Intermittent Fasting Inhibits High-Fat Diet–Induced Atherosclerosis by Ameliorating Hypercholesterolemia and Reducing Monocyte Chemoattraction

Figures

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  FIGURE 1. Inhibition of atherosclerosis in LDLR−/− mice by intermittent fasting treatment. (A) (experimental design): LDLR−/− mice were divided into four groups (n = 6), and received the following treatment for 7 or 14 weeks: Control groups, mice were fed HFD ad libitum; intermittent fasting (IF) groups, mice were fed HFD in cycles of 3-day HFD feeding and 1-day fasting. (B) The food intake in each cycle was determined and calculated as the daily average intake per mouse. *p < 0.05 [n = 12 (cycles) in 7 week groups, n = 23 (cycles) in 14 week groups]. (C,D) Lesions in the en face aortas (C) or sinus lesions in the aortic root (D) were determined by Oil Red O staining followed by quantitative analysis. Lesions in the en face aortas were expressed as a percentage of the whole aorta area, and sinus lesions were expressed as μm2. *p < 0.05, (n = 6). Ctrl: control; IF: intermittent fasting.

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  FIGURE 2. Intermittent fasting increases lesion plaque stability. The aortic root cross sections were used to complete the following assays: HE staining (A,C) with the quantitative analysis of necrotic core and fibrous cap areas (B,D). nc: necrotic cores marked by a black dotted line; fc: fibrous cap marked by a blue dotted line. The expression of SMA and CD68 (E,F) in plaques were determined by immunofluorescent staining with the quantification of SMA- or CD68-positive areas. *p < 0.05 (n = 6).

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  FIGURE 3. Intermittent fasting increases collagen content via inducing COL1A1 and COL3A1 expression. (A,B) The aortic root cross sections were used to determine collagen content by Picrosirius red staining with quantitative analysis of collagen positive areas. *p < 0.05 (n = 6). (C,D) The expression of COL1A1, COL3A1, MMP2, and MMP9 in plaques were determined by immunofluorescent staining with the quantification of positive staining areas. *p < 0.05, ns: not significant (n = 6). E: LDLR−/− mice were divided into the control or IF group (n = 6/group). After 2 weeks HFD feeding ad libitum or IF treatment, the mice aorta samples were collected. The expression of COL1A1, COL3A1, MMP2, and MMP9 were determined by qRT-PCR. *p < 0.05, ns: not significant (n = 6).

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  FIGURE 4. Intermittent fasting reduces hypercholesterolemia. Mouse serum samples isolated form LDLR-/- mice after 7 (A) or 14 weeks (B) IF feeding were used to detect levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) using an automatic biochemical analyzer. *p < 0.05, ns: not significant (n = 6). Ctrl: control; IF: intermittent fasting.

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  FIGURE 5. Intermittent fasting reduces HFD-induced lipid accumulation in the liver by reducing cholesterol synthesis. (A) Liver photos and HE staining of liver paraffin sections. Arrows indicate the vacuoles of lipid droplets after HE staining. (B)Liver TG and TC contents were quantitatively determined and normalized to protein content. (C): serum ALP and AST levels were determined using the enzymatic kits. (D,E) Expression of genes related to cholesterol synthesis [(D): HMGCR, HMGCS, and SREBP2], transport, and metabolism [(E): ABCA1, ABCG1, ABCG5, ABCG8, SR-BI, CYP7A1, apoB, and apoE] were determined by qRT-PCR. *p < 0.05, ns: not significant (n = 6).

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  FIGURE 6. Intermittent fasting suppresses CCL2 expression. (A): mouse serum CCL2, IL-1β, and IFN-γ levels were determined by the ELISA kits. (B,C): aortic root cross sections were used to determine CCL2 expression by immunofluorescent staining with the quantification of CCL2-positive area (C). *p < 0.05, ns: not significant (n = 6).

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  FIGURE 7. Intermittent fasting reduces circulating monocytes and their adhesion to endothelium. LDLR−/− mice were divided into the control or IF groups (n = 6/group). After 2 weeks of HFD feeding ad libitum or IF treatment, mouse peripheral blood and aorta samples were collected. Monocytes and T cells in blood samples were determined by FACS. (A–B): Inflammatory monocytes (CD11b + CD45 + Ly6Chigh) were determined and quantified in total CD11b + or CD45 + cells. (C–E) CD3+ (total T cells), CD4+ T cells, and CD8+ T cells. *p < 0.05, ns: not significant (n = 3). (F,G) Arch aorta, thoracic aorta, and abdominal aorta were dissected from the collected aortas. Each of them were then cultured in complete DMEM medium containing CFSE-labeled THP-1 cells for 1 h. The adhering THP-1 cells were then captured by a microscope and quantitively analyzed. *p < 0.05 (n = 6). (H) The aortic root cross sections from mice in Figure 1 was used to determine the expression of VCAM-1 and ICAM-1 in plaques by immunofluorescent staining with quantification of VCAM-1 and ICAM-1–positive areas. *p < 0.05 (n = 6).