Effects of kampo formulas on the progression of hypercholesterolemia and Fatty liver induced by high-cholesterol diet in rats

Abstract

Background: Bofutsushosan is a well known Kampo, traditional Japanese medicine, based on ancient Chinese medicine mainly used in the treatment of hypercholesterolemia in Japan. We selected two Kampo formulas, Boiogito and Keishibukuryogan mainly used in the treatment of hypercholesterolemia in China to compare with Bofutsushosan and cholesterol absorption inhibitor ezetimibe.

Methods: Hypercholesterolemia and fatty liver were induced by high cholesterol (containing 2% cholesterol and 0.5% cholic acid) diet in male Wistar rats for 6 and 12 weeks. Kampo formulas Boiogito, Bofutsushosan, Keishibukuryogan and ezetimibe were added to the high-cholesterol diet, respectively. After 6 and 12 weeks, body and liver weights, blood chemistry, cholesterol concentrations, fat-related and inflammatory-related factors were examined.

Results: High-cholesterol diet increased body and liver weights, and serum cholesterol concentrations. Boiogito and ezetimibe improved them. Serum ICAM-1 and RBP4 were increased in the high cholesterol diet group. Boiogito and ezetimibe improved them too. In the histological examinations of liver and adipose tissues, we observed a significant improvement after treatment. Immunostaining expression of ICAM-1 in aorta was improved by Boiogito, Bofutsushosan, Keishibukuryogan and ezetimibe. The mRNA expression of RBP4, HFABP, CFABP, MCP1 and CCR2 in liver and adipose tissue were decreased by Boiogito and ezetimibe.

Conclusion: Boiogito has a protective effect on the progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet in rats and more effective than Bofutsushosan and Keishibukuryogan. The lipid-lowering effect of Boiogito is not stronger than ezetimibe. But the anti-inflammatory (MCP1, CCR2) and anti-arteriosclerotic (ICAM-1) effects of Boiogito are more potent than ezetimibe.

Keywords: Bofutsushosan; Boiogito; Keishibukuryogan; fatty liver; hypercholesterolemia.

Fig. 1.

Histopathological examination of liver. A: Fatty degeneration (steatosis) of the liver is observed in the high-cholesterol diet-fed (H, HA, HB, HC and HE) groups, but not in Group C in hematoxylin-eosin stained tissues. Fatty changes increase with time to a greater extent in Group H than Groups HA, HB, HC and HE. B: Oil red O staining reveals more lipid droplets (stained red) to be accumulated in vacuoles in Group H than other groups. 6W, 6 weeks; 12W, 12 weeks. C, control: standard diet for 6 and 12 weeks (n = 5). H: high-cholesterol diet for 6 and 12 weeks (n = 5). HA: high-cholesterol diet with Boiogito for 6 and 12 weeks (n = 5). HB: high-cholesterol diet with Bofutsushosan for 6 and 12 weeks (n = 5). HC: high-cholesterol diet with Keishibukuryogan for 6 and 12 weeks (n = 5). HE: high-cholesterol diet with ezetimibe for 6 and 12 weeks (n = 5). Bars express 25 μm.

Fig. 2.

Histopathological examination of adipose tissue. The larger fat cells are observed in the high-cholesterol diet-fed (H, HA, HB, HC and HE) groups, but not in Group C (56.13 ± 11.51 and 64.88 ± 13.96 μm after 6 and 12 weeks, respectively) in hematoxylin-eosin stained tissues. Fat cells change with time to a greater extent in Group H (97.00 ± 20.14 and 104.63 ± 22.56 μm after 6 and 12 weeks, respectively) than Groups HA, HB, HC and HE (55.63 ± 5.85 and 78.88 ± 16.34 μm, 75.63 ± 11.53 and 78.13 ± 16.47 μm, 74.63 ± 13.94 and 88.13 ± 8.58 μm, 74.38 ± 13.21 and 83.13 ± 16.63 μm after 6 and 12 weeks, respectively). Bars express 50 μm.

Fig. 3.

Intercellular adhesion molecule-1 (ICAM-1) and retinol-binding protein 4 (RBP4) concentration in serum. ICAM-1 and RBP4 concentrations in control, standard diet group (C, n = 5), high-cholesterol diet group (H, n = 5), high-cholesterol diet with BOT group (HA, n = 5), high-cholesterol diet with BTS group (HB, n = 5), high-cholesterol diet with KBG group (HC, n = 5) and high-cholesterol diet with ezetimibe group (HE, n = 5) for 6 and 12 weeks. *P < 0.05 versus Group C. †P < 0.05 versus Group H.

Fig. 4.

Changes in mRNA expression with time in liver and adipose tissue. Levels of RBP4, HFABP, CFABP, MCP1 and CCR2 against beta-actin mRNA expression are shown in the above histograms. The left-side graphs show the expression in liver. The right-side graphs show the expression in adipose tissue around the left kidney.

Fig. 5.

Immunohistochemistry expression of ICAM-1 in aorta. A: Seldom ICAM-1 immunostaining (red-brown deposits indicate positive staining) is found on the whole layers of abdominal aortas in Group C. Bars express 25 μm. B: Mean optical density values of ICAM-1. The photographs generated were quantitatively analyzed the optical density of ICAM-1 with Image-Pro Plus version 6.0 software. *P < 0.05 versus Group C. †P < 0.05 versus Group H.