Asperlin Inhibits LPS-Evoked Foam Cell Formation and Prevents Atherosclerosis in ApoE-/- Mice

Abstract

Asperlin is a marine-derived natural product with antifungal and anti-inflammatory activities in vitro. In the present study, we isolated asperlin from a marine Aspergillus versicolor LZD4403 fungus and investigated its anti-atherosclerotic effects in vitro and in vivo. Asperlin significantly inhibited lipopolysaccharides (LPS)- but not oxidated low-density lipoprotein (oxLDL)-evoked foam cell formation and promoted cholesterol efflux in RAW264.7 macrophages. Supplementation with asperlin also suppressed LPS-elicited production of pro-inflammatory factors in RAW264.7 macrophages, decreased the expression levels of iNOS, IL-1β and TNFα, and increased the expression of IL-10 and IL-4, indicating a remarkable shift in M1/M2 macrophages polarization. In vivo experiments in high-fat diet (HFD)-fed ApoE^-/- mice showed that oral administration of asperlin for 12 weeks remarkably suppressed atherosclerotic plaque formation in the aorta, as revealed by the reduced aortic dilatation and decreased atherosclerotic lesion area. Asperlin also decreased serum levels of pro-inflammatory factors but showed little impact on blood lipids in ApoE^-/- atherosclerotic mice. These results suggested that asperlin is adequate to prevent atherosclerosis in vivo. It may exert atheroprotective function through suppressing inflammation rather than ameliorating dyslipidemia.

Keywords: M1/M2 polarization; asperlin; atherosclerosis; foam cell; inflammation; macrophage.

Figure 1

Structure of asperlin.

Figure 2

Asperlin suppressed lipopolysaccharide (LPS)-induced foam cell formation in RAW264.7 macrophages. (A) Typical images. (B) OD 358 nm after oil-red O staining (n = 8). (C) Intracellular levels of triglyceride (TG) (n = 6). Data are shown as means ± SD. ^# p < 0.05, ^## p < 0.01, LPS group vs. Blank group; * p < 0.05, ** p < 0.01, *** p < 0.001, test groups vs. LPS group. Blk: blank; Sim: simvastatin; Asp: asperlin.

Figure 3

Asperlin showed no influence on cell viability of RAW264.7 macrophages. (A) Normal conditions. (B) LPS-elicited conditions. Data are shown as means ± SD (n = 8). ^# p < 0.05, LPS group vs. Blank group.

Figure 4

Asperlin stimulated cholesterol efflux in LPS-treated RAW264.7 macrophages. (A) Cholesterol efflux rate. (B) Realtime PCR of cholesterol efflux-regulating genes. (C) Western blotting of cholesterol efflux-regulating proteins. (D) Quantitative analysis of the western blots grey. Data are shown as means ± SD. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, LPS group vs. Blank group; * p < 0.05, ** p < 0.01, *** p < 0.001, test groups vs. LPS group. Blk: blank; Rosi: rosiglitazone; Asp: asperlin.

Figure 5

Asperlin inhibited LPS-elicited production of inflammatory factor in RAW264.7 macrophages. (A) Nitrite (NO). (B) Interleukin-6 (IL-6). (C) Tumor necrosis factor α (TNF-α). (D) Monocyte chemotactic protein-1 (MCP-1). Data are shown as means ± SD. ^## p < 0.01, ^### p < 0.001, LPS group vs. Blank group; * p < 0.05, ** p < 0.01, *** p< 0.001, test groups vs. LPS group. Blk: blank; Asp: asperlin.

Figure 6

Asperlin regulated macrophage polarization. The transcriptional levels of M1 (A) and M2 (B) marker genes were determined after treatment with asperlin (10 μM) for 24 h. Data are shown as means ± SD. ^# p < 0.05, ^### p < 0.001, LPS group vs. Blank group; * p < 0.05, ** p < 0.01, test groups vs. LPS group. LPS group. Blk: blank; Asp: asperlin.

Figure 7

Asperlin did not inhibit foam cell formation induced by ox-LDL in RAW264.7 macrophages. (A) OD 358nm after oil-red O staining. (B) Intracellular levels of cholesterol (TC). (C) Time-dependent cholesterol uptake. Data are shown as means ± SD. ^## p < 0.01, ^### p < 0.001, ox-LDL group vs. Blank group; * p < 0.05, *** p < 0.001, test groups vs. ox-LDL group. Blk: blank; Rosi: rosiglitazone; Asp: asperlin.

Figure 8

Treatment with Asperlin for 12 weeks prevents high-fat diet (HFD)-induced atherosclerosis in apoE^−/− mice. (A) Atherosclerotic plaques (arrows) in aortic arches. (B) In vivo ultrasound. (C) Measurement of the diameter of the ascending aorta 2 cm above the aortic valve (AV, arrow), the brachiocephalic (BC) artery, left common carotid (LCC) artery and left subclavian (LS) artery. Data are shown as means ± SD. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, apoE^−/− group vs. normal group; * p < 0.05, ** p < 0.01, test groups vs. apoE^−/− group. Asp: asperlin.

Figure 9

Asperlin markedly ameliorates inflammation but shows weak influence on hyperlipidemia in HFD-induced atherosclerotic ApoE^−/− mice. (A) Serum levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1). (B) Serum levels of lipids. Data are shown as means ± SD. ^## p < 0.01, ^### p < 0.001, apoE^−/− group vs. normal group; * p < 0.05, ** p < 0.01, *** p < 0.001, test groups vs. apoE^−/− group. Asp: asperlin.