Ginkgo suppresses atherosclerosis through downregulating the expression of connexin 43 in rabbits

Abstract

Introduction: Ginkgo biloba extract (GBE) EGb761 is widely used for cardiovascular prevention. Here, we investigated the effects of GBE on atherosclerotic lesion development in rabbits with a high-fat diet.

Material and methods: Forty New Zealand white male rabbits were randomly divided into four groups. The first two were the normal diet group (C) and the high-fat group (HF). The remaining two groups were those who received a high cholesterol diet supplemented with either the standard drug (simvastatin 2 mg/kg/day) or GBE (3 mg/kg/day). At 12 weeks, histopathological and chemical analyses were performed.

Results: Plasma lipid measurement showed that GBE inhibited high-fat diet-induced increase of serum triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-C) by 59.1% (0.9 ±0.2 4 mmol/l vs. 2.2 ±0.4 mmol/l), 18.2% (31.1 ±1.4 mmol/l vs. 38.0 ±0.4 mmol/l) and 15% (28.9 ±1.3 mmol/l vs. 34.0±1.0 mmol/l), respectively, at 12 weeks (p < 0.01). The en face Sudan IV-positive lesion area of the aorta in the GBE group (51.7 ±3.1%) was significantly lower compared with that in the HF group (88.2 ±2.2%; p < 0.01). The mean atherosclerotic lesion area of the GBE group was reduced by 53.2% compared with the HF group (p < 0.01). Immunohistochemistry and western blot analysis showed that GBE markedly suppressed high-fat diet-induced upregulation of connexin 43 (Cx43) in rabbits (p < 0.01).

Conclusions: Thus, our study revealed that GBE prevented atherosclerosis progress through modulating plasma lipid, suppressing atherosclerotic lesion development, and attenuating the expression of Cx43 protein.

Keywords: Ginkgo biloba extract; atherosclerosis; connexin 43; high-fat diet; plasma lipid.

Figure 1

Effects of GBE on plaque formation in rabbits. Representative examples of en face dissection of thoracic and abdominal aorta, stained with Sudan IV, showed atherosclerotic lesions (red). Atherosclerotic involvement for each rabbit was quantitatively determined in en face sections by measuring the proportion of aortic intimal surface area occupied by Sudan IV positive lesions in total aorta C – normal diet control group, HF – high-fat diet group, S – simvastatin treated group, GBE – Ginkgo biloba extract treated group; results were expressed as mean ± SD; *p < 0.01 relative to normal diet control (C), #p < 0.01 relative to high-fat diet group (HF)

Figure 2

GBE inhibits atherosclerotic lesion development in the aorta of rabbits with high-fat diet. Representative hematoxylin and eosin stained histological cross sections from aorta of indicated groups Abbreviations – see Figure 1. Results were expressed as mean ± SD; *p < 0.01 relative to normal diet control (C), #p < 0.01 relative to high-fat diet group (HF). Magnification 100×

Figure 3

Expression of connexin 43 (Cx43) in atherosclerotic plaques in rabbits. Representative sections show immunohistochemistry for Cx43 (left). Sections from thoracic and abdominal aorta were stained for Cx43. Positive staining is shown as brown. Sections were counterstained with hematoxylin. Quantitative analysis (fold of normal diet control group) of Cx43 expression in different experimental groups was performed (right). The expression of Cx43 was significantly increased in the rabbits with a high-fat diet compared to that in the rabbits with a normal diet. The expression of Cx43 in both simvastatin and Ginkgo biloba extract groups was decreased relative to that of the high-fat diet control group The data are expressed in terms of the fold of the normal diet control group as mean ± SD; *p < 0.01 relative to normal diet control (C), #p < 0.01 relative to high-fat diet group (HF). Magnification 200×

Figure 4

Western blot analysis of connexin 43 (Cx43) expression. Top panel shows a representative blot of Cx43 protein. β-actin was used as an internal control. The normalized Cx43 protein levels are shown in the bottom panel Abbreviations – see Figure 1. Results were expressed as mean ± SD; *p < 0.01 relative to normal diet control (C), #p < 0.01 relative to high-fat diet group (HF)