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DOI 10.1002/mnfr.201500022
RESEARCH ARTICLE
3-polyunsaturated fatty acids suppress lipoprotein-associated phospholipase A2 expression in macrophages and animal models Zhuang Li∗ , Wenzhi Ren∗ , Xiaolei Han, Xingxing Liu, Gangqi Wang, Mingjun Zhang, Daxin Pang, Hongsheng Ouyang and Xiaochun Tang Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, China Scope: 3-polyunsaturated fatty acids (3-PUFAs) have beneficial effects on cardiovascular function, and lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with the risk of cardiovascular disease. Here, we investigated the effects of 3-PUFAs on Lp-PLA2 expression in vitro and in vivo and explored the mechanisms involved. Methods and results: Human monocyticcells (THP-1) were induced into macrophages in an in vitro model. 3-PUFAs suppressed Lp-PLA2 expression; the suppression induced by docosahexaenoic acid (DHA) was related to reduced inflammation. Tumor necrosis factor-␣ (TNF-␣) was employed to stimulate the phosphorylation of p38 mitogen-activated protein kinase (MAPK), nuclear factor-B (NF-B) p65 and Lp-PLA2 expression in macrophages. The stimulation was inhibited by DHA and the anti-inflammatory drug sodium salicylate. Moreover, the stimulation of Lp-PLA2 expression by TNF-␣ could be suppressed by NF-B and MAPK pathway inhibitors. Then, chronic inflammation was induced in an in vivo mouse model, resulting in an increase in Lp-PLA2 expression in peripheral blood mononuclear cells (PBMCs) and arteries. This increase was suppressed by 3-PUFAs. Inhibition of Lp-PLA2 transcription in PBMCs was also observed in 3-PUFA-enriched swine. Conclusion: Our results demonstrate that the protective effects of 3-PUFAs against cardiovascular events may be related to the suppression of Lp-PLA2 levels.
Received: January 9, 2015 Revised: April 25, 2015 Accepted: May 18, 2015
Keywords: Cardiovascular disease / Inflammation / Lipoprotein associated phospholipase A2 / 3-polyunsaturated fatty acids / Signaling pathways
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Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction
Marine 3-PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), exert a protective effect
Correspondence: Xiaochun Tang E-mail: [email protected] Abbreviations: AA, arachidonic acid; ALA, ␣-linolenic; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HFD, highfat diet; LA, linoleic acid; Lp-PLA2 , lipoprotein-associated phospholipase A2; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein 1; NF-B, nuclear factor-Kb; PMA, phorbol-12-myristate-13-acetate; 3-PUFAs, omega-3 polyunsaturated fatty acids; THP-1, human monocytic cell; TNF-␣, tumor necrosis factor-␣ C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
against cardiovascular events. A number of clinical studies has demonstrated that the consumption of 3-PUFAs improves lipoprotein metabolism, cardiac function, endothelial function, vascular reactivity and inflammatory responses in cardiovascular disease (CVD) [1–5]. The inflammation-related mechanisms of 3-PUFAs have been widely investigated, including the inhibition of the activation of NF-B following stimulation with LPS. This inhibition reduces the adhesion of neutrophils to endothelial cells and decreases the levels of inflammatory cytokines produced by activated neutrophils [6]. Additionally, 3-PUFAs suppress the phosphorylation of extracellular signal regulated protein kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2) in ∗ These
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macrophages [7, 8], while 3-PUFA-derived resolvin E1 promotes the clearance of apoptotic neutrophils in inflammatory tissues and reduces the expression of inflammatory cytokines and chemokines [9]. Lp-PLA2 , also known as platelet-activating factor acetylhydrolase (PAFAH), hydrolyzes platelet activating factor (PAF) or related oxidized phospholipids to eliminate inflammatory complexity in vitro and in rodent models [10, 11]. However, evidence has suggested that it binds to circulating lipoproteins and catalyzes the hydrolysis of oxidized low density lipoprotein (LDL) in humans with a truncated sn-2 acyl chain to release inflammatory products: oxidized fatty acids and lysophosphatidylcholine (LysoPC) [12, 13]. These products have been demonstrated to cause pro-inflammatory and proapoptotic effects [14]. Additionally, the plasma concentration of Lp-PLA2 showed a strong and positive association with the risk of coronary events that was first reported by the WOSCOPS (West of Scotland Coronary Prevention Study). This association was not confounded by age, systolic blood pressure, or lipoprotein levels [15], and the same results were obtained in subsequent clinical investigations [16, 17]. Although the data obtained in the first phase 3 clinical trial of darapladib (Lp-PLA2 inhibitor) challenged the notion that Lp-PLA2 was one of the therapeutic targets of CVD [18], investigations into the effects of CVD on biomarkers showed that darapladib decreased IL-6 and high-sensitivity C-reactive protein (hs-CRP) levels by 21.5 and 20.2%, respectively, in the within group comparison following the administration of a 160 mg dose daily for 12 weeks [19]. Additionally, darapladib halted the increase in the necrotic core volume significantly compared with a placebo after12 months of administration [20]. Previous clinical observations indicated that plasma Lp-PLA2 levels were decreased after 3-PUFA consumption in populations with or without CVD [21, 22]. However, there are few reports concerning the in vitro regulation and mechanisms involved. Here, we investigated the effects of 3-PUFAs on the expression of Lp-PLA2 in macrophages, characterized the signaling pathways associated with the inflammatory response and observed the in vivo effects of 3-PUFA consumption on Lp-PLA2 expression in mice and swine.
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Materials and methods
2.1 Reagents DHA, EPA, ALA, AA (arachidonic acid), LA (linoleic acid) and GW9508 were purchased from the Cayman Chemical Company Inc. (USA). Tumor necrosis factor (TNF)-␣ was obtained from Sino Bio Inc. (China). Na-Sal (Sodium salicylate), pyrrolidine dithiocarbamate (PDTC), SB203580 and antibodies against -actin and TNF-␣ were purchased from Abcam Biochemicals Inc. (UK). LPS was obtained from Sigma-Aldrich Inc. (USA). Antibodies against Lp-PLA2 , C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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p38 mitogen-activated protein kinase (MAPK), p-p38 MAPK, NF-B p65 and p-p65 NF-B were purchased from SantaCruz Biotechnology Inc. (USA).
2.2 Cell culture and treatment THP-1 cells (a human acute monocytic leukemia cell line) were maintained at 37⬚C under 5% CO2 in RPMI1640 (Gibco, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA). For the experiments, the RPMI 1640 medium with 10% FBS was replaced with RPMI 1640 medium with 0.5% FBS for 16 h. Then, the THP-1 cells were induced to become macrophages by treatment with 0.05 M phorbol-12-myristate-13-acetate (PMA) for 12 h. Subsequently, to investigate the down regulation of Lp-PLA2 expression by 3-PUFAs, the THP-1 derived macrophages were treated with media containing 0.5% FBS supplemented with DHA (20 M), EPA (20 M), ALA (20 M), AA (20 M) and LA (20 M) for 24 h. To explore the roles of Lp-PLA2 in 3-PUFAs-induced anti-inflammation, the Lp-PLA2 expression plasmid was transfected into THP1 derived macrophages with the FuGENE HD Transfection Reagent (Roche, CH) for 4 h, followed by culture in RPMI 1640 medium with 0.5% FBS for 20 h. Then, medium containing 0.5% FBS was supplemented with DHA, EPA and ALA for another 24 h. To investigate the inflammatory pathways involved in the regulation of Lp-PLA2 expression by 3PUFA supplementation, the macrophages were pretreated with DHA (20 M), NA-Sal (5 mM), GW9508 (2.5 M), PDTC (10 M) and SB203580 (10 M) for 1 h, then incubated with LPS or TNF-␣ (10 ng/ml) for another 24 h. Quantitative realtime PCR and western blot analyses were performed thereafter. Each treatment was performed in triplicate.
2.3 Animals Male C57BL/6J mice were purchased at 6 weeks of age from Yisheng Experimental Animal Science & Technology (Changchun, China) and housed in a 23 ± 3⬚C environment with free access to food and water. After acclimation for 1 week, 18 mice were divided into three groups randomly: a normal diet group as a control (10% of calories from fat, 20% of calories from protein and 70% of calories from carbohydrates); an HFD with casein (HC) group (60% of calories from fat, 20% of calories from protein and 20% of calories from carbohydrates); and an HC with 3-PUFAs group. 3-PUFA supplementation was 25 mg/kg/day on average. The mice in the HC and HC with 3-PUFAs groups received hypodermic injections of 10% casein (ICN Biochemicals, USA) dissolved in physiological saline three times per week, whereas the normal diet group only received physiological saline. Body weight was measured biweekly. Fasting and non-fasting plasma samples were obtained from tail vein blood samples and were collected using anticoagulant EDTA tubes. Plasma TNF-␣, www.mnf-journal.com
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MCP-1 and IL-6 levels were determined at 12 weeks using ELISA Kits (R & D Systems, USA), while fasting plasma total cholesterol (TC) and triglyceride (TG) levels were determined using TC and TG kits, respectively (BioSino, China). Peripheral blood mononuclear cells were isolated from whole blood with Histopaque-1083 (Sigma, USA). At the end of the experiments, the mice were sacrificed via cervical dislocation and their arteries were collected for the detection of Lp-PLA2 mRNA. hFat-1 transgenic swine (Songliao black pig, China), which accumulate higher levels of 3-PUFAs than wild-type swine, were obtained using somatic cell nuclear transfer technology. The hFat-1 transgenic swine were housed in a 23–28⬚C environment with free access to water and were fed three times per day with a normal diet (12% of calories from fat). All animal welfare and experimental procedures were performed strictly according to the guidelines for the care and use of laboratory animals (National Research Council of USA, 1996) and the related ethical regulations of Jilin University. All animal experiments were approved and guided by the Experimental Animal Management Center of Jilin University.
2.4 Quantitative real-time PCR analysis Total RNA from the treated macrophages, mouse and porcine peripheral blood mononuclear cells and mouse arteries was isolated using the TRIzol-A+ reagent (Tiangen, China) according to the manufacturer’s instructions. First-strand cDNAs were synthesized from 1 g of total RNA using the BioRT first-stand cDNA synthesis kit (Bioer, China), and quantitative PCR was performed using the Bioeasy SYBR Green I real-time PCR kit (Bioer, China) with the primers listed in Supporting Information Table S1.
2.5 Western blot analysis For western blot analysis, the treated macrophages were washed with cold phosphate-buffered saline (PBS) and lysed in lysis buffer (Beyotime, China) containing 100× phosphatase inhibitor cocktail and 1 mM phenylmethanesulfonylfluoride (PMSF) for 30 min, followed by centrifugation. Protein concentrations were determined using an enhanced BCA protein assay kit (Beyotime, China). Equal amounts of proteins were separated via 12% SDS–PAGE. Then, the separated proteins were transferred to a nitrocellulose membrane, blocked with 5% (wt/vol) nonfat milk in TBST, probed with antibodies overnight at 4⬚C, washed with TBST three times and incubated with the appropriate secondary antibodies for 1 h at room temperature. The resultant signal was visualized using the Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific, USA), scanned with the Azure c600 Western Blot Imaging System and quantified using Image J. Each treatment was performed in triplicate. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.6 Immunofluorescence Macrophages fixed in 4% paraformaldehyde and arteries in paraffin sections were exposed to primary antibodies against Lp-PLA2 (Abcam, UK) and CD68 (Abcam, UK) for 2 h at 37⬚C, then incubated with the appropriate secondary antibodies (FITC- or Cy3-conjugated IgG, Sigma, USA) for 1 h at 37⬚C. The nuclei were stained with DAPI (Sigma, USA). The immunostained macrophages or arteries were photographed using a Leica SP5 laser-scanning confocal microscope.
2.7 Statistical analysis Data from the macrophages and swine experiments were expressed as the mean ± SEM and analyzed using a two-tailed unpaired t-test with GraphPad Prism software. Data from the mouse experiments were expressed as medians with range and analyzed with One-way ANOVA with GraphPad Prism software. P < 0.05 was considered to be statistically significant.
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Results
3.1 3-PUFAs downregulate the expression of Lp-PLA2 Based on the clinical study demonstrating that 3-PUFA consumption decreased plasma Lp-PLA2 levels, we assessed the effects of 3-PUFAs on the expression of Lp-PLA2 . As shown in Fig. 1, the expression of Lp-PLA2 was suppressed by 3PUFAs (DHA, EPA and ALA), whereas the level of Lp-PLA2 was increased by 6-PUFAs (AA and LA) in THP-1 derived macrophages compared with the control cells (Fig. 1A and B). A mouse model of chronic inflammation was employed to confirm the effects of 3-PUFAs on Lp-PLA2 expression. The combination of HFD with casein injection (HC) stimulated Lp-PLA2 transcription in arteries and PBMCs; this stimulation was significantly suppressed by 3-PUFAs (Fig. 1C and D). Lp-PLA2 transcription was sharply decreased (Fig. 1E) in PBMCs from our previously developed hFat-1-transgenic swine (hFat-1 TG), which accumulated twofold higher 3PUFA levels compared with the wild-type controls (data not shown). These data suggest that -3 PUFAs can suppress the expression of Lp-PLA2 .
3.2 Effect of 3-PUFAs on inflammation occurs via an Lp-PLA2 -independent mechanism Previous reports indicated that 3-PUFAs reduced inflammatory responses in macrophages. We also observed a significant downregulation of TNF-␣, MCP-1and IL-6 in macrophages incubated with 3-PUFAs. Moreover, the www.mnf-journal.com
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Figure 1. 3-PUFAs downregulate the expression of Lp-PLA2 . (A and B) Human monocytic THP-1 cells were pretreated with PMA (0.05 M) for 12 h to induce their differentiation into macrophages, followed by incubation with 3-PUFAs or 6-PUFAs for 24 h in RPMI 1640 medium with 0.5% FBS. The effects of DHA (20M), EPA (20M), ALA (20M), AA (20M) and LA (20M) on Lp-PLA2 levels were detected through Western blotting (A) and immunofluorescence (B) (n = 3). (C and D) C57BL/6J mice we randomly divided into three groups: a normal diet (con) group, high fat diet together with casein injection (HC) group and an HC with 3-PUFAsupplementation (HC+3-PUFAs) group. Lp-PLA2 mRNA levels were determined in the arteries (C) and PBMCs (D). (E) Lp-PLA2 mRNA levels were determined in PBMCs isolated from hFat-1 transgenic swine (hFat-1 Tg, n = 4) and wild-type swine (n = 4). The data in A, B and E were expressed as the mean ± SEM and were analyzed using a two-tailed unpaired t-test with GraphPad Prism software.* P < 0.05, ** P < 0.005 and *** P < 0.0005. The data in C and D were expressed as the median with range and were analyzed with one-way ANOVA with GraphPad Prism software (n = 6). * P < 0.05 and ** P< 0.005.
Lp-PLA2 products acted on oxidized lipoproteins and exerted pro-inflammatory effects. Thus, we explored the effects of Lp-PLA2 on 3-PUFA-induced anti-inflammation in macrophages. As shown in Fig. 2, the concentrations of TNF-␣, MCP-1 and IL-6 decreased upon DHA treatment in THP-1 derived macrophages; however, Lp-PLA2 expression did not reverse the DHA-induced suppression effect (Fig. 2A– C). These results suggested that 3-PUFAs decreased the inflammatory response of macrophages through an Lp-PLA2 independent mechanism.
3.3 3-PUFA-modulated downregulation of Lp-PLA2 is associated with inflammation Previous reports by our group and other authors demonstrated that the NF-B and MAPK pathways mediated the regulation of Lp-PLA2 expression; these two pathways are closely related to inflammatory responses. Therefore, we examined the possibility that these inflammatory pathways were involved in the regulation of Lp-PLA2 expression by 3-PUFAs in THP-1 derived macrophages. LPS or TNF-␣ stimulated
Figure 2. The effect of 3-PUFAs on inflammation occurs via an Lp-PLA2 -independent mechanism. (A–C) The Lp-PLA2 expression plasmid was transiently transfected into PMA-induced macrophages using FuGENE HD for 24 h, followed by incubation with 3-PUFAs (20 M DHA, 20 M EPA and 20 M ALA) for another 24 h. The inflammatory cytokines (TNF-␣, MCP-1 and IL-6) secreted by macrophages into the medium were detected via ELISA. Data were expressed as the mean ± SEM and analyzed using a two-tailed unpaired t-test with GraphPad Prism software (n = 3). * P < 0.05, ** P < 0.005 and *** P < 0.0005. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. The 3-PUFA-modulated downregulation of Lp-PLA2 is associated with inflammation. Human monocytic THP-1 cells were pretreated with PMA (0.05 M) for 12 h, and then incubated with LPS (1 g/ml) or TNF-␣ (10 ng/ml) for 1 h prior to treatment with DHA (20 M), Na-Sal (5 mM), GW9508 (2.5 M), PDTC (10 M) and SB203580 (10 M). (A and B) Lp-PLA2 and TNF-␣ protein levels were detected by Western blotting and quantified with Image J. (C) Effects of DHA and Na-Salon TNF-␣-induced Lp-PLA2 mRNA levels. (D and E) Effects of DHA and different signaling pathway inhibitors (GW9508/PDTC/SB203580) on TNF-␣-induced Lp-PLA2 mRNA or protein levels. (F) Effects of DHA and Na-Sal on the TNF-␣-induced protein levels of p-p38 MAPK, p-p65 NF-B, total p38 MAPK and p65 NF-B. Data were expressed as the mean ± SEM and analyzed using a two-tailed unpaired t-test with GraphPad Prism software (n = 3). # P < 0.05, ## P < 0.005 versus con. * P < 0.05, ** P < 0.005 and *** P < 0.0005 versus vehicle.
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Table 1. Effects of 3-PUFAs on inflammation in a mouse model
Control
HFD + casein (HC)
HC + 3- PUFAs
Body weight 2.35 ± 0.22 3.53 ± 0.03*** 2.68 ± 0.08### gain (g) Plasma TC 1.24 ± 0.03 1.76 ± 0.05** 1.45 ± 0.04# (mmol/L) Plasma TG 2.65 ± 0.07 4.51 ± 0.45* 3.53 ± 0.04 (mmol/L) Plasma IL-6 282.6 ± 20.7 299.1 ± 37.3 174.7 ± 4.23## (pg/mL) 44.2 ± 19.2 351.9 ± 19.2* Plasma 63.4 ± 38.4# MCP-1 (pg/mL) 352.5 ± 12.5 577.5 ± 37.5* 365.0 ± 25.0# Plasma TNF-␣ (pg/mL) Data are expressed as medians with range; statistical analysis were performed with one-way ANOVA with GraphPad Prism software (n = 6). * P < 0.05, ** P < 0.005 and *** P < 0.0005 versus control group; # P< 0.05, ## P < 0.005 and ### P < 0.0005 versus HC group.
the expression of Lp-PLA2 , and this stimulation could be suppressed by DHA and the anti-inflammatory drug-Na-Sal (Fig. 3A–C, Supporting Information Figs. S1–3). The stimulation of Lp-PLA2 expression by TNF-␣ was also inhibited by the MAPK inhibitor SB203580 and the NF-B inhibitor PDTC, but not by the GPR120 inhibitor GW9508 (Fig. 3D and E). Further analysis showed that TNF-␣ stimulated the phosphorylation of MAPK p38 and NF-B p65. Additionally, DHA and Na-Sal blocked the phosphorylation of MAPK p38 and NF-B p65 induced by TNF-␣ (Fig. 3F).These results indicate that these inflammatory pathways are involved in the 3-PUFA-induced suppression of Lp-PLA2 .
3.4 3-PUFAs inhibit Lp-PLA2 expression in a chronic inflammatory mouse model A mouse model of chronic inflammation was established to explore the effects of 3-PUFAs on Lp-PLA2 expression levels in vivo. HFD together with casein injection (HC) increased body weight, TC and TG levels compared with the controls, whereas 3-PUFA supplementation (HC with 3-PUFAs group) significantly inhibited body weight gain and TC levels compared with the HC group (Table 1, Fig. 4A–D). Lp-PLA2 expression and the concentrations of TNF-␣ and MCP-1 in plasma were increased significantly in the HC group (1D, 4E and F). Moreover, the HC with 3-PUFAs group exhibited reduced expression levels of inflammatory factors and Lp-PLA2 compared with the HC group (Figs. 1D, 4E–G). Immunofluorescence staining revealed that macrophages that stained positive for Lp-PLA2 accumulated in the arteries of the HC C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
group, while both Lp-PLA2 expression and macrophage accumulation were reduced in the HC with 3-PUFAs group (Fig. 4H). These results indicate that 3-PUFA supplementation inhibits inflammatory cytokines and Lp-PLA2 expression in our mouse model of chronic inflammation.
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Discussion
Here, we report that Lp-PLA2 expression was suppressed by 3-PUFAs (DHA, EPA and ALA) in THP-1 derived macrophages compared with the control. Both our findings and other reports indicate that 3-PUFAs reduce inflammatory responses in macrophages. However, in the present study the inhibition of inflammatory responses by 3-PUFAs occurred via an Lp-PLA2 -independent mechanism. The 3-PUFA-induced suppression of Lp-PLA2 expression in macrophages was associated with inflammation, and the suppression process was mediated by the NF-B and MAPK inflammatory pathways. Moreover, we confirmed the suppressive effects of 3-PUFAs on Lp-PLA2 expression in our chronic inflammatory mouse model as well as in a 3-PUFA-enriched porcine model. In the mouse model, Lp-PLA2 expression was induced by inflammation within macrophages in the arteries, and the increased levels of LpPLA2 and inflammation could be cosuppressed by 3-PUFA supplementation. In vitro studies, animal experiments and observational studies have indicated that the consumption of 3-PUFAs from fish or fish oil improves cardiovascular risk factors such as plasma triglycerides, heart rate and blood pressure and has potential beneficial effects on CVD outcomes. Moreover, EPA and DHA or fish oil inhibit the stimulation of IL-6, TNF-␣ and IL-1 in vitro and in vivo [7, 23, 24]. In the present study, we also observed reduced inflammatory effects of 3-PUFAs in macrophages and in a chronic inflammatory mouse model induced by an HFD together with casein injection. A number of prospective epidemiological studies have reported that the activity or mass of circulating Lp-PLA2 is associated with the risk of vascular disease.Lp-PLA2 binds to circulating lipoproteins and catalyzes the hydrolysis of oxidized low density lipoprotein (LDL) with a truncated sn-2 acyl chain to release oxidized fatty acids and lysophosphatidylcholine (LysoPC) [12, 13]. These products have been demonstrated to exert pro-inflammatory and pro-apoptotic effects. Lp-PLA2 is also considered to be an independent inflammatory predictor of CVD that is not correlated with traditional CVD predictors (i.e. C-reactive protein, white-cell counts and fibrinogen) [15]. 3-PUFA consumption was not found to have any effect on plasma C-reactive protein levels in a previous study [25]. However, plasma Lp-PLA2 levels were significantly decreased by 3-PUFA supplementation (fish or fish oil), and this suppression exerted dose-dependent effects in CVD patients [21, 22]. In the present study, we confirmed that 3-PUFAs suppressed Lp-PLA2 expression both in vitro www.mnf-journal.com
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Figure 4. 3-PUFAs inhibit Lp-PLA2 expression in a chronic inflammatory mouse model. C57BL/6J mice were randomly divided into three different groups: a normal diet (con) group; a high fat diet together with casein injection (HC) group; and HC with 3-PUFA supplementation (HC+3-PUFAs) group. (A and B) Body weight was monitored from the beginning of the diet until the end of the experiment. (C and D) The plasma TC and TG levels in mice were determined at week 12. (E–G) Plasma levels of inflammatory cytokines (TNF-␣, MCP-1 and IL-6) in mice determined by ELISA. (H) Immunofluorescence of CD68 (red) and Lp-PLA2 (green) in the arteries of mice. Data were expressed as medians with range and were analyzed with one-way ANOVA with GraphPad Prism software (n = 6) * P < 0.05, ** P < 0.005 and *** P < 0.0005.
and in vivo. Although Lp-PLA2 exhibited pro-inflammatory effects, it was not involved in inflammatory suppression by 3-PUFAs in macrophages. Conversely, inflammation was involved in the regulation of Lp-PLA2 expression by 3PUFAs in macrophages. We and other researchers have previously reported that TNF-␣ or LPS stimulates Lp-PLA2 expression via the MAPK and NF-B pathways, which play important roles in inflammatory responses [26]. In this study, TNF-␣ was found to stimulate the phosphorylation of MAPK p38 and NF-B p65 and the expression of Lp-PLA2 in macrophages, and this stimulation was suppressed by DHA as well as the anti-inflammatory chemical agent Na-Sal. Moreover, the MAPK and NF-B inhibitors SB203580 and PDTC, respectively, exhibited suppressive effects on Lp-PLA2 expression. TNF-␣ and MCP-1 levels in plasma were significantly increased in our HFD together with casein injection-induced mouse model of chronic inflammation, and this increase was suppressed by 3-PUFA supplementation. IL-6 was not C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
significantly increased, but was still inhibited by 3-PUFAs (Fig. 4G). Immunofluorescence staining also confirmed that Lp-PLA2 was mainly released by inflammatory cells. Lp-PLA2 transcription in PBMCs was dramatically suppressed in our hFat-1 transgenic swine, which accumulate twofold higher levels of 3-PUFA compared to wild-type swine. This result illustrated that suppression of Lp-PLA2 levels by 3-PUFAs was a widely existing phenomenon. Although the Lp-PLA2 inhibitor darapladib did not exhibit efficacy in reducing the risk of the primary composite end point of cardiovascular death in a first phase three clinical trial [18], it decreased IL-6 and high-sensitivity C-reactive protein (hs-CRP) levels by 21.5 and 20.2%, respectively, when a dose of 160 mg was administered daily for 12 weeks [19]. Additionally, darapladib significantly halted the increase in the necrotic core volume compared with a placebo at 12 months [20]. Decreased plasma Lp-PLA2 levels have been observed after the intake of statins and 3-PUFAs in clinical www.mnf-journal.com
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studies [21, 27]. Thus, the investigation of Lp-PLA2 expression and regulation in further studies is essential. In summary, we observed that 3-PUFAs inhibited the expression of Lp-PLA2 in THP-1 cell-derived macrophages as well as in a mouse model of chronic inflammation and in hFat-1-transgenic (hFat-1 TG) swine. Furthermore, we identified the inflammation pathways involved in the suppression process. The findings of this study may contribute to the understanding of the beneficial effects of fish, fish oil or 3-PUFAs on the vascular system. This work was supported by grants from the China National Natural Science Foundation (31472053), the National Natural Science Foundation (31201874) and the China National Key Basic Research Program (973 Program, 2011CBA01000). We thank Li Shen, Guangyao Ran, Shengnan Sun, Yu Zhang and Yunqing Ma for assistance with the experimental animal model. The authors have declared no conflict of interest.
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References
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[9] El Kebir, D., Gjorstrup, P., Filep, J. G., Resolvin E1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation. Proc. Natl Acad. Sci. U S A 2012, 109, 14983–14988. [10] Watson, A. D., Navab, M., Hama, S. Y., Sevanian, A. et al., Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J. Clin. Invest. 1995, 95, 774–782. [11] Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M. et al., Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature 1995, 374, 549–553. [12] Zalewski, A., Macphee, C., Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 923–931. [13] Rosenson, R. S., Stafforini, D. M., Modulation of oxidative stress, inflammation, and atherosclerosis by lipoproteinassociated phospholipase A2. J. Lipid Res. 2012, 53, 1767– 1782. [14] Matsumoto, T., Kobayashi, T., Kamata, K., Role of lysophosphatidylcholine (LPC) in atherosclerosis. Curr. Med. Chem. 2007, 14, 3209–3220. [15] Packard, C. J., O’Reilly, D. S., Caslake, M. J., McMahon, A. D. et al., Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. N. Engl. J. Med. 2000, 343, 1148–1155. [16] Hatoum, I. J., Cook, N. R., Nelson, J. J., Rexrode, K. M., s, Lipoprotein-associated phospholipase A2 activity improves risk discrimination of incident coronary heart disease among women. Am. Heart J. 2011, 161, 516–522. [17] Sabatine, M. S., Morrow, D. A., O’Donoghue, M., Jablonksi, K. A. et al., Prognostic utility of lipoprotein-associated phospholipase A2 for cardiovascular outcomes in patients with stable coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 2463–2469. [18] Investigators, S., White, H. D., Held, C., Stewart, R. et al., Darapladib for preventing ischemic events in stable coronary heart disease. N. Engl. J. Med. 2014, 370, 1702–1711. [19] Mohler, E. R., 3rd, Ballantyne, C. M., Davidson, M. H., Hanefeld, M. et al., The effect of darapladib on plasma lipoproteinassociated phospholipase A2 activity and cardiovascular biomarkers in patients with stable coronary heart disease or coronary heart disease risk equivalent: the results of a multicenter, randomized, double-blind, placebo-controlled study. J. Am. Coll. Cardiol. 2008, 51, 1632–1641. [20] Serruys, P. W., Garcia-Garcia, H. M., Buszman, P., Erne, P. et al., Effects of the direct lipoprotein-associated phospholipase A(2) inhibitor darapladib on human coronary atherosclerotic plaque. Circulation 2008, 118, 1172–1182. [21] Gajos, G., Zalewski, J., Mostowik, M., Konduracka, E. et al., Polyunsaturated omega-3 fatty acids reduce lipoprotein-associated phospholipase A(2) in patients with stable angina. Nutr. Metab. Cardiovasc. Dis 2014, 24, 434–439.
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[22] Steffen, B. T., Steffen, L. M., Liang, S., Tracy, R. et al., n-3 and n-6 Fatty acids are independently associated with lipoprotein-associated phospholipase A2 in the Multi-Ethnic Study of Atherosclerosis. Br. J. Nutr. 2013, 110, 1664–1671. [23] De Caterina, R., Liao, J. K., Libby, P., Fatty acid modulation of endothelial activation. Am. J. Clin. Nutr. 2000, 71, 213S-223S. [24] Kiecolt-Glaser, J. K., Belury, M. A., Andridge, R., Malarkey, W. B. et al., Omega-3 supplementation lowers inflammation and anxiety in medical students: a randomized controlled trial. Brain Behav. Immun. 2011, 25, 1725–1734. [25] Madsen, T., Christensen, J. H., Schmidt, E. B., C-reactive protein and n-3 fatty acids in patients with a previous
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1779 myocardial infarction: a placebo-controlled randomized study. Eur. J. Nutr. 2007, 46, 428–430. [26] Wang, G., Ji, Y., Li, Z., Han, X. et al., Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFkappaB pathways. Sci. Rep. 2014, 4, 4905. [27] Song, J. X., Ren, J. Y., Chen, H., Simvastatin reduces lipoprotein-associated phospholipase A2 in lipopolysaccharide-stimulated human monocyte-derived macrophages through inhibition of the mevalonate-geranylgeranyl pyrophosphate-RhoA-p38 mitogen-activated protein kinase pathway. J. Cardiovasc. Pharmacol. 2011, 57, 213–222.
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DOI 10.1002/mnfr.201500022
RESEARCH ARTICLE
3-polyunsaturated fatty acids suppress lipoprotein-associated phospholipase A2 expression in macrophages and animal models Zhuang Li∗ , Wenzhi Ren∗ , Xiaolei Han, Xingxing Liu, Gangqi Wang, Mingjun Zhang, Daxin Pang, Hongsheng Ouyang and Xiaochun Tang Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun, China Scope: 3-polyunsaturated fatty acids (3-PUFAs) have beneficial effects on cardiovascular function, and lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with the risk of cardiovascular disease. Here, we investigated the effects of 3-PUFAs on Lp-PLA2 expression in vitro and in vivo and explored the mechanisms involved. Methods and results: Human monocyticcells (THP-1) were induced into macrophages in an in vitro model. 3-PUFAs suppressed Lp-PLA2 expression; the suppression induced by docosahexaenoic acid (DHA) was related to reduced inflammation. Tumor necrosis factor-␣ (TNF-␣) was employed to stimulate the phosphorylation of p38 mitogen-activated protein kinase (MAPK), nuclear factor-B (NF-B) p65 and Lp-PLA2 expression in macrophages. The stimulation was inhibited by DHA and the anti-inflammatory drug sodium salicylate. Moreover, the stimulation of Lp-PLA2 expression by TNF-␣ could be suppressed by NF-B and MAPK pathway inhibitors. Then, chronic inflammation was induced in an in vivo mouse model, resulting in an increase in Lp-PLA2 expression in peripheral blood mononuclear cells (PBMCs) and arteries. This increase was suppressed by 3-PUFAs. Inhibition of Lp-PLA2 transcription in PBMCs was also observed in 3-PUFA-enriched swine. Conclusion: Our results demonstrate that the protective effects of 3-PUFAs against cardiovascular events may be related to the suppression of Lp-PLA2 levels.
Received: January 9, 2015 Revised: April 25, 2015 Accepted: May 18, 2015
Keywords: Cardiovascular disease / Inflammation / Lipoprotein associated phospholipase A2 / 3-polyunsaturated fatty acids / Signaling pathways
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Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction
Marine 3-PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), exert a protective effect
Correspondence: Xiaochun Tang E-mail: [email protected] Abbreviations: AA, arachidonic acid; ALA, ␣-linolenic; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HFD, highfat diet; LA, linoleic acid; Lp-PLA2 , lipoprotein-associated phospholipase A2; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein 1; NF-B, nuclear factor-Kb; PMA, phorbol-12-myristate-13-acetate; 3-PUFAs, omega-3 polyunsaturated fatty acids; THP-1, human monocytic cell; TNF-␣, tumor necrosis factor-␣ C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
against cardiovascular events. A number of clinical studies has demonstrated that the consumption of 3-PUFAs improves lipoprotein metabolism, cardiac function, endothelial function, vascular reactivity and inflammatory responses in cardiovascular disease (CVD) [1–5]. The inflammation-related mechanisms of 3-PUFAs have been widely investigated, including the inhibition of the activation of NF-B following stimulation with LPS. This inhibition reduces the adhesion of neutrophils to endothelial cells and decreases the levels of inflammatory cytokines produced by activated neutrophils [6]. Additionally, 3-PUFAs suppress the phosphorylation of extracellular signal regulated protein kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2) in ∗ These
authors contributed equally to this work. www.mnf-journal.com
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macrophages [7, 8], while 3-PUFA-derived resolvin E1 promotes the clearance of apoptotic neutrophils in inflammatory tissues and reduces the expression of inflammatory cytokines and chemokines [9]. Lp-PLA2 , also known as platelet-activating factor acetylhydrolase (PAFAH), hydrolyzes platelet activating factor (PAF) or related oxidized phospholipids to eliminate inflammatory complexity in vitro and in rodent models [10, 11]. However, evidence has suggested that it binds to circulating lipoproteins and catalyzes the hydrolysis of oxidized low density lipoprotein (LDL) in humans with a truncated sn-2 acyl chain to release inflammatory products: oxidized fatty acids and lysophosphatidylcholine (LysoPC) [12, 13]. These products have been demonstrated to cause pro-inflammatory and proapoptotic effects [14]. Additionally, the plasma concentration of Lp-PLA2 showed a strong and positive association with the risk of coronary events that was first reported by the WOSCOPS (West of Scotland Coronary Prevention Study). This association was not confounded by age, systolic blood pressure, or lipoprotein levels [15], and the same results were obtained in subsequent clinical investigations [16, 17]. Although the data obtained in the first phase 3 clinical trial of darapladib (Lp-PLA2 inhibitor) challenged the notion that Lp-PLA2 was one of the therapeutic targets of CVD [18], investigations into the effects of CVD on biomarkers showed that darapladib decreased IL-6 and high-sensitivity C-reactive protein (hs-CRP) levels by 21.5 and 20.2%, respectively, in the within group comparison following the administration of a 160 mg dose daily for 12 weeks [19]. Additionally, darapladib halted the increase in the necrotic core volume significantly compared with a placebo after12 months of administration [20]. Previous clinical observations indicated that plasma Lp-PLA2 levels were decreased after 3-PUFA consumption in populations with or without CVD [21, 22]. However, there are few reports concerning the in vitro regulation and mechanisms involved. Here, we investigated the effects of 3-PUFAs on the expression of Lp-PLA2 in macrophages, characterized the signaling pathways associated with the inflammatory response and observed the in vivo effects of 3-PUFA consumption on Lp-PLA2 expression in mice and swine.
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Materials and methods
2.1 Reagents DHA, EPA, ALA, AA (arachidonic acid), LA (linoleic acid) and GW9508 were purchased from the Cayman Chemical Company Inc. (USA). Tumor necrosis factor (TNF)-␣ was obtained from Sino Bio Inc. (China). Na-Sal (Sodium salicylate), pyrrolidine dithiocarbamate (PDTC), SB203580 and antibodies against -actin and TNF-␣ were purchased from Abcam Biochemicals Inc. (UK). LPS was obtained from Sigma-Aldrich Inc. (USA). Antibodies against Lp-PLA2 , C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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p38 mitogen-activated protein kinase (MAPK), p-p38 MAPK, NF-B p65 and p-p65 NF-B were purchased from SantaCruz Biotechnology Inc. (USA).
2.2 Cell culture and treatment THP-1 cells (a human acute monocytic leukemia cell line) were maintained at 37⬚C under 5% CO2 in RPMI1640 (Gibco, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA). For the experiments, the RPMI 1640 medium with 10% FBS was replaced with RPMI 1640 medium with 0.5% FBS for 16 h. Then, the THP-1 cells were induced to become macrophages by treatment with 0.05 M phorbol-12-myristate-13-acetate (PMA) for 12 h. Subsequently, to investigate the down regulation of Lp-PLA2 expression by 3-PUFAs, the THP-1 derived macrophages were treated with media containing 0.5% FBS supplemented with DHA (20 M), EPA (20 M), ALA (20 M), AA (20 M) and LA (20 M) for 24 h. To explore the roles of Lp-PLA2 in 3-PUFAs-induced anti-inflammation, the Lp-PLA2 expression plasmid was transfected into THP1 derived macrophages with the FuGENE HD Transfection Reagent (Roche, CH) for 4 h, followed by culture in RPMI 1640 medium with 0.5% FBS for 20 h. Then, medium containing 0.5% FBS was supplemented with DHA, EPA and ALA for another 24 h. To investigate the inflammatory pathways involved in the regulation of Lp-PLA2 expression by 3PUFA supplementation, the macrophages were pretreated with DHA (20 M), NA-Sal (5 mM), GW9508 (2.5 M), PDTC (10 M) and SB203580 (10 M) for 1 h, then incubated with LPS or TNF-␣ (10 ng/ml) for another 24 h. Quantitative realtime PCR and western blot analyses were performed thereafter. Each treatment was performed in triplicate.
2.3 Animals Male C57BL/6J mice were purchased at 6 weeks of age from Yisheng Experimental Animal Science & Technology (Changchun, China) and housed in a 23 ± 3⬚C environment with free access to food and water. After acclimation for 1 week, 18 mice were divided into three groups randomly: a normal diet group as a control (10% of calories from fat, 20% of calories from protein and 70% of calories from carbohydrates); an HFD with casein (HC) group (60% of calories from fat, 20% of calories from protein and 20% of calories from carbohydrates); and an HC with 3-PUFAs group. 3-PUFA supplementation was 25 mg/kg/day on average. The mice in the HC and HC with 3-PUFAs groups received hypodermic injections of 10% casein (ICN Biochemicals, USA) dissolved in physiological saline three times per week, whereas the normal diet group only received physiological saline. Body weight was measured biweekly. Fasting and non-fasting plasma samples were obtained from tail vein blood samples and were collected using anticoagulant EDTA tubes. Plasma TNF-␣, www.mnf-journal.com
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MCP-1 and IL-6 levels were determined at 12 weeks using ELISA Kits (R & D Systems, USA), while fasting plasma total cholesterol (TC) and triglyceride (TG) levels were determined using TC and TG kits, respectively (BioSino, China). Peripheral blood mononuclear cells were isolated from whole blood with Histopaque-1083 (Sigma, USA). At the end of the experiments, the mice were sacrificed via cervical dislocation and their arteries were collected for the detection of Lp-PLA2 mRNA. hFat-1 transgenic swine (Songliao black pig, China), which accumulate higher levels of 3-PUFAs than wild-type swine, were obtained using somatic cell nuclear transfer technology. The hFat-1 transgenic swine were housed in a 23–28⬚C environment with free access to water and were fed three times per day with a normal diet (12% of calories from fat). All animal welfare and experimental procedures were performed strictly according to the guidelines for the care and use of laboratory animals (National Research Council of USA, 1996) and the related ethical regulations of Jilin University. All animal experiments were approved and guided by the Experimental Animal Management Center of Jilin University.
2.4 Quantitative real-time PCR analysis Total RNA from the treated macrophages, mouse and porcine peripheral blood mononuclear cells and mouse arteries was isolated using the TRIzol-A+ reagent (Tiangen, China) according to the manufacturer’s instructions. First-strand cDNAs were synthesized from 1 g of total RNA using the BioRT first-stand cDNA synthesis kit (Bioer, China), and quantitative PCR was performed using the Bioeasy SYBR Green I real-time PCR kit (Bioer, China) with the primers listed in Supporting Information Table S1.
2.5 Western blot analysis For western blot analysis, the treated macrophages were washed with cold phosphate-buffered saline (PBS) and lysed in lysis buffer (Beyotime, China) containing 100× phosphatase inhibitor cocktail and 1 mM phenylmethanesulfonylfluoride (PMSF) for 30 min, followed by centrifugation. Protein concentrations were determined using an enhanced BCA protein assay kit (Beyotime, China). Equal amounts of proteins were separated via 12% SDS–PAGE. Then, the separated proteins were transferred to a nitrocellulose membrane, blocked with 5% (wt/vol) nonfat milk in TBST, probed with antibodies overnight at 4⬚C, washed with TBST three times and incubated with the appropriate secondary antibodies for 1 h at room temperature. The resultant signal was visualized using the Pierce enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo Scientific, USA), scanned with the Azure c600 Western Blot Imaging System and quantified using Image J. Each treatment was performed in triplicate. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.6 Immunofluorescence Macrophages fixed in 4% paraformaldehyde and arteries in paraffin sections were exposed to primary antibodies against Lp-PLA2 (Abcam, UK) and CD68 (Abcam, UK) for 2 h at 37⬚C, then incubated with the appropriate secondary antibodies (FITC- or Cy3-conjugated IgG, Sigma, USA) for 1 h at 37⬚C. The nuclei were stained with DAPI (Sigma, USA). The immunostained macrophages or arteries were photographed using a Leica SP5 laser-scanning confocal microscope.
2.7 Statistical analysis Data from the macrophages and swine experiments were expressed as the mean ± SEM and analyzed using a two-tailed unpaired t-test with GraphPad Prism software. Data from the mouse experiments were expressed as medians with range and analyzed with One-way ANOVA with GraphPad Prism software. P < 0.05 was considered to be statistically significant.
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Results
3.1 3-PUFAs downregulate the expression of Lp-PLA2 Based on the clinical study demonstrating that 3-PUFA consumption decreased plasma Lp-PLA2 levels, we assessed the effects of 3-PUFAs on the expression of Lp-PLA2 . As shown in Fig. 1, the expression of Lp-PLA2 was suppressed by 3PUFAs (DHA, EPA and ALA), whereas the level of Lp-PLA2 was increased by 6-PUFAs (AA and LA) in THP-1 derived macrophages compared with the control cells (Fig. 1A and B). A mouse model of chronic inflammation was employed to confirm the effects of 3-PUFAs on Lp-PLA2 expression. The combination of HFD with casein injection (HC) stimulated Lp-PLA2 transcription in arteries and PBMCs; this stimulation was significantly suppressed by 3-PUFAs (Fig. 1C and D). Lp-PLA2 transcription was sharply decreased (Fig. 1E) in PBMCs from our previously developed hFat-1-transgenic swine (hFat-1 TG), which accumulated twofold higher 3PUFA levels compared with the wild-type controls (data not shown). These data suggest that -3 PUFAs can suppress the expression of Lp-PLA2 .
3.2 Effect of 3-PUFAs on inflammation occurs via an Lp-PLA2 -independent mechanism Previous reports indicated that 3-PUFAs reduced inflammatory responses in macrophages. We also observed a significant downregulation of TNF-␣, MCP-1and IL-6 in macrophages incubated with 3-PUFAs. Moreover, the www.mnf-journal.com
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Figure 1. 3-PUFAs downregulate the expression of Lp-PLA2 . (A and B) Human monocytic THP-1 cells were pretreated with PMA (0.05 M) for 12 h to induce their differentiation into macrophages, followed by incubation with 3-PUFAs or 6-PUFAs for 24 h in RPMI 1640 medium with 0.5% FBS. The effects of DHA (20M), EPA (20M), ALA (20M), AA (20M) and LA (20M) on Lp-PLA2 levels were detected through Western blotting (A) and immunofluorescence (B) (n = 3). (C and D) C57BL/6J mice we randomly divided into three groups: a normal diet (con) group, high fat diet together with casein injection (HC) group and an HC with 3-PUFAsupplementation (HC+3-PUFAs) group. Lp-PLA2 mRNA levels were determined in the arteries (C) and PBMCs (D). (E) Lp-PLA2 mRNA levels were determined in PBMCs isolated from hFat-1 transgenic swine (hFat-1 Tg, n = 4) and wild-type swine (n = 4). The data in A, B and E were expressed as the mean ± SEM and were analyzed using a two-tailed unpaired t-test with GraphPad Prism software.* P < 0.05, ** P < 0.005 and *** P < 0.0005. The data in C and D were expressed as the median with range and were analyzed with one-way ANOVA with GraphPad Prism software (n = 6). * P < 0.05 and ** P< 0.005.
Lp-PLA2 products acted on oxidized lipoproteins and exerted pro-inflammatory effects. Thus, we explored the effects of Lp-PLA2 on 3-PUFA-induced anti-inflammation in macrophages. As shown in Fig. 2, the concentrations of TNF-␣, MCP-1 and IL-6 decreased upon DHA treatment in THP-1 derived macrophages; however, Lp-PLA2 expression did not reverse the DHA-induced suppression effect (Fig. 2A– C). These results suggested that 3-PUFAs decreased the inflammatory response of macrophages through an Lp-PLA2 independent mechanism.
3.3 3-PUFA-modulated downregulation of Lp-PLA2 is associated with inflammation Previous reports by our group and other authors demonstrated that the NF-B and MAPK pathways mediated the regulation of Lp-PLA2 expression; these two pathways are closely related to inflammatory responses. Therefore, we examined the possibility that these inflammatory pathways were involved in the regulation of Lp-PLA2 expression by 3-PUFAs in THP-1 derived macrophages. LPS or TNF-␣ stimulated
Figure 2. The effect of 3-PUFAs on inflammation occurs via an Lp-PLA2 -independent mechanism. (A–C) The Lp-PLA2 expression plasmid was transiently transfected into PMA-induced macrophages using FuGENE HD for 24 h, followed by incubation with 3-PUFAs (20 M DHA, 20 M EPA and 20 M ALA) for another 24 h. The inflammatory cytokines (TNF-␣, MCP-1 and IL-6) secreted by macrophages into the medium were detected via ELISA. Data were expressed as the mean ± SEM and analyzed using a two-tailed unpaired t-test with GraphPad Prism software (n = 3). * P < 0.05, ** P < 0.005 and *** P < 0.0005. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. The 3-PUFA-modulated downregulation of Lp-PLA2 is associated with inflammation. Human monocytic THP-1 cells were pretreated with PMA (0.05 M) for 12 h, and then incubated with LPS (1 g/ml) or TNF-␣ (10 ng/ml) for 1 h prior to treatment with DHA (20 M), Na-Sal (5 mM), GW9508 (2.5 M), PDTC (10 M) and SB203580 (10 M). (A and B) Lp-PLA2 and TNF-␣ protein levels were detected by Western blotting and quantified with Image J. (C) Effects of DHA and Na-Salon TNF-␣-induced Lp-PLA2 mRNA levels. (D and E) Effects of DHA and different signaling pathway inhibitors (GW9508/PDTC/SB203580) on TNF-␣-induced Lp-PLA2 mRNA or protein levels. (F) Effects of DHA and Na-Sal on the TNF-␣-induced protein levels of p-p38 MAPK, p-p65 NF-B, total p38 MAPK and p65 NF-B. Data were expressed as the mean ± SEM and analyzed using a two-tailed unpaired t-test with GraphPad Prism software (n = 3). # P < 0.05, ## P < 0.005 versus con. * P < 0.05, ** P < 0.005 and *** P < 0.0005 versus vehicle.
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Table 1. Effects of 3-PUFAs on inflammation in a mouse model
Control
HFD + casein (HC)
HC + 3- PUFAs
Body weight 2.35 ± 0.22 3.53 ± 0.03*** 2.68 ± 0.08### gain (g) Plasma TC 1.24 ± 0.03 1.76 ± 0.05** 1.45 ± 0.04# (mmol/L) Plasma TG 2.65 ± 0.07 4.51 ± 0.45* 3.53 ± 0.04 (mmol/L) Plasma IL-6 282.6 ± 20.7 299.1 ± 37.3 174.7 ± 4.23## (pg/mL) 44.2 ± 19.2 351.9 ± 19.2* Plasma 63.4 ± 38.4# MCP-1 (pg/mL) 352.5 ± 12.5 577.5 ± 37.5* 365.0 ± 25.0# Plasma TNF-␣ (pg/mL) Data are expressed as medians with range; statistical analysis were performed with one-way ANOVA with GraphPad Prism software (n = 6). * P < 0.05, ** P < 0.005 and *** P < 0.0005 versus control group; # P< 0.05, ## P < 0.005 and ### P < 0.0005 versus HC group.
the expression of Lp-PLA2 , and this stimulation could be suppressed by DHA and the anti-inflammatory drug-Na-Sal (Fig. 3A–C, Supporting Information Figs. S1–3). The stimulation of Lp-PLA2 expression by TNF-␣ was also inhibited by the MAPK inhibitor SB203580 and the NF-B inhibitor PDTC, but not by the GPR120 inhibitor GW9508 (Fig. 3D and E). Further analysis showed that TNF-␣ stimulated the phosphorylation of MAPK p38 and NF-B p65. Additionally, DHA and Na-Sal blocked the phosphorylation of MAPK p38 and NF-B p65 induced by TNF-␣ (Fig. 3F).These results indicate that these inflammatory pathways are involved in the 3-PUFA-induced suppression of Lp-PLA2 .
3.4 3-PUFAs inhibit Lp-PLA2 expression in a chronic inflammatory mouse model A mouse model of chronic inflammation was established to explore the effects of 3-PUFAs on Lp-PLA2 expression levels in vivo. HFD together with casein injection (HC) increased body weight, TC and TG levels compared with the controls, whereas 3-PUFA supplementation (HC with 3-PUFAs group) significantly inhibited body weight gain and TC levels compared with the HC group (Table 1, Fig. 4A–D). Lp-PLA2 expression and the concentrations of TNF-␣ and MCP-1 in plasma were increased significantly in the HC group (1D, 4E and F). Moreover, the HC with 3-PUFAs group exhibited reduced expression levels of inflammatory factors and Lp-PLA2 compared with the HC group (Figs. 1D, 4E–G). Immunofluorescence staining revealed that macrophages that stained positive for Lp-PLA2 accumulated in the arteries of the HC C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
group, while both Lp-PLA2 expression and macrophage accumulation were reduced in the HC with 3-PUFAs group (Fig. 4H). These results indicate that 3-PUFA supplementation inhibits inflammatory cytokines and Lp-PLA2 expression in our mouse model of chronic inflammation.
4
Discussion
Here, we report that Lp-PLA2 expression was suppressed by 3-PUFAs (DHA, EPA and ALA) in THP-1 derived macrophages compared with the control. Both our findings and other reports indicate that 3-PUFAs reduce inflammatory responses in macrophages. However, in the present study the inhibition of inflammatory responses by 3-PUFAs occurred via an Lp-PLA2 -independent mechanism. The 3-PUFA-induced suppression of Lp-PLA2 expression in macrophages was associated with inflammation, and the suppression process was mediated by the NF-B and MAPK inflammatory pathways. Moreover, we confirmed the suppressive effects of 3-PUFAs on Lp-PLA2 expression in our chronic inflammatory mouse model as well as in a 3-PUFA-enriched porcine model. In the mouse model, Lp-PLA2 expression was induced by inflammation within macrophages in the arteries, and the increased levels of LpPLA2 and inflammation could be cosuppressed by 3-PUFA supplementation. In vitro studies, animal experiments and observational studies have indicated that the consumption of 3-PUFAs from fish or fish oil improves cardiovascular risk factors such as plasma triglycerides, heart rate and blood pressure and has potential beneficial effects on CVD outcomes. Moreover, EPA and DHA or fish oil inhibit the stimulation of IL-6, TNF-␣ and IL-1 in vitro and in vivo [7, 23, 24]. In the present study, we also observed reduced inflammatory effects of 3-PUFAs in macrophages and in a chronic inflammatory mouse model induced by an HFD together with casein injection. A number of prospective epidemiological studies have reported that the activity or mass of circulating Lp-PLA2 is associated with the risk of vascular disease.Lp-PLA2 binds to circulating lipoproteins and catalyzes the hydrolysis of oxidized low density lipoprotein (LDL) with a truncated sn-2 acyl chain to release oxidized fatty acids and lysophosphatidylcholine (LysoPC) [12, 13]. These products have been demonstrated to exert pro-inflammatory and pro-apoptotic effects. Lp-PLA2 is also considered to be an independent inflammatory predictor of CVD that is not correlated with traditional CVD predictors (i.e. C-reactive protein, white-cell counts and fibrinogen) [15]. 3-PUFA consumption was not found to have any effect on plasma C-reactive protein levels in a previous study [25]. However, plasma Lp-PLA2 levels were significantly decreased by 3-PUFA supplementation (fish or fish oil), and this suppression exerted dose-dependent effects in CVD patients [21, 22]. In the present study, we confirmed that 3-PUFAs suppressed Lp-PLA2 expression both in vitro www.mnf-journal.com
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Figure 4. 3-PUFAs inhibit Lp-PLA2 expression in a chronic inflammatory mouse model. C57BL/6J mice were randomly divided into three different groups: a normal diet (con) group; a high fat diet together with casein injection (HC) group; and HC with 3-PUFA supplementation (HC+3-PUFAs) group. (A and B) Body weight was monitored from the beginning of the diet until the end of the experiment. (C and D) The plasma TC and TG levels in mice were determined at week 12. (E–G) Plasma levels of inflammatory cytokines (TNF-␣, MCP-1 and IL-6) in mice determined by ELISA. (H) Immunofluorescence of CD68 (red) and Lp-PLA2 (green) in the arteries of mice. Data were expressed as medians with range and were analyzed with one-way ANOVA with GraphPad Prism software (n = 6) * P < 0.05, ** P < 0.005 and *** P < 0.0005.
and in vivo. Although Lp-PLA2 exhibited pro-inflammatory effects, it was not involved in inflammatory suppression by 3-PUFAs in macrophages. Conversely, inflammation was involved in the regulation of Lp-PLA2 expression by 3PUFAs in macrophages. We and other researchers have previously reported that TNF-␣ or LPS stimulates Lp-PLA2 expression via the MAPK and NF-B pathways, which play important roles in inflammatory responses [26]. In this study, TNF-␣ was found to stimulate the phosphorylation of MAPK p38 and NF-B p65 and the expression of Lp-PLA2 in macrophages, and this stimulation was suppressed by DHA as well as the anti-inflammatory chemical agent Na-Sal. Moreover, the MAPK and NF-B inhibitors SB203580 and PDTC, respectively, exhibited suppressive effects on Lp-PLA2 expression. TNF-␣ and MCP-1 levels in plasma were significantly increased in our HFD together with casein injection-induced mouse model of chronic inflammation, and this increase was suppressed by 3-PUFA supplementation. IL-6 was not C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
significantly increased, but was still inhibited by 3-PUFAs (Fig. 4G). Immunofluorescence staining also confirmed that Lp-PLA2 was mainly released by inflammatory cells. Lp-PLA2 transcription in PBMCs was dramatically suppressed in our hFat-1 transgenic swine, which accumulate twofold higher levels of 3-PUFA compared to wild-type swine. This result illustrated that suppression of Lp-PLA2 levels by 3-PUFAs was a widely existing phenomenon. Although the Lp-PLA2 inhibitor darapladib did not exhibit efficacy in reducing the risk of the primary composite end point of cardiovascular death in a first phase three clinical trial [18], it decreased IL-6 and high-sensitivity C-reactive protein (hs-CRP) levels by 21.5 and 20.2%, respectively, when a dose of 160 mg was administered daily for 12 weeks [19]. Additionally, darapladib significantly halted the increase in the necrotic core volume compared with a placebo at 12 months [20]. Decreased plasma Lp-PLA2 levels have been observed after the intake of statins and 3-PUFAs in clinical www.mnf-journal.com
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studies [21, 27]. Thus, the investigation of Lp-PLA2 expression and regulation in further studies is essential. In summary, we observed that 3-PUFAs inhibited the expression of Lp-PLA2 in THP-1 cell-derived macrophages as well as in a mouse model of chronic inflammation and in hFat-1-transgenic (hFat-1 TG) swine. Furthermore, we identified the inflammation pathways involved in the suppression process. The findings of this study may contribute to the understanding of the beneficial effects of fish, fish oil or 3-PUFAs on the vascular system. This work was supported by grants from the China National Natural Science Foundation (31472053), the National Natural Science Foundation (31201874) and the China National Key Basic Research Program (973 Program, 2011CBA01000). We thank Li Shen, Guangyao Ran, Shengnan Sun, Yu Zhang and Yunqing Ma for assistance with the experimental animal model. The authors have declared no conflict of interest.
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