Chlorogenic acid protects against atherosclerosis in ApoE-/- mice and promotes cholesterol efflux from RAW264.7 macrophages

Abstract

Chlorogenic acid (CGA) is one of the most abundant polyphenols in the human diet and is suggested to be a potential antiatherosclerotic agent due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate the effect of CGA on atherosclerosis development in ApoE(-/-) mice and its potential mechanism. ApoE(-/-) mice were fed a cholesterol-rich diet without (control) or with CGA (200 and 400 mg/kg) or atorvastatin (4 mg/kg) for 12 weeks. During the study plasma lipid and inflammatory parameters were determined. Treatment with CGA (400 mg/kg) reduced atherosclerotic lesion area and vascular dilatation in the aortic root, comparable to atorvastatin. CGA (400 mg/kg) also significantly decreased plasma levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol as well as inflammatory markers. Supplementation with CGA or CGA metabolites-containing serum suppressed oxidized low-density lipoprotein (oxLDL)-induced lipid accumulation and stimulated cholesterol efflux from RAW264.7 cells. CGA significantly increased the mRNA levels of PPARγ, LXRα, ABCA1 and ABCG1 as well as the transcriptional activity of PPARγ. Cholesterol efflux assay showed that three major metabolites, caffeic, ferulic and gallic acids, significantly stimulated cholesterol efflux from RAW264.7 cells. These results suggest that CGA potently reduces atherosclerosis development in ApoE(-/-) mice and promotes cholesterol efflux from RAW264.7 macrophages. Caffeic, ferulic and gallic acids may be the potential active compounds accounting for the in vivo effect of CGA.

Figure 1. Chlorogenic acid (CGA) reduces atherosclerosis development.

Slides of the valve area of the aortic root were stained with hematoxylin and eosin (H&E) (A), and plaque coverage percentage of the total vessel surface area (B) and lesion area (C) were calculated in four sections per mouse starting from the appearance of open aortic valve leaflets. Values are means±SEM (n = 24). *p<0.05, **p<0.01 vs. ApoE^−/− group. Atorv.  =  atorvastatin, CGA  =  Chlorogenic acid.

Figure 2. Treatment with Chlorogenic acid (CGA) reduces aortic dilatation.

(A) In vivo ultrasound for measurement of the diameter of the ascending aorta 2 cm above the aortic valve (AV, arrow) and at the origin of the brachiocephalic (BC) artery were quantified in B and C (n  = 6). Values are means±SEM. *p<0.05, **p<0.01 vs. ApoE^−/− group. Atorv.  =  atorvastatin, CGA  =  Chlorogenic acid.

Figure 3. Treatment with chlorogenic acid (CGA) for 12 weeks reduces the serum levels of interleukin-6 (IL-6) (A), interleukin-8 (IL-8) (B), tumor necrosis factor α (TNFα) (C) and monocyte chemotactic protein-1 (MCP-1) (D) in ApoE^−/− mice.

Values are means±SEM. *p<0.05, **p<0.01, ***p<0.001 vs. ApoE^−/− group. Atorv.  =  atorvastatin, CGA  =  Chlorogenic acid.

Figure 4. Treatment with chlorogenic acid (CGA) reduces intracellular levels of IL-1β (A), IL-6 (B) and TNFα (C) elicited by LPS in RAW264.7 cells.

Values are means ± SEM of at least three experiments. ^### p<0.001 LPS+vehicle group vs. vehicle group; *p<0.05, **p<0.01, ***p<0.001 test group vs. LPS+vehicle group. Atorv.  =  atorvastatin, CGA  =  Chlorogenic acid, LPS  =  lipopolysaccharides.

Figure 5. Chlorogenic acid (CGA) inhibits oxidized low-density lipoprotein (oxLDL)-elicited foam cell formation in RAW264.7 cells.

RAW264.7 cells were elicited by oxLDL for 24 h with or without supplementation of CGA or atorvastatin. Cells were then stained with Oil red O, and the representative staining pictures (A), the aborptance at 358 nm (B), and intracellular totale cholesterol content (C) were acquired. Bar  =  50 µm. Values are means ± SEM of at least three experiments. ^### p<0.001 oxLDL group vs. blank group; *p<0.05, **p<0.01 test group vs. oxLDL group. Atorv.  =  atorvastatin, CGA  =  Chlorogenic acid,

Figure 6. Chlorogenic acid (CGA) stimulates NBD-cholesterol efflux to HDL (A) and ApoA1 (B) in RAW264.7 macrophages.

Cells were equilibrated with NBD-cholesterol for 12 h then incubated in serum-free DMEM medium containing HDL or ApoA1 and indicated concentration of CGA for 6 h. Cholesterol efflux was expressed as a percentage of fluorescence in the medium relative to the total amounts of fluorescence detected in cells and the medium. Rosiglitazone (5 µM) was used as positive control while PPARγ inhibitor GW-9662 (20 µM) was used to test the role of PPARγ in CGA-elicited cholesterol efflux. Values are means ± SEM of at least three experiments. *p<0.05, **p<0.01, ***p<0.001 vs. control. Rosigli  =  rosiglitazone, CGA  =  Chlorogenic acid, GW  =  GW-9662.

Figure 7. Chlorogenic acid (CGA) upregulates the transcriptional expression of PPARγ, LXRα, ABCA1 and ABCG1 in RAW264.7 cells.

Real-time PCR was conducted with gene specific oligonucleotide primers. The amplification of β-actin served as the internal control. Values are means ± SEM of at least three experiments. *p<0.05, **p<0.01, ***p<0.001 vs. control.

Figure 8. Chlorogenic acid (CGA) increases the transcriptional activity of PPARγ.

The transcriptional activity of PPARγ was assessed by transactivation reporter assay in 293T cells. Rosiglitazone (5 µM) was used as positive control. Values are means ± SEM of at least three experiments. *p<0.05, **p<0.01 vs. control. Rosigli  =  rosiglitazone, CGA  =  Chlorogenic acid.

Figure 9. Chlorogenic acid (CGA) metabolites-containing serum from CGA-treated normal mice decreases lipid accumulation and stimulates cholesterol efflux from RAW264.7 cells.

Normal C57BL/6J mice were orally gavaged with 400 mg/kg of CGA or equal volume of distilled water for 3 days and blood was collected at 45 min after the final treatment. 1% (v/v) of serum from CGA-treated normal mice (S_CGA) significantly decreased oxLDL-induced neutral lipid accumulation (A) and total cholesterol (B), and stimulates cholesterol efflux (C) in RAW264.7 cells as compared with that from distilled water-treated animals (S_NC). Values are means ± SEM of at least three experiments. **p<0.01, ***p<0.001.

Figure 10. Caffeic acid, ferulic acid and gallic acid are active in promoting cholesterol efflux from RAW264.7 cell mediated by HDL.

Cells were equilibrated with NBD-cholesterol for 12 h then incubated in serum-free DMEM medium containing HDL and 10 µM of respective compound for 6 h. Cholesterol efflux was expressed as a percentage of fluorescence in the medium relative to the total amounts of fluorescence detected in cells and the medium. Rosiglitazone (5 µM) was used as positive control. Values are means ± SEM of at least three experiments. *p<0.05, **p<0.01, ***p<0.001 vs. control. Rosigli  =  rosiglitazone.