Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in Cuff-Induced Vascular Injury Mouse Model

<div><p>Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuff-induced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% <i>Citrus unshiu</i> (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% <i>Citrus iyo</i> (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.</p></div

Effect of drinking citrus fruit juice on immune cell infiltration.

<p>Comparison of filtration of macrophage and neutrophils in injured femoral artery 7 days after cuff placement. Representative photos of injured femoral artery in cross-sections after immunofluorescent staining using antibodies against a macrophage marker, F4/80 and a neutrophil marker, LY-6G/-6C. CU; <i>Citrus unshiu</i> juice, CI; <i>Citrus iyo</i> juice.</p

Comparison of contents and ingredients between <i>Citrus unshiu</i> and <i>Citrus iyo</i> juice.

<p>Comparison of contents and ingredients between <i>Citrus unshiu</i> and <i>Citrus iyo</i> juice.</p

Effect of drinking citrus fruit juice on inflammatory cytokines.

<p>Expression of TNF-α, MCP-1, IL-6, and IL-1β determined by real-time quantitative RT-PCR in femoral artery 7 days after cuff placement. Tissue samples were prepared from cuffed arteries 7 days after operation. CU; <i>Citrus unshiu</i> juice, CI; <i>Citrus iyo</i> juice. Values are mean ± SEM (n = 4 for each group).</p

Effect of drinking citrus fruit juice on neointima formation.

<p>Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% <i>Citrus unshiu</i> (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% <i>Citrus iyo</i> (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Samples were prepared from cuffed-femoral arteries of C57BL/6J mice as described in Methods. A, Representative photos of neointimal area in cross-sections of femoral artery with elastic van Gieson staining 14 days after cuff placement at 100x magnification. B, Higher magnified photos at 400x magnification described as squares in Figure A. Scale bars show 50 μm in each photo. C, Quantitative analysis of neointimal area in injured femoral artery. Values are mean ± SEM (n = 6 for Cuff (-), n = 8 for other groups). *p<0.05, **p<0.01 vs. Cuff (+) Control, †p<0.05 vs. administration of juice of different citrus fruit at same %.</p

Effect of drinking citrus fruit juice on superoxide anion production induced in injured artery.

<p>Tissue samples were prepared from cuffed arteries 7 days after operation. Representative photos of injured femoral artery in cross-sections after dihydroethidium staining (A) and fluorescence intensity in intima and media (B). CU; <i>Citrus unshiu</i> juice, CI; <i>Citrus iyo</i> juice. **p<0.01 vs. Cuff (+) Control. Values are mean ± SEM (n = 7 to 8 for each group).</p

Comparison of flavonoid concentrations between <i>Citrus unshiu</i> and <i>Citrus iyo</i> juice.

<p>Comparison of flavonoid concentrations between <i>Citrus unshiu</i> and <i>Citrus iyo</i> juice.</p

Effect of drinking citrus fruit juice on ERK activation.

<p>Comparison of ERK activation in injured femoral artery 7 days after cuff placement. A, Representative photos of injured femoral artery in cross-sections after phospho-ERK staining. Blue arrows indicate phosphor-ERK positive nuclei. B, Quantitative analysis of positive stained cell ratio in neointima. Values are mean ± SEM (n = 5 for Cuff (-), n = 5 for Cuff (+) Control and CU10% group and n = 3 for CI10% group). **p<0.01 vs. Cuff (+) Control. C, Representative immunoblots of ERK in pooled samples are shown. CU; <i>Citrus unshiu</i> juice, CI; <i>Citrus iyo</i> juice.</p

Effect of drinking citrus fruit juice on cell proliferation in injured femoral artery after cuff placement.

<p>Representative photos of injured femoral artery in cross-sections after PCNA staining (A) (200x and 400x magnification) and PCNA labeling index in intima (B) and media (C). Higher magnified photos described as squares in Figure A. Cuff placement around the femoral artery was performed, and PCNA was detected as described in Methods. Cell proliferation was measured as the ratio of PCNA-positive nuclei to total nuclei in the femoral artery 7 days after cuff placement. CU; <i>Citrus unshiu</i> juice, CI; <i>Citrus iyo</i> juice. a; adventitia, m; media, i; intima. Values are mean ± SEM (n = 6 for Cuff (-), n = 4 for other groups). *p<0.05, **p<0.01 vs. Cuff (+) Control, †p<0.05 vs. administration of juice of different citrus fruit at same %.</p