Yellow Wine Polyphenolic Compounds Inhibit Matrix Metalloproteinase-2, -9 Expression and Improve Atherosclerotic Plaque in LDL-Receptor– Knockout Mice
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J Pharmacol Sci 125, 132 – 141 (2014) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Yellow Wine Polyphenolic Compounds Inhibit Matrix Metalloproteinase-2, -9 Expression and Improve Atherosclerotic Plaque in LDL-Receptor– Knockout Mice Xiaoya Zhai1,2,§, Jufang Chi1,§, Weiliang Tang1, Zheng Ji1, Fei Zhao1,2, Chengjian Jiang1,2, Haitao Lv1, and Hangyuan Guo1,* 1Department of Cardiology, Shaoxing People’s Hospital, Shaoxing Hospital of Zhejiang University, 568 Zhongxing North Road, Shaoxing, Zhejiang 312000, China 2Wenzhou Medical University, Wenzhou City, Zhejiang 325000, China Received December 16, 2013; Accepted February 28, 2014 Abstract. Many epidemiological studies have strongly suggested an inverse correlation between dietary polyphenol consumption and reduced risks of cardiovascular diseases. Yellow rice wine is a Chinese specialty and one of the three most ancient wines in the world (Shaoxing rice wine, beer, and grape wine). There is a large amount of polyphenol substances in yellow rice wine. This experiment was designed to study the potential beneficial effects of yellow wine polyphenolic compounds (YWPC) from yellow rice wine on progression of atherosclerosis in vivo and to further explore its underlying mechanisms. Six-week-old male LDL-receptor–knockout mice were treated with high-fat diet to establish the mouse model with atherosclerosis. Animals received 10, 30, or 50 mg/kg per day of YWPC or 10 mg/kg per day rosuvastatin or water (vehicle) for 14 weeks. The results indicated that YWPC and rosuvastatin significantly decreased circulating total cholesterol and low-density lipoprotein cholesterol. Compared to the control group, the atherosclerosis lesion area in the rosuvastatin-intervention group and YWPC at doses of 10, 30, and 50 mg/kg per day intervention groups decreased by 74.14%, 18.51%, 40.09%, and 38.42%, respectively. YWPC and rosuvastatin decreased the expression and activity of matrix metalloproteinases (MMP)-2, 9, whereas the expression of the endogenous inhibitors of these proteins, namely, tissue inhibitors of matrix metalloproteinases (TIMP)-1, 2, increased when compared to the control group. It can be concluded that the YWPC is similar to the benefic effects of rosuvastatin on cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions by lowering lipid and modulating the activity and expression of MMP-2, 9 and TIMP-1, 2. Keywords: yellow wine polyphenolic compound, atherosclerosis, lipid-lowering, matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase Introduction consumption of red wine is a major cause of the “French paradox” and further confirmed that the protective effect The French people have a lower mortality rate from of red wine consumption against cardiovascular heart cardiovascular disease than the other Nordic countries disease (CHD) might be attributable, at least in part, despite a high consumption of saturated fatty acids in the to polyphenols, including catechins, epicatechin, gallic daily diet, which is namely the famous “French paradox”. acid, ferulic acid, caffeic acid, anthocyanins, and Renaud and de Lorgeril (1) demonstrated that the resveratrol (2, 3). Yellow rice wine is one of the three ancient wines in the world (Shaoxing rice wine, beer, and grape wine) which is derived from Shaoxing, China. It §These authors contributed equally to this work. *Corresponding author. [email protected] is fermented from glutinous rice, wheat, and aspergillus Published online in J-STAGE on May 23, 2014 yeast over 80 days (4). Yellow rice wine is also rich in doi: 10.1254/jphs.13263FP polyphenols, which is mainly from glutinous rice and 132 YWPC Improve Atherosclerosis 133 wheat, including catechin, epicatechin, rutin, quercetin, astatin is a common dosage in clinical practice (18) protocatechuic, chlorogenic acid, caffeic acid, syringic and has been widely used in animal studies. It has been acid, ferulic acid, p-incense beans, and so on. The total found that this dose is effective in the prevention of phenolic content comes to 1.023 mg/ml, which can be atherosclerosis in the LDLR−/− mice (19). So we chose comparable with polyphenol content in the red wine the dose of 10 mg/kg per day of rosuvastatin as the (4 – 6). In previous studies, we have observed that intervention dosage in the present study. Chinese yellow wine could prevent the progression of The present study was designed to investigate the early atherosclerotic lesions in vivo (7) and inhibit effects of YWPC on the activity and expression of the activity of MMP-2 induced by homocysteine (HCY) MMP-2, 9 and to evaluate the preventive effects against in cultured rat vascular smooth muscle cells (VSMCs) atherogenesis and compare them with those of rosuvas- (8). Atherosclerotic plaque area and matrix metallo tatin, further promoting our understanding of the protec- proteinase-2 expression and activity significantly decrease tive effects of this compound on CHD. For this purpose, in the yellow wine group (high fat diet + 3% rice wine) we used the LDLR−/− mice, which are a more physiologic and red wine group (high fat diet + 3% red wine) com- model to study mechanisms involved in atherogenesis pared to those in the control group (high fat diet + sterile in comparison to Apo-E–deficient mice as demonstrated water) and alcohol group (high fat diet + 3% alcohol) in by previous studies (20). In this model an absence of vivo (7). We hypothesized that something in the yellow functional low density lipoprotein (LDL) receptors wine, possibly the yellow wine polyphenols compounds leads to marked accumulation of cholesterol-rich very (YWPC), besides the alcohol may be responsible for low-density lipoprotein (VLDL), intermediate density the protective effect on the cardiovascular system. lipoproteins (IDL), and LDL in plasma and a secondary The formation and progression of atherosclerotic deposition of cholesteryl esters in macrophage foam lesions are lipid-driven chronic inflammatory processes cells along with massive atherosclerosis (21, 22). associated with excessive vascular remodeling, which is mainly regulated by matrix metalloproteinases (MMPs) Materials and Methods through degradation and synthesis of extracellular matrix (ECM) (9). The present study found that MMP-2 Materials and reagents contributed to monocyte migration and macrophage YWPC in dry powder form from yellow rice wine proliferation into the intima, resulting in increased (Shaoxing, China) were provided by national engineer- atherosclerotic plaque lesions (10, 11). Growing evidence ing and research center for traditional Chinese medicine suggested that MMP-9 deficiency reduced athero- (Shanghai, China). The procedures used to prepare and sclerosis progression or promoted a stable plaque analyze YWPC have been described previously (4). phenotype in apoE−/− mice (11). These findings suggest Rosuvastatin was provided by Astrazeneca (Macclesfield, strongly that inhibiting MMP-2 and MMP-9 production UK). Antibodies against MMP-2, 9 and TIMP-2 were should have a protective effect against plaque growth purchased from Abcam (Cambridge, MA, USA), and and remodeling. The activity of MMPs is controlled by, antibodies against TIMP-1 were purchased from Abbiotec an endogenous inhibitor, tissue inhibitors of metallo- (San Diego, CA, USA). Anti-MMP-2, MMP-9, TIMP-1, proteinases (TIMPs) (9). and TIMP-2 and horseradish peroxidase–conjugated Statins are a family of compounds that competitively goat anti-mouse and goat anti-rabbit IgG antibody were inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reduc- purchased from Jackson Immunoresearch Laboratories tase (HMG-CoA reductase), the rate-controlling enzyme (West Grove, PA, USA). Gelatin was purchased in cholesterol synthesis (12). At present, rosuvastatin from Abcam (Cambridge, MA). The other reagents of or atorvastatin has become the main compound among immuno blot assay were purchased from Beyotime the statins used clinically for lipid lowering therapy to (Jiangsu, China). All other chemicals were of reagent prevent cardiovascular disease (12, 13). Furthermore, grade or were of the highest grade commercially available. recent investigations have found that statins have pleio- tropic effects that besides lipid lowering, may directly Animals or indirectly limit atherosclerosis progression. The Animal studies were treated in compliance with the pleiotropic effects of statins include relieving vascular Guide for the Care and Use of Laboratory Animals inflammation, improving the function of endothelial and were approved by the Institutional Animal Care and cells, inhibiting the proliferation and migration of Use Committee of Wenzhou Medical University. Six- VSMCs, reducing the expression of MMPs, increasing week-old male LDL-receptor–knockout (LDLR−/−) mice the expression of TIMPs, and stabilizing atherosclerotic with the genetic background back-crossed ten genera- plaques (14 – 17). The dose of 10 mg/kg per day rosuv- tions into C57BL/6J (n = 40, body weight 20 – 25 g) 134 X Zhai et al were obtained from Model Animal Research Center of Morphological and atherosclerotic lesion area of the Nanjing University (Nanjing, China). The LDLR−/− aorta artery mice can spontaneously develop atherosclerosis with After blood collection, the mice were perfused through plaque size (20) and components similar to those of the left cardiac ventricle with ice-cold phosphate humans with a high fat diet, so these mice are a more buffered saline (PBS). After removal of the surrounding