Rebamipide ameliorates atherosclerosis by controlling lipid metabolism and inflammation

Abstract

The oral administration of rebamipide decreased plaque formation in atherosclerotic lesions as well as the markers of metabolic disorder in ApoE-deficient mice with atherosclerosis. Pro-inflammatory cytokines were also suppressed by rebamapide. In addition, the population of Th17 was decreased, whereas Treg was increased in the spleen of rebamipide-treated ApoE deficient mice. Rebamipide also ameliorated the severity of obese arthritis and has the capability to reduce the development of atherosclerosis by controlling the balance between Th17 and Treg cells. Thus, rebamipide could be a therapeutic agent to improve the progression of inflammation in metabolic diseases.

Fig 1. Rebamipide decreased atherosclerotic lesions in ApoE-KO mice.

(A) To induce atherosclerosis, eight-week old male ApoE-KO mice were fed a western diet with rebamipide (100 mg/kg/day) or statin (25mg/kg/day) every day for 8 weeks. (B) Aorta were prepared from the ApoE-KO mice at the end of study and stained with Oil-red-O to determine the accumulation of lipid. (C) Longitudinal section of aorta from rebamipide or statin administrated mouse was stained with Oil red O and lesion area was analyzed.

Fig 2. Rebamipide therapy decreased serum lipid in the ApoE KO.

(A)-(E) Metabolic parameters from atherosclerosis-induced ApoE-KO mice that were orally administrated with or without rebamipide or statin were measured using blood at the 8^th week. Aspartate aminotransferase (AST), Alanine aminotrasferase (ALT). NFD (non-fat diet).

Fig 3. Inhibitory effect of rebamipide on ox-LDL induced foam cell formation.

(A) THP-1 cells were incubated with PMA for 1 day and then with oxLDL followed by co-treatment with rebamipide at a dose of 100, 250, 500 and 1000 μM as indicated. Twenty-four hours later, the accumulation of lipid droplets was observed using oil red O staining. The graph shows the number of oil red O stained cells counted. (B) The cell viability of THP-1 cells was also determine through MTT assay. Graph shows O.D. for each indicated rebamipide concentration.

Fig 4. Regulation of IL-17 and Treg cells and suppression of cell adhesion.

(A) The aorta isolated from ApoE-KO mice treated with or without rebamipide were subjected to immunochemical staining for IL-17, Foxp3 or VCAM-1. Scale bars: 200um. (B) IL-17, Foxp3 and VCAM-1 expression in atherosclerotic lesions from ApoE-KO mice treated with or without rebamipide. The number of cells was counted in four independent quadrants.

Fig 5. Rebamipide suppresses inflammatory cytokines.

Normal B6 cells were isolated from the spleen, treated with LPS (100ng/ml) and incubated with or without rebamipide at the indicated dose for 3 days. Then, enzyme-linked immunosorbent assay (ELISA) was performed using the supernatants to measure the inflammatory cytokines TNF-α, IL-6.

Fig 6. Rebamipide modified Th17/Treg balance.

(A) Total splenocytes were isolated from rebamipide treated western-dieted ApoE-KO mice or control ApoE-KO mice. For FACS analysis, cells were stained with anti-CD4 and anti-IL-17 antibodies for Th17 cells, and with anti-CD4, CD25 and FoxP3 for Treg cells. (B) Spleen tissues from western-dieted ApoE-KO mice were also stained for Th17 or Treg cells and confocal microscope images were obtained. Bar indicates 10μm.

Fig 7. Rebamipide effect on obese arthritis in vivo.

(A) B6 (7 weeks old) mice were divided into 2 groups (N = 5) and were fed a high fat diet and injected with type II collagen to induce obesity-rheumatoid arthritis (obese CIA). Mice were treated with rebamipide (100mg/kg) orally once a week for 6 weeks. The graphs show disease score and incidence of arthritis. (B) Joint tissues from obese CIA mice with or without rebamipide were stained with H&E, safranin O or Toluidine blue to determine the destruction of the joints. The graph of inflammation score and cartilage score show lymphocyte infiltration and joint destruction in obese CIA mice with or without rebamipide, respectively.