Apigenin attenuates the atherosclerotic lesions through enhancing selective autophagy/lipophagy and promoting RCT process

Abstract

Context: Apigenin, a naturally flavonoid, is reported to have protective effects in chronic and metabolic diseases. But the therapeutic or ameliorative effects of apigenin on atherosclerosis are not known.

Objective: Our study aimed to elucidate the underlying mechanism of apigenin on preventing atherosclerosis by enhancing selective autophagy/lipophagy and promoting RCT process.

Materials and methods: ApoE^-/- mice fed with a high-fat diet (HFD) for 18 weeks were used to establish atherosclerosis model. Oil-Red-O staining of the plaques in the aorta and the heart was used to determine the severity of atherosclerosis. The autophagy flux was evaluated by western blot and reverse transcription quantitative PCR (RT-qPCR). Then triton WR-1339 (TWR) was injected into muscles of C57BL/6 mice, and the role of autophagy was assessed by autophagy inhibitor LY294002 intervention. The transmission electron microscopy (TEM) and immunofluorescence microscopy analysis (IFM) were used to elucidate the lipid-lowering mechanism of apigenin.

Results: In HFD-induced mice, apigenin inhibited the dangerous progression of atherosclerosis through decreasing lipid deposition in plaques, lowering serum and liver lipid contents, activating autophagy and promoting reverse cholesterol transport (RCT). In TWR-induced mice, apigenin reduced the serum and liver lipid levels, enhanced the autophagy flux and increased RCT, but the above effects of apigenin were weakened by LY294002. The TEM and IFM images revealed that apigenin promoted the formation of autophagosomes and the co-localization between autophagy proteins with lipid protein.

Discussion and conclusions: The lipid-lowering effects of apigenin were mediated through promoting RCT and enhancing selective lipophagy, meanwhile it provided a potential therapeutic option for atherosclerosis.

Keywords: Apigenin; dyslipidemia in atherosclerosis; reverse cholesterol transport; selective autophagy/lipophagy.

Figure 1.

Apigenin alleviated the atherosclerotic lesions in ApoE^−/− mice. (A) Flowchart of HFD induced animal model. (B) Histology of oil Red O staining of thoracic aorta lesion (left). Atherosclerotic plaques were visualized by red color. Quantification analyses of atherosclerotic plaque of thoracic aorta lesion was shown in the right (n = 6). (C) Histology of oil Red O-stained coronal sections of heart (left). Quantification analyses of coronal sections in heart (n = 6). *p < 0.05 and **p < 0.01.

Figure 2.

Apigenin reduced the lipid levels in HFD-induced mice. (A) Serum TC, TG and LDL-C concentrations (n = 10). (B) Liver TC, TG and LDL-C concentrations (n = 10). (C) The liver histopathology was analyzed by H&E staining (200× magnification). Morphological investigation includes observation of lipid droplet accumulation and hepatocellular ballooning degeneration (indicated by white arrow). (D) Histological scoring of liver (n = 4). #p < 0.05, ##p < 0.01 vs NC group; *p < 0.05 and **p < 0.01.

Figure 3.

Apigenin activated and promoted autophagy in ApoE^−/− mice. (A) Western blot analysis of ULK1, UVRAG and beclin-1 were shown in left, and graph on the right represented the quantification analysis of autophagy proteins normalized by β-actin (n = 3). (B) Western blot analysis of ATG3, ATG5 and LC3 were shown in left, and graph on the right represented the quantification analysis of autophagy proteins normalized by α-tubulin (n = 3). (C) The mRNA expression levels of UVRAG, LC3B, Beclin-1 and ATG14 were examined by RT-qPCR (n = 10). #p < 0.05, ##p < 0.01 vs NC group; *p < 0.05 and **p < 0.01.

Figure 4.

Apigenin facilitated RCT process in ApoE^−/− mice. (A) Western blot analysis of ABCG5 and SR-BI in ApoE^−/− mice were shown in left, and graph on the right represented the quantification analysis of the proteins normalized by α-tubulin (n = 3). (B) The mRNA expression level of CD36 was examined by RT-qPCR (n = 10). #p < 0.05, ##p < 0.01 vs NC group; *p < 0.05 and **p < 0.01.

Figure 5.

Apigenin alleviated lipid accumulation in TWR-induced mice. (A) Flowchart of TWR-induced animal model. (B) Serum TC, TG and LDL-C concentrations (n = 10). (C) Liver TC, TG and LDL-C concentrations (n = 10). #p < 0.05, ##p < 0.01 vs NC group; *p < 0.05 and **p < 0.01.

Figure 6.

Apigenin alleviated lipid accumulation in TWR-induced mice. (A) The liver histopathology was analyzed by H&E staining in C57BL/6 mice (200× magnification). Morphological investigation includes observation of lipid droplet accumulation (a) and hepatocellular ballooning degeneration (b). (B) Histological scoring of liver (n = 4). #p < 0.05, ##p < 0.01 vs NC group; *p < 0.05 and **p < 0.01.

Figure 7.

Apigenin promoted the formation of autophagosomes in TWR-induced mice. (A) The mRNA expression levels of UVRAG, LC3B, Beclin-1 and ATG5 were examined by RT-qPCR. (B) Transmission electron microscope images of hepatocyte in C57BL/6 mice (10 μm for the left images, 2 μm for the right images). Enlarged inserts at the right side of each image show lipid drop or damage organelle (red arrow), autophagosome (white arrow). #p < 0.05, ##p < 0.01vs NC group; *p < 0.05 and **p < 0.01.

Figure 8.

Apigenin promoted RCT process and inducted the co-localization of UVRAG/LC3B and ADRP. (A) The co-localization of LC3B/ADRP and UVRAG/ADRP under a confocal microscope (left). Quantification analyses of the co-localization of autophagy protein and lipid protein were shown in the right. (B) The expressions of ABCG1, CD36 and SR-BI were examined by RT-qPCR (n = 10). #p < 0.05, ##p < 0.01 vs NC group; *p < 0.05 and **p < 0.01.