The combined effects of the Korean traditional medicine DF and metformin or atorvastatin on nonalcoholic fatty liver disease and atherosclerosis in Paigen-diet fed C57BL/6J mice

다국어 초록 (Multilingual Abstract)

Nonalcoholic fatty liver disease (NAFLD) is diverse, ranging from simple fatty liver through nonalcoholic steatohepatitis to fibrosis, cirrhosis and hepatocellular carcinoma. NAFLD is closely related to the metabolic syndrome, such as obesity, insulin resistance, type 2 diabetes and dyslipidemia, and may be caused by the prolonged high-fat or high-cholesterol intake. Consumption of a high-cholesterol diet induces hypercholesterolemia, which can lead to many features of metabolic syndrome including NAFLD and atherosclerosis. Current potential drugs used for the treatment of NAFLD include an antidiabetic drug metformin (MF) and a hypolipidemic drug atorvastatin (AS). The herbal composition DF from Ephedra intermedia, Rheum palmatum, and Lithospermum erythrorhizon has been used to treat obesity and hepatic steatosis in Korean local clinics, but its mechanism of action is unknown. Therefore, this study was undertaken to investigate whether DF may enhance the inhibitory effects of MF and AS on NAFLD and atherosclerosis in wild-type C57BL/6J mice fed a high-fat/high-cholesterol diet (Paigen diet), an animal model of NAFLD and atherosclerosis.

1) A Paigen diet did not increase body weights.
2) As decreased liver weights, which were increased by a Paigen diet. Liver weights were further decreased when AS was co-administered with DF compared with individual treatments.
3) The lipid droplets in the liver were increased by a Paigen diet. However, hepatic lipid droplets were decreased by MF or AS alone compared with a Paigen diet and further reduced by concurrent treatments with MF or AS and DF compared with each treatments. DFAS(1) and DFAS(2) most effectively inhibited hepatic lipid accumulation. In parallel with the histological data, a Paigen diet increased mRNA expression of lipogenic genes (PPARγ, C/EBPδ, FAS and ACC1) and decreased mRNA expression of fatty acid-oxidizing genes (AMPKα1, AMPKα2, VLCAD and CPT-1). However, this expression pattern was improved by MF or AS alone and expression of the above genes except for AMPKα1 and AMPKα2 was more effectively improved by combination of MF or AS with DF.
4) Inflammatory mast cells were increased in the liver of Paigen diet-fed mice. However, mast cells were reduced by MF or AS compared with a Paigen diet and further reduced by co-treatments of MF or AS with DF compared with each treatments. Of all treatments, DFAS(2) most effectively reduced mast cells. Consistent with the above results, mRNA levels of inflammatory genes (ICAM-1, VCAM-1, MCP-1 and CD68) were decreased by MF or AS alone and further decreased by their combined treatments with DF.
5) Hepatic collagen levels and α-SMA-positive cells were increased in Paigen diet-fed mice. In contrast, they were decreased by MF or AS compared with a Paigen diet and further decreased by concomitant treatments with DF compared with each treatments. Collagen levels were effectively reduced by DFAS(1) and DFAS(2) whereas α-SMA-positive cells were significantly decreased by DFMF(1), DFMF(2) and DFAS(2). In addition, mRNA expression of fibrogenic genes (α-SMA and TGFβ1) was decreased by MF and AS as well as combinations of MF or AS with DF.
6) Hepatic apoptotic cells were increased by a Paigen diet. However, apoptotic cells were decreased by MF or AS compared with a Paigen diet and further decreased when MF or AS were co-administered with DF compared with each treatments. The mRNA expression of an anti-apoptotic gene Bcl-2 was increased by MF, AS and combinations of MF or AS with DF.
7) The accumulation of aortic plaques, thickness of arterial walls and plaque size of aortic roots were increased by a Paigen diet. However, they were decreased by MF or AS compared with a Paigen diet and further decreased by concurrent treatments of MF or AS with DF compared with each treatments. DFMF(2) effectively reduced the accumulation of aortic plaque whereas DFAS(2) significantly reduced thickness of arterial walls and plaque size of aortic roots. The mRNA expression of aortic inflammatory genes (IL-6, MCP-1 and TGFβ1), which was increased by a Paigen diet, was reduced by MF or AS and more effectively reduced by co-treatments of MF or AS with DF.
In conclusion, these results indicate that DF may potentiate the inhibitory effects of MF and AS on high cholesterol-induced hepatic steatosis, inflammation, fibrosis and apoptosis as well as arterial inflammation in a mouse model of NAFLD and atherosclerosis.

목차 (Table of Contents)

• CONTENTS ⅰ
• LIST OF FIGURES ⅳ
• LIST OF TABLES ⅵ
• ABBREVIATIONS ⅶ
• ABSTRACT ⅹ
• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 34
• 1. Preparation of DF 34
• 2. Animal treatments 36
• 3. Blood analysis 41
• 4. Histological analysis 41
• 5. Immunohistochemistry 42
• 6. En face preparation of aorta and quantification of atherosclerotic lesion 42
• 7. Atherosclerotic lesion assessment at the aortic roots 43
• 8. Reverse transcription polymerase chain reaction (RT-PCR) 43
• 9. Statistics 44
• Ⅲ. RESULTS 47
• 1. Effects of DF, metformin and atorvastatin on body and liver weights 47
• 2. Effects of DF, metformin and atorvastatin on circulating 47
• levels of liver damage markers and lipids
• 3. Effects of DF, metformin and atorvastatin on hepatic steatosis 52
• 3.1 Histological analysis 52
• 3.2 Expression of genes involved in fatty acid β-oxidation 52
• 3.3 Expression of lipogenesis genes 56
• 3.4 Expression of genes involved in cholesterol metabolism 57
• 4. Effects of DF, metformin and atorvastatin on hepatic inflammation 58
• 4.1 Histological analysis 58
• 4.2 Expression of genes related to hepatic inflammation 61
• 5. Effects of DF, metformin and atorvastatin on hepatic fibrosis 63
• 5.1 Histological analysis 63
• 5.2 Immunohistochemical analysis : α-SMA 63
• 5.3 Expression of genes related to hepatic fibrosis 65
• 6. Effects of DF, metformin and atorvastatin on hepatic apoptosis 68
• 6.1 Histological analysis 68
• 6.2 Expression of genes related to hepatic apoptosis 68
• 7. Effects of DF, metformin and atorvastatin on atherosclerosis 70
• 7.1 Effects of DF, metformin and atorvastatin on 70
• atherosclerotic lesion formation in aortas
• 7.2 Expression of genes related to atherosclerosis 73
• 7.3 Expression of inflammatory genes in hearts 77
• 7.4 Expression of lipogenic genes in hearts 77
• Ⅳ. DISCUSSION 80
• Ⅴ. REFERENCES 89