Isorhamnetin attenuates atherosclerosis by inhibiting macrophage apoptosis via PI3K/AKT activation and HO-1 induction

Abstract

Background and purpose: Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet.

Methods and results: Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions.

Conclusion: In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction.

Fig 1. Chemical structure of Iso.

Fig 2. Protective effects of Iso against ox-LDL-induced THP-1-derived macrophage cell injury.

THP-1 monocytes were incubated with PMA (160 nM) to induce differentiation into macrophages. (A) Effects of ox-LDL on macrophage cell viability. Cells were treated with increasing concentrations of ox-LDL (20, 40, 60, 80, 100, 120, and 140 μg/ml) for 24 h, and cell viability was determined by MTT assay. (B) Effects of Iso on ox-LDL-induced macrophage cell viability. Macrophages were pretreated with indicated doses of Iso and LOV for 8 h before exposure to 100 μg/ml ox-LDL for 24 h. Cell viability was measured by MTT assay. (C) Effects of Iso on macrophage cell viability. Cells were incubated with the indicated doses of Iso and LOV for 8 h. Cell viability was measured by MTT assay (A, B and C, expressed as the percentage of control). All results are expressed as the mean ± SD of three independent experiments. ^## P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. ox-LDL-treated cells.

Fig 3. Iso reduced lipid deposition in THP-1-derived macrophages.

THP-1-derived macrophages were pretreated with indicated doses of Iso for 8 h and then exposed to ox-LDL (100 μg/ml) for 24 h. (A) Representative images for each condition showing THP-1-derived macrophages stained with oil red O. (B) Area of the average size of lipid deposits. The results were obtained from three independent experiments. ^## P < 0.01 vs. control; *P < 0.05, **P < 0.01 vs. ox-LDL-treated cells.

Fig 4. Iso mitigates the pro-apoptotic effects of ox-LDL on mitochondrial injuries, caspase-3 activation, and TUNEL-positive cells in THP-1-derived macrophages.

THP-1-derived macrophages were pretreated with the indicated doses of Iso for 8 h and then exposed to ox-LDL (100 μg/ml) for 24 h. (A) Representative images showing macrophages stained with JC-1 dye as observed by fluorescence microscopy. (B) Internucleosomal DNA fragmentation was determined by TUNEL assay. (C) MTP in each group was calculated as the ratio of red to green fluorescence. (D) The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total cells. (E) Caspase-3 activity was detected using a fluorimetric assay. Results are represented as the mean ± SD from three independent experiments. ^## P < 0.01 vs. control; *P < 0.05, **P < 0.01 vs. ox-LDL-treated cells.

Fig 5. Iso prevents ox-LDL-induced THP-1-derived macrophage oxidative stress injuries.

THP-1-derived macrophages were pretreated with the indicated doses of Iso for 8 h and then exposed to ox-LDL (100 μg/ml) for 24 h. (A) The intracellular ROS levels were measured with a fluorimetric assay. (B) MPO activity was determined by a colorimetric activity assay kit. (C) The GSH-px activity was measured using a commercial kit. (D) NOX activity was tested by a commercial kit. (E, F) THP-1-derived macrophages were pre-incubated with 10 μM ZnPP for 48 h or 20 μM LY290042 for 30 min, and then pretreated with Iso (20 μM) for 8 h followed by ox-LDL (100 μg/ml) for 24 h. Results are represented as the mean ± SD from three independent experiments. ^## P < 0.01 vs. control; *P < 0.05, **P < 0.01 vs. ox-LDL-treated cells; ^$ P < 0.05, ^$$ P < 0.01 vs. ox-LDL and Iso-treated cells.

Fig 6. Iso modulates the expression of apoptosis-related proteins.

After the indicated treatments, cells were harvested and lysed to measure Apaf-1, Cyt-c, Bcl-2, Bax, caspase-3, and caspase-9, and β-actin protein levels by western blot analysis. The blots are representative of three independent experiments, and values are presented as the mean ± SD from three independent experiments. ^## P < 0.01 vs. control; *P < 0.05, **P < 0.01 vs. ox-LDL-treated cells.

Fig 7. Iso activates PI3K/AKT and HO-1 induction in THP-1-derived macrophages induced by ox-LDL.

THP-1-derived macrophages were exposed to ox-LDL (100 μg/ml) in the presence or absence of Iso (5, 10, and 20 μM) for 8 h. Expression levels of AKT, p-AKT, HO-1, and Nrf2 in the cytoplasm and nucleus were investigated by western blot. (A, D) Representative images of HO-1 and Nrf2 expression levels and statistical results relative to control group. (B, E) Representative images of p-AKT/AKT expression level and statistical results relative to control group. (C, F) Representative images of HO-1 and p-AKT/AKT expression level and statistical results relative to control group. (G, H, I, J) Representative images of Bax, Bcl-2, caspase-3, nucleus Nrf2 and β-actin expression level and statistical results relative to control group. The blots are representative of three independent experiments, and values are presented as the mean ± SD from three independent experiments. ^## P < 0.01 vs. control; *P < 0.05, **P < 0.01 vs. ox-LDL-treated cells; ^$ P < 0.05, ^$$ P < 0.01 vs. ox-LDL and Iso-treated cells.

Fig 8. Iso relieves AS in ApoE-/- mice.

All mice were fed HFC. ApoE-/- mice were treated with Iso (20 mg/kg, i.g.), LOV (3 mg/kg, i.g.), or its vehicle (as model group) for eight weeks; C57 mice were used as the control group. (A) Levels of serum lipids (TC, HDL-C, LDL-C, and TG) in mice at 0, 4, and 8 weeks of administration. (B, C) Representative light photomicrographs and morphometric analysis of oil red O-stained sections from the aortic root. (D, E) Representative images of Sudan IV-stained en face preparations of the proximal aorta and morphometric analysis of aortic AS expressed as a fraction of the total aortic area. (F, G) Representative images and morphometric analysis of CD68 staining from the aortic root. Values (n = 10 per group) are expressed as the mean ± SD. ^## P < 0.01 compared with the C57 control group; *P < 0.05, **P < 0.01, compared with the vehicle-treated ApoE-/- model group.

Fig 9. Iso showed inhibitory effects on macrophage apoptosis in atherosclerotic lesions.

Mice were fed an HFC diet. ApoE-/- mice were treated with Iso (20 mg/kg, i.g.), LOV (3 mg/kg, i.g.), or its vehicle (as model group) for eight weeks; C57 mice were used as the control group. (A, B) Representative images of caspase-3 expression under immunofluorescent staining. (C) Representative images of the TUNEL assay. Macrophage nuclei (DAPI, blue), nuclei+TUNEL-positive staining (green), and merged images of macrophage cytoplasm (red). (D, E) Quantity of TUNEL-positive cells and ratio of macrophage-associated TUNEL-positive cells. Values (n = 10 per group) are expressed as the mean ± SD. ^## P < 0.01 compared with the C57 control group; *P < 0.05, **P < 0.01, compared with the vehicle-treated ApoE-/- model group.

Fig 10. Schematic of Iso mechanism of preventing ox-LDL-induced apoptosis by activation of PI3K/AKT signaling and up-regulation of HO-1 in THP-1-derived macrophages.