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Enhanced CD36 expression changes the role of Nrf2 activation from anti-atherogenic to pro-atherogenic in apoE-deficient mice
Atherosclerosis 225 (2012) 83e90
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Enhanced CD36 expression changes the role of Nrf2 activation from anti-atherogenic to pro-atherogenic in apoE-deficient mice Hirotaka Sawada, Yoshiro Saito, Noriko Noguchi* Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakodani, Tatara, Kyotanabe, Kyoto 610-0394, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 May 2012 Received in revised form 31 July 2012 Accepted 14 August 2012 Available online 24 August 2012
Oxidative stress has been implicated as a causative factor of atherosclerosis. Defense systems against oxidative stress are maintained by radical scavenging antioxidants and/or by regulating the expression of antioxidant genes by activating oxidative stress-sensitive transcription factor: nuclear factor (erythroidderived 2)-like 2 (Nrf2). We investigated the anti-atherogenic effects of three synthesized compounds (shogaol A: radical scavenging antioxidant activity; shogaol N: Nrf2-activating activity; shogaol N þ A: both activities) and curcumin (both activities) in apolipoprotein E (apoE)-deficient mice. We expected compounds with both activities to have additive or synergistic anti-atherogenic effects; however, atherosclerosis was exacerbated significantly by curcumin and slightly by shogaol N þ A. Shogaol A, shogaol N, and shogaol N þ A showed no significant effect on atherosclerosis development. Immunohistochemical analysis of the aorta revealed that expression of CD36, an Nrf2-regulated gene, was strongly induced by treatment with curcumin. The total antioxidant capacity of plasma collected from mice administered the three compounds was evaluated using a hydrophilic probe, pyranine. Shogaol N or shogaol N þ A significantly enhanced the antioxidant capacity of plasma, whereas shogaol A and curcumin did not show this activity. The concentrations of the three shogaol derivatives in plasma were similar (approximately 100 nM), while that of curcumin was much lower. These results suggest that plasma antioxidant capacity is maintained at high levels via Nrf2 activation and that CD36 expression enhances atherosclerosis development. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Oxidative stress Radical scavenging antioxidant Nrf2 CD36 Atherosclerosis ApoE deficient
1. Introduction Oxidative modification of low-density lipoprotein (LDL) is a key initial event in the pathogenesis of atherosclerosis [1]. Monocytederived macrophages actively uptake oxidized LDL via scavenger receptors, resulting in the formation of foam cells and fatty streaks. In vivo there are two complementary antioxidant systems that function to prevent LDL oxidation. One is the direct radical scavenging activity of low-molecular weight antioxidants and the other is indirect antioxidant activity [2], which enhances the expression of antioxidative enzymes and cytoprotective proteins through activation of the oxidative-stress sensing transcription factor Nrf2 [3]. Several natural and synthetic antioxidants have been shown to prevent LDL oxidation both in vitro and in vivo [4]. Therapeutic approaches aimed at suppressing atherosclerosis development by inhibiting LDL oxidation have also been examined. Radical scavenging activity is attributed to the phenolic group of antioxidants * Corresponding author. Tel./fax: þ81 774 65 6262. E-mail addresses: [email protected] (H. Sawada), ysaito@ mail.doshisha.ac.jp (Y. Saito), [email protected] (N. Noguchi). 0021-9150/$ e see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2012.08.023
[5]. Natural phenolic antioxidants such as vitamin E and vitamin C are known to inhibit LDL oxidation; however, the results of several long-term clinical trials have been controversial regarding the effect of vitamin E and/or vitamin C intake on the risk of cardiovascular events [6,7]. In contrast, synthetic radical scavenging antioxidants such as probucol have been shown to inhibit atherosclerosis by inhibiting LDL oxidation [8]. Nrf2, a redox-sensing transcription factor, is inactivated in the cytosol when bound to Kelch-like ECH-associated protein 1 (Keap1) under normal conditions. Keap1 is redox-sensitive because its cysteine groups can be easily oxidized, resulting in dissociation of Nrf2 from Keap1 [9], allowing translocation of Nrf2 to the nucleus [10]. The promoter regions of several genes encoding antioxidative enzymes and cytoprotective proteins, such as heme oxygenase 1 (HO-1), glutamate cysteine ligase catalysis and modifier (GCLC, GCLM), and solute carrier family 7A number 11 (xCT), contain antioxidant response elements (ARE) to which Nrf2 can bind [3]. Some dietary compounds, such as sulforaphane and curcumin, contain isothiocyanate and a,b-unsaturated carbonyl functional groups, respectively, which covalently bind to cysteine residues on Keap1 to enhance expression of Nrf2-regulated genes [2,11].
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Vascular endothelial cells are constantly subjected to mechanical shear stress due to blood flow. Atherosclerotic lesions are likely to develop focally at bifurcations and branch points in vessels [12]. Most atherosclerosis-prone regions are exposed to nonunidirectional, disturbed, or oscillatory flow; in contrast, atherosclerosis-resistant regions are exposed to unidirectional laminar flow [13,14]. Nrf2 is activated at atherosclerosis-resistant regions exposed to unidirectional and laminar flow, whereas Nrf2 is diffuse in atherosclerosis-prone regions exposed to disturbed flow [15]. On the other hand, the formation of atherosclerotic lesions was attenuated in apoE/Nrf2 double-deficient mice compared to in apoE-deficient mice. Enhanced expression of the scavenger receptor CD36 via Nrf2 activation has been suggested to contribute to atherogenesis [16e18]. Thus, the role of Nrf2 in the pathology of atherosclerosis remains unclear. To evaluate the importance of radical scavenging activity and Nrf2-activating activity in atherogenesis, we investigated the effects of administration of three synthesized compounds (shogaol A: radical scavenging antioxidant activity; shogaol N: Nrf2activating activity; shogaol N þ A: both activities) and curcumin (both activities) on atherosclerosis development and total antioxidant capacity in apoE-deficient mice. 2. Materials and methods
accordance with the regulations established by the Ethics Review Committee for Animal Experiments of Doshisha University. ApoEdeficient mice were divided into 5 groups at 5 weeks of age, and 12 of each group were placed on a pellet diet containing 0.5 w/w% shogaol N, shogaol A, shogaol N þ A, curcumin, or no drug (control group) for 15 weeks (approximately 100 days). The preparation of diet is described in the Supplement section. 2.5. Determination of plasma concentration Blood samples were collected from the caudal vein and plasma concentrations of compounds were determined using liquid chromatography/tandem mass spectrometry (LC-MS/MS) as described in the Supplement section. 2.6. Quantification of atherosclerotic lesions Atherosclerotic lesion severity in aortas was assessed as described previously [20] and in the Supplement section. 2.7. Immunohistochemistry Paraffin-embedded sections (5 mm) of each left common carotid artery were stained immunohistochemically with an anti-CD36 mAb as described previously [18] and in the Supplement section.
2.1. Chemicals 2.8. Statistical analysis Shogaol derivatives (E)-12-(3-methoxy-4-(methoxymethoxy) phenyl)-10-oxododec-8-enoic acid ethyl ester (shogaol N, Fig. 1A), 8-(3-(4-hydroxy-3-methoxyphenyl) propanamido) octanoic acid ethyl ester (shogaol A, Fig. 1B), (E)-12-(4-hydroxy-3methoxyphenyl)-10-oxododec-8-enoic acid ethyl ester (shogaol N þ A, Fig. 1C), and their de-esterified (de-ethylated) compounds were provided by Toagosei (Nagoya, Japan). Curcumin (Fig. 1D) and pyranine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Other chemicals were obtained as described in the Supplement. 2.2. THP-1 cell culture, RNA isolation, and determination of antioxidant genes THP-1 cells, a human monocyte leukemia cell line, were cultured in RPMI-1640 medium supplemented with 10% heatinactivated FBS, penicillin (100 U/mL), streptomycin (0.1 mg/mL) in a humidified atmosphere of 5% CO2 and 95% air at 37 C. To induce differentiation of THP-1 monocytes into macrophages, cells (2 106 cells/6-cm dish) were treated with PMA (0.1 mM) for 48 h. Expression of target genes was determined using real-time polymerase chain reaction (PCR) as described previously [16] and in the Supplement section. 2.3. Measurement of radical scavenging activity in plasma Free radical scavenging activity of each compound was assessed as described previously [19] and in the Supplement section. 2.4. Animals Four-week-old male C57BL/6 mice and apolipoprotein E (apoE)deficient mice (C57BL/6.KOR-Apoeshl) were obtained from SLC Japan (Shizuoka, Japan) and fed a normal rodent chow diet obtained from Oriental Yeast (Tokyo, Japan) and water for 1 week after purchase. Mice were maintained under controlled temperature and humidity, with 15e20 fresh air changes per hour and a 12-h light/dark cycle. All experimental procedures and protocols were performed in
Data are reported as the mean, standard error, and number of experiments. Statistical significance of the difference between experiments was calculated using analysis of variance (ANOVA) with Dunnett’s test used for multiple comparisons. Values of p < 0.05 were considered significant. 3. Results 3.1. Structure and stability of compounds used in this study Structures of the compounds used in this study are shown in Fig. 1AeD. Shogaol N (Fig. 1A), but not shogaol A (Fig. 1B), is classified as an a,b-unsaturated carbonyl compound. Shogaol A, but not shogaol N, contains an antioxidative phenolic group. Shogaol N þ A and curcumin possess both structures (Fig. 1C and D). For preliminary evaluation of biological stability, these compounds were incubated in C57BL/6 mouse plasma at 37 C for 4 h. Shogaol N, shogaol A, and shogaol N þ A were rapidly hydrolyzed to their deesterified (de-ethylated) compounds in vitro with the halftimes of 0.966 min, 0.531 min, and 0.564 min, respectively (Fig. S1). Shogaol derivatives and these de-ethylated compounds were used in the present study. 3.2. Characterization of Nrf2 activating activity of shogaol derivatives and curcumin To identify the Nrf2-activating activity of the a,b-unsaturated carbonyl functional group present in shogaol derivatives and curcumin, we investigated the effect of this group on the up-regulation of Nrf2-regulated cell defense genes such as HO-1 and xCT in PMAdifferentiated THP-1 macrophages. Cells were treated with each deethylated shogaol derivative and curcumin at 10 mM for 6 h. PGJ2 was used as a positive control to activate the Keap1-Nrf2 pathway. Real-time PCR analysis revealed that de-ethylated shogaol N, de-ethylated shogaol N þ A, and curcumin, which possess an a,bunsaturated carbonyl functional group, significantly increased HO-1 and xCT mRNA expression in PMA-differentiated THP-1
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Fig. 1. Chemical structures of shogaol derivatives (AeC) and curcumin (D). (E, F) Change in HO-1 (E) and xCT (F) expression by shogaol derivatives and curcumin in PMAdifferentiated THP-1 macrophages. Macrophages were incubated with each compound or PGJ2 at 10 mM for 6 h. Changes in HO-1 (E) and xCT (F) expression were measured using real-time PCR (mean SEM, n ¼ 6) (*: p < 0.05; ***: p < 0.001; Dunnett, ANOVA).
macrophages (Fig. 1E and F). In contrast, de-ethylated shogaol A, which is not an a,b-unsaturated carbonyl compound, had no effects on HO-1 and xCT expression. NQO-1 mRNA expression increased following treatment with de-ethylated shogaol N and de-ethylated shogaol N þ A; however, GCLM expression did not change significantly (Fig. S2). These results indicate that the a,b-unsaturated carbonyl functional group of de-ethylated shogaol N, de-ethylated shogaol N þ A, and curcumin is essential for the up-regulation of Nrf2-regulated genes.
3.3. Characterization of radical scavenging activity of shogaol derivatives and curcumin The antioxidant effects of shogaol derivatives, curcumin, and atocopherol against peroxyl radicals were assessed in an in vitro system using mouse plasma. Pyranine was used as a reference probe to evaluate the radical scavenging activity of the tested compounds. Pyranine is a convenient probe because it produces a clear lag phase in the presence of antioxidants due to its low reactivity toward free
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radicals compared to other antioxidants. In the absence of plasma, pyranine was consumed at a constant rate with no lag phase when free radicals were generated through thermal decomposition of AAPH (Fig. 2A). Addition of mouse plasma (10 vol% in the system) completely inhibited pyranine consumption and produced a clear lag phase because endogenous antioxidants present in the plasma scavenged free radicals faster than scavenging by pyranine. After the lag phase, pyranine was consumed at the same rate as that without plasma. The lag phase is directly proportional to the amount of radical scavenging antioxidant present. In fact, a-tocopherol extended the lag phase in a concentration-dependent manner (data not shown). In accordance with the activity of a-tocopherol, addition of shogaol A or shogaol N þ A, which contain an antioxidative phenolic group, completely inhibited pyranine consumption and produced a clear lag phase. In contrast, shogaol N, with no phenolic group, did not exhibit antioxidant activity. The lag phase of each compound at 10 and 100 mM is summarized in Fig. 2B. These results clearly indicate that shogaol A, shogaol N þ A, and their de-ethylated compounds possess radical scavenging activity, whereas this activity was not detected for shogaol N. The antioxidant activity of curcumin, which reportedly has radical scavenging activity [21], could not be determined using this system because of its low solubility in the range of concentrations evaluated.
3.4. Determination of plasma concentrations of shogaol derivatives and curcumin Plasma was collected from apoE-deficient mice fed a normal rodent chow diet containing 0.5 w/w% shogaol derivatives or
curcumin, and plasma concentrations of each compound were determined using LC-MS/MS analysis. Shogaol derivatives and their de-ethylated compounds were detected 18 h after feeding. Plasma concentrations of these compounds were constant over the entire sampling interval (Fig. 3AeC). Although the concentration ratio of shogaol derivatives to their de-ethylated compounds was not consistent, total concentration was approximately 100 nM in the plasma. The plasma concentration of curcumin was maintained at approximately 20 nM, which was 5 times lower than that of the concentration of shogaol derivatives (Fig. 3D).
3.5. Evaluation of radical scavenging activity in plasma of apoEdeficient mice fed on a diet containing shogaol derivatives or curcumin To evaluate the in vivo antioxidant activity of shogaol derivatives and curcumin, plasma collected from apoE-deficient mice fed a normal diet containing the compounds was assessed as described above. Plasma collected from the shogaol N- and shogaol N þ Atreated groups had longer lag times than that of the control group (Fig. 3E). In contrast, the shogaol A- and curcumin-treated groups showed no apparent increase in lag time compared to the control group. 3.6. Effects of shogaol derivatives and curcumin on the development of atherosclerotic lesions in apoE-deficient mice To evaluate the anti-atherogenic effects of shogaol derivatives and curcumin, atherosclerotic lesions in apoE-deficient mice treated with each compound were evaluated using Sudan III staining. Protective effects of shogaol derivatives against atherosclerosis development were not observed at 15 weeks after feeding of a normal diet containing each compound. Against our expectations, atherosclerosis was exacerbated significantly by curcumin and slightly by shogaol N þ A (Fig. 4AeE). The quantitative analysis of atherosclerosis lesion in the aortic root, ascending aorta, and aortic arch was conducted. As a result, statistically significant difference between control and curcumin was observed in the plaque area visualized by en face Sudan III staining (1.94 0.79 mm2 vs. 4.83 2.85 mm2, p < 0.05) (Fig. 4F). A significant increase in the percentage of atherosclerotic plaques in the curcumin-treated group was also observed in the aortic root plaque area (Fig. 4G).
3.7. Effects of curcumin on CD36 expression in the aortic sinus and THP-1 macrophages
Fig. 2. In vitro evaluation of radical scavenging activities of shogaol derivatives. (A) The reaction was initiated by the addition of 50 mM AAPH at 37 C. Bleaching of pyranine with or without antioxidants was conducted as described in Materials and methods and the results were plotted as the ratio of absorbance at time t to time zero. (B) The lag time produced by the indicated concentrations of shogaol derivatives was plotted.
The scavenger receptor CD36, an Nrf2-regulated gene, is reportedly responsible for the uptake of oxidized LDL in macrophages, and thus is associated with atherosclerotic plaque development in the aortic sinus. Therefore, we identified the presence of CD36 in the left common carotid artery using immunohistochemical analysis. Expression of CD36 was not observed in the aortic sinus obtained in mice fed a normal diet containing vehicle (control) and shogaol derivatives (Fig. 5AeD). In contrast, CD36positive areas were observed in the vascular endothelium of the curcumin-treated group (white arrows; Fig. 5E). The effects of curcumin on CD36 expression were further examined using PMAdifferentiated THP-1 macrophages. Treatment of PMAdifferentiated THP-1 macrophages with curcumin significantly increased CD36 expression (1.0 vs. 2.16 1.15, ratio of arbitrary unit to the control group, p < 0.05) (Fig. 5F).
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Fig. 3. Plasma concentrations of shogaol derivatives and curcumin and their radical scavenging antioxidant capacity in the plasma of apoE-deficient mice fed with diet containing these compounds. (AeD) Plasma concentrations of shogaol derivatives (AeC), and curcumin (D) were determined as described in Materials and methods (n ¼ 2). (E) The radical scavenging antioxidant capacity in each plasma sample was evaluated based on pyranine consumption induced by AAPH. Mean values of estimated lag time are shown (n ¼ 2).
4. Discussion We investigated the anti-atherogenic effects of compounds with radical-scavenging and/or Nrf2-activating activities in apoEdeficient mice fed a normal rodent chow diet. Evaluated compounds showed no significant suppressive effects on atherosclerosis development. Surprisingly, atherosclerotic lesions were increased by administration of curcumin, which has both radical scavenging and Nrf2-activating activities. Previous studies demonstrated the inhibitory effects of curcumin on atherosclerosis in mice deficient in the LDL receptor [22], apoE [23], and both the LDL receptor and apoE [24]. In these studies, mice were fed a highfat diet, whereas in our study, mice were fed a normal chow diet. The amount of curcumin used in this study was significantly higher than that used in previous studies (10e300 vs. 1000 mg/kg/day, respectively). Changes in plasma levels of triglycerides and total cholesterol were not significant in this study, which is in contrast to the results of previous studies [23,25] (Fig. S3). Since the body
weight of all mice used in this study increased at a good rate (Fig. S4) and there were not significant changes in plasma levels of alanine aminotransferase and aspartate aminotransferase in these mice (data not shown), the toxic effects of all compounds including curcumin seemed not to be serious. It is very interesting to compare the atherogenic effects of curcumin at several different doses using apoE-deficient mice with a normal diet and a high-fat diet. CD36 expression was found to be enhanced by treatment with curcumin in some atherosclerotic lesions. Further, it was shown that CD36 was significantly up-regulated in PMA-differentiated THP-1 macrophages treated with curcumin. CD36 has been shown to contribute to atherosclerosis development via Nrf2 activation in apoE/Nrf2 double-deficient mice [17,18]. We confirmed the importance of CD36 up-regulation in atherogenesis through administration of an Nrf2-activating compound to apoE-deficient mice. Expression of CD36 is regulated by not only Nrf2 [26] but also by PPARg [27] or FOXO3a [22]. Since curcumin has been reported to contribute to activation of PPARg [28] and FOXO3a [22],
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Fig. 4. Effects of shogaol derivatives and curcumin on atherosclerotic lesion development in apoE-deficient mice. (AeE) Atherosclerotic lesions were evaluated using Sudan III staining in apoE-deficient mice fed a normal pelleted diet supplemented without the drug (A) as a control or with shogaol N (B), shogaol A (C), shogaol N þ A (D), or curcumin (E) for 15 weeks. (F, G) The extent of Sudan III-positive areas was measured (F) and expressed as percentage of the total area, including the descending thoracic aorta (G) (mean SEM, n ¼ 6). (*: p < 0.05; Dunnett, ANOVA).
CD36 expression in atherosclerotic lesions may be enhanced via these routes. Similarly to curcumin, shogaol N þ A has both radical scavenging and Nrf2-activating activities and increased the number of atherosclerotic plaques; however, this increase was not significant. In contrast, shogaol N treatment induced neither CD36 expression nor atherosclerosis. This may be due to the high antioxidant capacity of plasma during feeding of a diet containing shogaol N or shogaol N þ A (Fig. 3E). It is well known that LDL oxidation is an initial event in atherogenesis and that oxLDL is taken up by macrophages via scavenger receptors [1]. Furthermore, oxLDL is known to induce CD36 expression [29]. These results suggest that radical scavenging antioxidant capacity is effectively increased via Nrf2 activation. Therefore, LDL oxidation was inhibited in mice treated with shogaol N or shogaol N þ A. The concentration of curcumin was likely not sufficient to induce antioxidative enzymes
via Nrf2 activation. We confirmed the poor bioavailability of curcumin in a Caco-2 cell monolayer permeability study which was an established method for predicting intestinal absorption, although no metabolites of curcumin were identified in the present study. The permeability coefficients of shogaol N, shogaol A, shogaol N þ A, and curcumin were 53.7, 16.6, 47.2, and 2.69 106 cm/s, respectively. It is important to identify the plasma components responsible for the radical scavenging antioxidant capacity of mice treated with shogaol N or shogaol N þ A. Shogaol A scavenges free radicals, indicating that administration of shogaol A may extend the lag time when measuring antioxidant capacity. However, plasma concentration levels of shogaol A were too low (0.2e0.3 mM, shown in Fig. 3B) to increase the lag time since at least 1 mM was required for evaluation in the present assay. This implies that direct radical scavenging compounds should be maintained at higher concentrations in plasma than Nrf2-activating
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Fig. 5. Effects of shogaol derivatives and curcumin on CD36 expression in aortic sinus and THP-1 macrophages. (AeE) Expression of CD36 was evaluated using immunohistochemistry in the aortic sinus obtained from apoE-deficient mice fed a normal pelleted diet supplemented without the drug (A) as a control or with shogaol N (B), shogaol A (C), shogaol N þ A (D), or curcumin (E-1, 2) for 15 weeks. The white arrows represent regions yielding high fluorescence intensity on enlarged views (E-2). (F) Altered CD36 expression by shogaol derivatives and curcumin in PMA-differentiated THP-1 macrophages was measured using quantitative PCR (mean SEM, n ¼ 6). Macrophages were incubated with each compound at 10 mM for 6 h and harvested for RNA isolation and real-time PCR analysis (*: p < 0.05; ***: p < 0.001; Dunnett, ANOVA).
compounds to suppress atherosclerosis in vivo. Thus, radical scavenging antioxidant capacity can be maintained by keeping a low concentration of Nrf2-activating drugs in the plasma. 5. Conclusions In conclusion, the present study demonstrated the importance of maintaining a high antioxidant capacity in the plasma and low CD36 expression in vascular cells by selective activation of Nrf2 to suppress atherosclerosis development. Acknowledgment We thank Dr. Hisatoyo Kato of Toagosei Co., Ltd. for his kind supply of shogaol derivatives. This study was partly supported by the Academic Frontier Research Project on “New Frontier of Biomedical Engineering Research” of the Ministry of Education, Culture, Sports, Science and Technology. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. atherosclerosis.2012.08.023.
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Enhanced CD36 expression changes the role of Nrf2 activation from anti-atherogenic to pro-atherogenic in apoE-deficient mice Hirotaka Sawada, Yoshiro Saito, Noriko Noguchi* Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakodani, Tatara, Kyotanabe, Kyoto 610-0394, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 May 2012 Received in revised form 31 July 2012 Accepted 14 August 2012 Available online 24 August 2012
Oxidative stress has been implicated as a causative factor of atherosclerosis. Defense systems against oxidative stress are maintained by radical scavenging antioxidants and/or by regulating the expression of antioxidant genes by activating oxidative stress-sensitive transcription factor: nuclear factor (erythroidderived 2)-like 2 (Nrf2). We investigated the anti-atherogenic effects of three synthesized compounds (shogaol A: radical scavenging antioxidant activity; shogaol N: Nrf2-activating activity; shogaol N þ A: both activities) and curcumin (both activities) in apolipoprotein E (apoE)-deficient mice. We expected compounds with both activities to have additive or synergistic anti-atherogenic effects; however, atherosclerosis was exacerbated significantly by curcumin and slightly by shogaol N þ A. Shogaol A, shogaol N, and shogaol N þ A showed no significant effect on atherosclerosis development. Immunohistochemical analysis of the aorta revealed that expression of CD36, an Nrf2-regulated gene, was strongly induced by treatment with curcumin. The total antioxidant capacity of plasma collected from mice administered the three compounds was evaluated using a hydrophilic probe, pyranine. Shogaol N or shogaol N þ A significantly enhanced the antioxidant capacity of plasma, whereas shogaol A and curcumin did not show this activity. The concentrations of the three shogaol derivatives in plasma were similar (approximately 100 nM), while that of curcumin was much lower. These results suggest that plasma antioxidant capacity is maintained at high levels via Nrf2 activation and that CD36 expression enhances atherosclerosis development. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Oxidative stress Radical scavenging antioxidant Nrf2 CD36 Atherosclerosis ApoE deficient
1. Introduction Oxidative modification of low-density lipoprotein (LDL) is a key initial event in the pathogenesis of atherosclerosis [1]. Monocytederived macrophages actively uptake oxidized LDL via scavenger receptors, resulting in the formation of foam cells and fatty streaks. In vivo there are two complementary antioxidant systems that function to prevent LDL oxidation. One is the direct radical scavenging activity of low-molecular weight antioxidants and the other is indirect antioxidant activity [2], which enhances the expression of antioxidative enzymes and cytoprotective proteins through activation of the oxidative-stress sensing transcription factor Nrf2 [3]. Several natural and synthetic antioxidants have been shown to prevent LDL oxidation both in vitro and in vivo [4]. Therapeutic approaches aimed at suppressing atherosclerosis development by inhibiting LDL oxidation have also been examined. Radical scavenging activity is attributed to the phenolic group of antioxidants * Corresponding author. Tel./fax: þ81 774 65 6262. E-mail addresses: [email protected] (H. Sawada), ysaito@ mail.doshisha.ac.jp (Y. Saito), [email protected] (N. Noguchi). 0021-9150/$ e see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2012.08.023
[5]. Natural phenolic antioxidants such as vitamin E and vitamin C are known to inhibit LDL oxidation; however, the results of several long-term clinical trials have been controversial regarding the effect of vitamin E and/or vitamin C intake on the risk of cardiovascular events [6,7]. In contrast, synthetic radical scavenging antioxidants such as probucol have been shown to inhibit atherosclerosis by inhibiting LDL oxidation [8]. Nrf2, a redox-sensing transcription factor, is inactivated in the cytosol when bound to Kelch-like ECH-associated protein 1 (Keap1) under normal conditions. Keap1 is redox-sensitive because its cysteine groups can be easily oxidized, resulting in dissociation of Nrf2 from Keap1 [9], allowing translocation of Nrf2 to the nucleus [10]. The promoter regions of several genes encoding antioxidative enzymes and cytoprotective proteins, such as heme oxygenase 1 (HO-1), glutamate cysteine ligase catalysis and modifier (GCLC, GCLM), and solute carrier family 7A number 11 (xCT), contain antioxidant response elements (ARE) to which Nrf2 can bind [3]. Some dietary compounds, such as sulforaphane and curcumin, contain isothiocyanate and a,b-unsaturated carbonyl functional groups, respectively, which covalently bind to cysteine residues on Keap1 to enhance expression of Nrf2-regulated genes [2,11].
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Vascular endothelial cells are constantly subjected to mechanical shear stress due to blood flow. Atherosclerotic lesions are likely to develop focally at bifurcations and branch points in vessels [12]. Most atherosclerosis-prone regions are exposed to nonunidirectional, disturbed, or oscillatory flow; in contrast, atherosclerosis-resistant regions are exposed to unidirectional laminar flow [13,14]. Nrf2 is activated at atherosclerosis-resistant regions exposed to unidirectional and laminar flow, whereas Nrf2 is diffuse in atherosclerosis-prone regions exposed to disturbed flow [15]. On the other hand, the formation of atherosclerotic lesions was attenuated in apoE/Nrf2 double-deficient mice compared to in apoE-deficient mice. Enhanced expression of the scavenger receptor CD36 via Nrf2 activation has been suggested to contribute to atherogenesis [16e18]. Thus, the role of Nrf2 in the pathology of atherosclerosis remains unclear. To evaluate the importance of radical scavenging activity and Nrf2-activating activity in atherogenesis, we investigated the effects of administration of three synthesized compounds (shogaol A: radical scavenging antioxidant activity; shogaol N: Nrf2activating activity; shogaol N þ A: both activities) and curcumin (both activities) on atherosclerosis development and total antioxidant capacity in apoE-deficient mice. 2. Materials and methods
accordance with the regulations established by the Ethics Review Committee for Animal Experiments of Doshisha University. ApoEdeficient mice were divided into 5 groups at 5 weeks of age, and 12 of each group were placed on a pellet diet containing 0.5 w/w% shogaol N, shogaol A, shogaol N þ A, curcumin, or no drug (control group) for 15 weeks (approximately 100 days). The preparation of diet is described in the Supplement section. 2.5. Determination of plasma concentration Blood samples were collected from the caudal vein and plasma concentrations of compounds were determined using liquid chromatography/tandem mass spectrometry (LC-MS/MS) as described in the Supplement section. 2.6. Quantification of atherosclerotic lesions Atherosclerotic lesion severity in aortas was assessed as described previously [20] and in the Supplement section. 2.7. Immunohistochemistry Paraffin-embedded sections (5 mm) of each left common carotid artery were stained immunohistochemically with an anti-CD36 mAb as described previously [18] and in the Supplement section.
2.1. Chemicals 2.8. Statistical analysis Shogaol derivatives (E)-12-(3-methoxy-4-(methoxymethoxy) phenyl)-10-oxododec-8-enoic acid ethyl ester (shogaol N, Fig. 1A), 8-(3-(4-hydroxy-3-methoxyphenyl) propanamido) octanoic acid ethyl ester (shogaol A, Fig. 1B), (E)-12-(4-hydroxy-3methoxyphenyl)-10-oxododec-8-enoic acid ethyl ester (shogaol N þ A, Fig. 1C), and their de-esterified (de-ethylated) compounds were provided by Toagosei (Nagoya, Japan). Curcumin (Fig. 1D) and pyranine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Other chemicals were obtained as described in the Supplement. 2.2. THP-1 cell culture, RNA isolation, and determination of antioxidant genes THP-1 cells, a human monocyte leukemia cell line, were cultured in RPMI-1640 medium supplemented with 10% heatinactivated FBS, penicillin (100 U/mL), streptomycin (0.1 mg/mL) in a humidified atmosphere of 5% CO2 and 95% air at 37 C. To induce differentiation of THP-1 monocytes into macrophages, cells (2 106 cells/6-cm dish) were treated with PMA (0.1 mM) for 48 h. Expression of target genes was determined using real-time polymerase chain reaction (PCR) as described previously [16] and in the Supplement section. 2.3. Measurement of radical scavenging activity in plasma Free radical scavenging activity of each compound was assessed as described previously [19] and in the Supplement section. 2.4. Animals Four-week-old male C57BL/6 mice and apolipoprotein E (apoE)deficient mice (C57BL/6.KOR-Apoeshl) were obtained from SLC Japan (Shizuoka, Japan) and fed a normal rodent chow diet obtained from Oriental Yeast (Tokyo, Japan) and water for 1 week after purchase. Mice were maintained under controlled temperature and humidity, with 15e20 fresh air changes per hour and a 12-h light/dark cycle. All experimental procedures and protocols were performed in
Data are reported as the mean, standard error, and number of experiments. Statistical significance of the difference between experiments was calculated using analysis of variance (ANOVA) with Dunnett’s test used for multiple comparisons. Values of p < 0.05 were considered significant. 3. Results 3.1. Structure and stability of compounds used in this study Structures of the compounds used in this study are shown in Fig. 1AeD. Shogaol N (Fig. 1A), but not shogaol A (Fig. 1B), is classified as an a,b-unsaturated carbonyl compound. Shogaol A, but not shogaol N, contains an antioxidative phenolic group. Shogaol N þ A and curcumin possess both structures (Fig. 1C and D). For preliminary evaluation of biological stability, these compounds were incubated in C57BL/6 mouse plasma at 37 C for 4 h. Shogaol N, shogaol A, and shogaol N þ A were rapidly hydrolyzed to their deesterified (de-ethylated) compounds in vitro with the halftimes of 0.966 min, 0.531 min, and 0.564 min, respectively (Fig. S1). Shogaol derivatives and these de-ethylated compounds were used in the present study. 3.2. Characterization of Nrf2 activating activity of shogaol derivatives and curcumin To identify the Nrf2-activating activity of the a,b-unsaturated carbonyl functional group present in shogaol derivatives and curcumin, we investigated the effect of this group on the up-regulation of Nrf2-regulated cell defense genes such as HO-1 and xCT in PMAdifferentiated THP-1 macrophages. Cells were treated with each deethylated shogaol derivative and curcumin at 10 mM for 6 h. PGJ2 was used as a positive control to activate the Keap1-Nrf2 pathway. Real-time PCR analysis revealed that de-ethylated shogaol N, de-ethylated shogaol N þ A, and curcumin, which possess an a,bunsaturated carbonyl functional group, significantly increased HO-1 and xCT mRNA expression in PMA-differentiated THP-1
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Fig. 1. Chemical structures of shogaol derivatives (AeC) and curcumin (D). (E, F) Change in HO-1 (E) and xCT (F) expression by shogaol derivatives and curcumin in PMAdifferentiated THP-1 macrophages. Macrophages were incubated with each compound or PGJ2 at 10 mM for 6 h. Changes in HO-1 (E) and xCT (F) expression were measured using real-time PCR (mean SEM, n ¼ 6) (*: p < 0.05; ***: p < 0.001; Dunnett, ANOVA).
macrophages (Fig. 1E and F). In contrast, de-ethylated shogaol A, which is not an a,b-unsaturated carbonyl compound, had no effects on HO-1 and xCT expression. NQO-1 mRNA expression increased following treatment with de-ethylated shogaol N and de-ethylated shogaol N þ A; however, GCLM expression did not change significantly (Fig. S2). These results indicate that the a,b-unsaturated carbonyl functional group of de-ethylated shogaol N, de-ethylated shogaol N þ A, and curcumin is essential for the up-regulation of Nrf2-regulated genes.
3.3. Characterization of radical scavenging activity of shogaol derivatives and curcumin The antioxidant effects of shogaol derivatives, curcumin, and atocopherol against peroxyl radicals were assessed in an in vitro system using mouse plasma. Pyranine was used as a reference probe to evaluate the radical scavenging activity of the tested compounds. Pyranine is a convenient probe because it produces a clear lag phase in the presence of antioxidants due to its low reactivity toward free
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radicals compared to other antioxidants. In the absence of plasma, pyranine was consumed at a constant rate with no lag phase when free radicals were generated through thermal decomposition of AAPH (Fig. 2A). Addition of mouse plasma (10 vol% in the system) completely inhibited pyranine consumption and produced a clear lag phase because endogenous antioxidants present in the plasma scavenged free radicals faster than scavenging by pyranine. After the lag phase, pyranine was consumed at the same rate as that without plasma. The lag phase is directly proportional to the amount of radical scavenging antioxidant present. In fact, a-tocopherol extended the lag phase in a concentration-dependent manner (data not shown). In accordance with the activity of a-tocopherol, addition of shogaol A or shogaol N þ A, which contain an antioxidative phenolic group, completely inhibited pyranine consumption and produced a clear lag phase. In contrast, shogaol N, with no phenolic group, did not exhibit antioxidant activity. The lag phase of each compound at 10 and 100 mM is summarized in Fig. 2B. These results clearly indicate that shogaol A, shogaol N þ A, and their de-ethylated compounds possess radical scavenging activity, whereas this activity was not detected for shogaol N. The antioxidant activity of curcumin, which reportedly has radical scavenging activity [21], could not be determined using this system because of its low solubility in the range of concentrations evaluated.
3.4. Determination of plasma concentrations of shogaol derivatives and curcumin Plasma was collected from apoE-deficient mice fed a normal rodent chow diet containing 0.5 w/w% shogaol derivatives or
curcumin, and plasma concentrations of each compound were determined using LC-MS/MS analysis. Shogaol derivatives and their de-ethylated compounds were detected 18 h after feeding. Plasma concentrations of these compounds were constant over the entire sampling interval (Fig. 3AeC). Although the concentration ratio of shogaol derivatives to their de-ethylated compounds was not consistent, total concentration was approximately 100 nM in the plasma. The plasma concentration of curcumin was maintained at approximately 20 nM, which was 5 times lower than that of the concentration of shogaol derivatives (Fig. 3D).
3.5. Evaluation of radical scavenging activity in plasma of apoEdeficient mice fed on a diet containing shogaol derivatives or curcumin To evaluate the in vivo antioxidant activity of shogaol derivatives and curcumin, plasma collected from apoE-deficient mice fed a normal diet containing the compounds was assessed as described above. Plasma collected from the shogaol N- and shogaol N þ Atreated groups had longer lag times than that of the control group (Fig. 3E). In contrast, the shogaol A- and curcumin-treated groups showed no apparent increase in lag time compared to the control group. 3.6. Effects of shogaol derivatives and curcumin on the development of atherosclerotic lesions in apoE-deficient mice To evaluate the anti-atherogenic effects of shogaol derivatives and curcumin, atherosclerotic lesions in apoE-deficient mice treated with each compound were evaluated using Sudan III staining. Protective effects of shogaol derivatives against atherosclerosis development were not observed at 15 weeks after feeding of a normal diet containing each compound. Against our expectations, atherosclerosis was exacerbated significantly by curcumin and slightly by shogaol N þ A (Fig. 4AeE). The quantitative analysis of atherosclerosis lesion in the aortic root, ascending aorta, and aortic arch was conducted. As a result, statistically significant difference between control and curcumin was observed in the plaque area visualized by en face Sudan III staining (1.94 0.79 mm2 vs. 4.83 2.85 mm2, p < 0.05) (Fig. 4F). A significant increase in the percentage of atherosclerotic plaques in the curcumin-treated group was also observed in the aortic root plaque area (Fig. 4G).
3.7. Effects of curcumin on CD36 expression in the aortic sinus and THP-1 macrophages
Fig. 2. In vitro evaluation of radical scavenging activities of shogaol derivatives. (A) The reaction was initiated by the addition of 50 mM AAPH at 37 C. Bleaching of pyranine with or without antioxidants was conducted as described in Materials and methods and the results were plotted as the ratio of absorbance at time t to time zero. (B) The lag time produced by the indicated concentrations of shogaol derivatives was plotted.
The scavenger receptor CD36, an Nrf2-regulated gene, is reportedly responsible for the uptake of oxidized LDL in macrophages, and thus is associated with atherosclerotic plaque development in the aortic sinus. Therefore, we identified the presence of CD36 in the left common carotid artery using immunohistochemical analysis. Expression of CD36 was not observed in the aortic sinus obtained in mice fed a normal diet containing vehicle (control) and shogaol derivatives (Fig. 5AeD). In contrast, CD36positive areas were observed in the vascular endothelium of the curcumin-treated group (white arrows; Fig. 5E). The effects of curcumin on CD36 expression were further examined using PMAdifferentiated THP-1 macrophages. Treatment of PMAdifferentiated THP-1 macrophages with curcumin significantly increased CD36 expression (1.0 vs. 2.16 1.15, ratio of arbitrary unit to the control group, p < 0.05) (Fig. 5F).
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Fig. 3. Plasma concentrations of shogaol derivatives and curcumin and their radical scavenging antioxidant capacity in the plasma of apoE-deficient mice fed with diet containing these compounds. (AeD) Plasma concentrations of shogaol derivatives (AeC), and curcumin (D) were determined as described in Materials and methods (n ¼ 2). (E) The radical scavenging antioxidant capacity in each plasma sample was evaluated based on pyranine consumption induced by AAPH. Mean values of estimated lag time are shown (n ¼ 2).
4. Discussion We investigated the anti-atherogenic effects of compounds with radical-scavenging and/or Nrf2-activating activities in apoEdeficient mice fed a normal rodent chow diet. Evaluated compounds showed no significant suppressive effects on atherosclerosis development. Surprisingly, atherosclerotic lesions were increased by administration of curcumin, which has both radical scavenging and Nrf2-activating activities. Previous studies demonstrated the inhibitory effects of curcumin on atherosclerosis in mice deficient in the LDL receptor [22], apoE [23], and both the LDL receptor and apoE [24]. In these studies, mice were fed a highfat diet, whereas in our study, mice were fed a normal chow diet. The amount of curcumin used in this study was significantly higher than that used in previous studies (10e300 vs. 1000 mg/kg/day, respectively). Changes in plasma levels of triglycerides and total cholesterol were not significant in this study, which is in contrast to the results of previous studies [23,25] (Fig. S3). Since the body
weight of all mice used in this study increased at a good rate (Fig. S4) and there were not significant changes in plasma levels of alanine aminotransferase and aspartate aminotransferase in these mice (data not shown), the toxic effects of all compounds including curcumin seemed not to be serious. It is very interesting to compare the atherogenic effects of curcumin at several different doses using apoE-deficient mice with a normal diet and a high-fat diet. CD36 expression was found to be enhanced by treatment with curcumin in some atherosclerotic lesions. Further, it was shown that CD36 was significantly up-regulated in PMA-differentiated THP-1 macrophages treated with curcumin. CD36 has been shown to contribute to atherosclerosis development via Nrf2 activation in apoE/Nrf2 double-deficient mice [17,18]. We confirmed the importance of CD36 up-regulation in atherogenesis through administration of an Nrf2-activating compound to apoE-deficient mice. Expression of CD36 is regulated by not only Nrf2 [26] but also by PPARg [27] or FOXO3a [22]. Since curcumin has been reported to contribute to activation of PPARg [28] and FOXO3a [22],
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Fig. 4. Effects of shogaol derivatives and curcumin on atherosclerotic lesion development in apoE-deficient mice. (AeE) Atherosclerotic lesions were evaluated using Sudan III staining in apoE-deficient mice fed a normal pelleted diet supplemented without the drug (A) as a control or with shogaol N (B), shogaol A (C), shogaol N þ A (D), or curcumin (E) for 15 weeks. (F, G) The extent of Sudan III-positive areas was measured (F) and expressed as percentage of the total area, including the descending thoracic aorta (G) (mean SEM, n ¼ 6). (*: p < 0.05; Dunnett, ANOVA).
CD36 expression in atherosclerotic lesions may be enhanced via these routes. Similarly to curcumin, shogaol N þ A has both radical scavenging and Nrf2-activating activities and increased the number of atherosclerotic plaques; however, this increase was not significant. In contrast, shogaol N treatment induced neither CD36 expression nor atherosclerosis. This may be due to the high antioxidant capacity of plasma during feeding of a diet containing shogaol N or shogaol N þ A (Fig. 3E). It is well known that LDL oxidation is an initial event in atherogenesis and that oxLDL is taken up by macrophages via scavenger receptors [1]. Furthermore, oxLDL is known to induce CD36 expression [29]. These results suggest that radical scavenging antioxidant capacity is effectively increased via Nrf2 activation. Therefore, LDL oxidation was inhibited in mice treated with shogaol N or shogaol N þ A. The concentration of curcumin was likely not sufficient to induce antioxidative enzymes
via Nrf2 activation. We confirmed the poor bioavailability of curcumin in a Caco-2 cell monolayer permeability study which was an established method for predicting intestinal absorption, although no metabolites of curcumin were identified in the present study. The permeability coefficients of shogaol N, shogaol A, shogaol N þ A, and curcumin were 53.7, 16.6, 47.2, and 2.69 106 cm/s, respectively. It is important to identify the plasma components responsible for the radical scavenging antioxidant capacity of mice treated with shogaol N or shogaol N þ A. Shogaol A scavenges free radicals, indicating that administration of shogaol A may extend the lag time when measuring antioxidant capacity. However, plasma concentration levels of shogaol A were too low (0.2e0.3 mM, shown in Fig. 3B) to increase the lag time since at least 1 mM was required for evaluation in the present assay. This implies that direct radical scavenging compounds should be maintained at higher concentrations in plasma than Nrf2-activating
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Fig. 5. Effects of shogaol derivatives and curcumin on CD36 expression in aortic sinus and THP-1 macrophages. (AeE) Expression of CD36 was evaluated using immunohistochemistry in the aortic sinus obtained from apoE-deficient mice fed a normal pelleted diet supplemented without the drug (A) as a control or with shogaol N (B), shogaol A (C), shogaol N þ A (D), or curcumin (E-1, 2) for 15 weeks. The white arrows represent regions yielding high fluorescence intensity on enlarged views (E-2). (F) Altered CD36 expression by shogaol derivatives and curcumin in PMA-differentiated THP-1 macrophages was measured using quantitative PCR (mean SEM, n ¼ 6). Macrophages were incubated with each compound at 10 mM for 6 h and harvested for RNA isolation and real-time PCR analysis (*: p < 0.05; ***: p < 0.001; Dunnett, ANOVA).
compounds to suppress atherosclerosis in vivo. Thus, radical scavenging antioxidant capacity can be maintained by keeping a low concentration of Nrf2-activating drugs in the plasma. 5. Conclusions In conclusion, the present study demonstrated the importance of maintaining a high antioxidant capacity in the plasma and low CD36 expression in vascular cells by selective activation of Nrf2 to suppress atherosclerosis development. Acknowledgment We thank Dr. Hisatoyo Kato of Toagosei Co., Ltd. for his kind supply of shogaol derivatives. This study was partly supported by the Academic Frontier Research Project on “New Frontier of Biomedical Engineering Research” of the Ministry of Education, Culture, Sports, Science and Technology. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. atherosclerosis.2012.08.023.
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