transporter A1. Biochemical and Biophysical Research Communications 353, 650–654. Webb, N.R., Moore, K.J., 2007. Macrophage-derived foam cells in atherosclerosis: lessons from murine models and implications for therapy. Current Drug Targets 8, 1249–1263. Yancey, P.G., Bortnick, A.E., Kellner-Weibel, G., de la Llera-Moya, M., Phillips, M.C., Rothblat, G.H., 2003. Importance of different pathways of cellular cholesterol efflux. Arteriosclerosis Thrombosis and Vascular Biology 23, 712–719. Yang, L., Yang, J.B., Chen, J., Yu, G.Y., Zhou, P., Lei, L., Wang, Z.Z., Cy Chang, C., Yang, X.Y., Chang, T.Y., Li, B.L., 2004. Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone. Cell Research 14, 315–323. Zhang, Y.G., Zhang, H.G., Zhang, G.Y., Fan, J.S., Li, X.H., Liu, Y.H., Li, S.H., Lian, X.M., Tang, Z., 2008. Panax notoginseng saponins attenuate atherosclerosis in rats by regulating the blood lipid profile and an anti-inflammatory action. Clinical and Experimental Pharmacology and Physiology 35, 1238–1244.