J Pharmacol Sci 125, 132 – 141 (2014) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Yellow 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 wine is a Chinese specialty and one of the three most ancient in the world (Shaoxing , , 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 immuno­blot 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 physiologic model to study mechanisms involved in adventitial fat and fascia tissue, the aortas were opened atherogenesis. longitudinally from the aortic root to the renal artery and fixed in formalin (10%) for 24 h. The aortas were Methods then rinsed in 70% alcohol for 30 s, stained with Mice were maintained on a 12 h light/dark cycle, in IV for 15 min, differentiated in 80% alcohol for 20 min, a temperature (25°C ± 2°C) and humidity (55% ± 3%) and washed in running water for 1 h. The aortas were controlled environment. Forty mice were randomly photographed by a digital camera connected to a divided into 5 groups (n = 8, each group). After one week dissection microscope. The extent of atherosclerosis was of an adaptation, at age 7 weeks, the mice were fed an analyzed with Image-pro plus 6.0 (Media Cybernetics experimental high-fat diet containing 10% fat and 1.25% Inc., Bethesda, MD, USA). Data are reported as the cholesterol for the entire study. YWPC and rosuvastatin percentage of the aortic surface covered by lesions were dissolved in drinking water as follows: group 1, (total surface area of the atherosclerotic lesions divided high-fat diet (control group); group 2, high-fat diet plus by the total surface area of the aorta) (25). 10 mg/kg per day rosuvastatin; group 3, high-fat diet plus 10 mg/kg per day YWPC; group 4, high-fat diet plus Western blotting analysis 30 mg/kg per day YWPC; group 5, high-fat diet plus Aorta protein extracts were prepared by homogenizing 50 mg/kg per day YWPC. They were allowed free access 100 mg of aorta in 500 ml radio-immunoprecipitation to food and water. Weights, fluid intake, and food con- assay buffer (RIPA Lysis Buffer; Beyotime, Jiangsu, China) sumption were monitored weekly throughout the study. containing freshly dissolved protease inhibitors (1 mM The Food and Drug Administration (FDA) recommend phenylmethanesulfonyl fluoride, PMSF; Beyotime). that daily moderate alcohol consumption is up to 12 – 24 g Homogenates were centrifuged at 12, 000 × g for 10 min of alcohol/day, so 200 mL yellow wine of 12% ethanol at 4°C. The supernatant was collected and assayed for per day is moderate drinking. Considering that the YWPC protein concentrations using the BCA method (BCA content of yellow wine was 1.0 mg/ml, the average Protein assay kit, Beyotime). The supernatant was mixed person gets about 200 mg of YWPC a day. The concen- with 5 × SDS sample buffer and heated in a boiling water tration in the intake YWPC is 2.86 mg/kg in a 70-kg bath for 5 min. Denatured protein (30 mg total proteins man. This translates into 26 mg/kg of YWPC in 20 – 25 g for MMPs, 50 mg total proteins for TIMPs) was subjected mice according to the coefficient of 9.1 (23). According to SDS-PAGE on a 10% (for MMP-2 and MMP-9) or to the previous experiments, treatment with polyphenols 12% (for TIMP-1 and TIMP-2) polyacrylamide gel at a dose of 10 mg/kg per day attenuated the progression and transferred to PVDF blotting membranes. After the of atherosclerosis (24). In our experiment, we chose transfer, membranes were blocked with blocking buffer doses ranging from 10 to 50 mg/kg per day, and 30 mg/kg for 1 h at room temperature and then incubated with per day was used as a middle dose. the respective primary antibody overnight at 4°C. After being washed with TBS-T (3 times for 10 min), the Plasma analysis membranes were incubated with horseradish peroxidase– At the end of the 14-week experimental period, these linked secondary antibody at room temperature for 1 h. mice were deprived of food for 12 h and sacrificed by Finally, antigen was detected by using the standard inhalant 1.5% isoflurane anesthesia. Blood was drawn chemical luminescence method (Beyotime). The mem- from the right ventricle into EDTA containing tube and branes were exposed to Kodak X-Omat AR film then plasma was isolated by centrifugation at 2000 × g (Eastman Kodak Co., Rochester, NY, USA). Finally, for 15 min at 4°C and stored at −80°C until analysis. the films were scanned on a densitograph and measured Total serum cholesterol (TC), triacylglycerol (TG), using Quantity One 4.4 (Bio-Rad, Hercules, CA, USA). low-density lipoprotein cholesterol (LDL-C), and high- density lipoprotein cholesterol (HDL-C) levels were Gelatin zymography analysis measured by the standard enzymatic method, using a The activity of MMP-2 and MMP-9 in the supernatants fully automatic clinical biochemical analyzer (Abbott was assessed by zymography. Equal amounts (30 mg) of Labs, Abbott Park, IL, USA). total protein extracts were mixed with 5 × SDS sample buffer without a reducing agent and loaded onto the YWPC Improve Atherosclerosis 135

SDS-PAGE gel (8% polyacrylamide gel containing 0.1% Plaque area gelatin). After electrophoresis, the gels were washed at Their aortas were analyzed for the extent of athero- room temperature for 30 min × 2 in a solution containing sclerosis. Extensive atherosclerotic lesions were ob- 2.5% Triton X-100 to remove the SDS and then incu- served proximal to the aortas in LDLR−/− mice, especially bated in renaturation buffer (pH 7.5, 50 mM Tris-HCl, in the control group (Fig. 1). Yellow wine polyphenolic 10 mM CaCl2, 0.02% NaN3) for 42 h at 37°C. The compounds reduced the lesion area: even the lowest gels were stained for 2 h with 0.1% Coomassie blue dose (10 mg/kg per day) reduced the lesion area by R-250 in 10% glacial acetic acid / 45% methanol and 18.51% compared with the control group. Similarly, then destained with 10% glacial acetic acid / 45% 30 and 50 mg/kg per day YWPC reduced the lesion methanol. Gelatinolytic activity was visualized as clear area by 40.09% and 38.42%, respectively; and stronger bands against the blue background of the stained gelatin. inhibition, 74.14%, was observed in the rosuvastatin The strength of each band was quantified by means of densitometric scanning using a digital camera and measured using Quantity One 4.4 (Bio-Rad).

Statistical analyses Results are presented as the mean ± S.D. The statistical significance of differences between groups was deter- mined by one-way analysis of variance (ANOVA) followed by Tukey’s Studentized Range (HSD) post-hoc test for multiple comparisons. A value of P < 0.05 was considered significant.

Results

Serum cholesterol levels After the mice were treated for 14 weeks with YWPC or rosuvastatin, there were no significant differences in food intake, water intake, and body weight in the 5 groups. The levels of plasma TC, LDL-C, and TG in the rosuvastatin group were significantly compared to those in the control group. YWPC at 10, 30, and 50 mg/kg per day significantly decreased the plasma TC and LDL-C levels compared with those in the control group. YWPC tended to decrease the serum TG level, although the effect was not significant. The levels of plasma TC, LDL-C, and TG were each significantly different between the YWPC groups and the rosuvastatin intervention group. There was no significant difference in the level of plasma HDL-C among the groups (Table 1). Fig. 1. Morphology of the aorta artery of the 5 groups.

Table 1. Effects of rosuvastatin and 10, 30, and 50 mg/kg per day YWPC on plasma lipid concentration in LDLR−/− mice treated with high-fat diet after 14 weeks Group TG (mmol/L) TC (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) Rosuvs 2.76 ± 0.67# 14.32 ± 2.45# 9.59 ± 1.81# 5.57 ± 0.67 Control 4.52 ± 0.93 30.73 ± 2.92 22.99 ± 3.50 5.03 ± 0.65 YWPC 10 mg/kg per day 4.17 ± 0.77* 25.28 ± 2.46*,# 18.98 ± 2.17*,# 5.21 ± 0.41 YWPC 30 mg/kg per day 3.75 ± 0.77* 18.26 ± 2.89*,#,& 13.47 ± 1.94*,#,& 5.48 ± 0.92 YWPC 50 mg/kg per day 3.86 ± 0.90* 20.09 ± 3.48*,#,& 15.23 ± 2.58*,#,& 5.36 ± 0.72 Data are expressed as the mean ± S.D. *P < 0.05 vs. rosuvs, #P < 0.01 vs. control, &P < 0.05 vs. YWPC (10 mg/kg per day). Rosuvs, rosuvastatin. 136 X Zhai et al group. Yellow wine polyphenolic compounds and rosu- and protein expression in comparison with the control vastatin significantly reduced the atherosclerosis lesion group and YWPC groups. (Figs. 3, 4) area (Fig. 2). Effects of YWPC on TIMP-1 and TIMP-2 protein Effects of YWPC on MMP-2 and MMP-9 protein expres- expression sion and activity The physiological activities of MMPs are related MMPs degrade the vascular ECM, which have been closely to that of their specific endogenous tissue involved in atherosclerotic plaque growth and instability, inhibitors of metalloproteinase. We further examined leading to an increased risk of cardiovascular events. In protein expression of TIMP-1 and TIMP-2, the key order to determine the impact of YWPC supplementation inhibitors of MMP-9 and MMP-2. We found that the on MMP-2 and MMP-9, we analyzed their protein expression of TIMP-1 and TIMP-2 in mice treated with expression by western blotting and activity, by gelatin 10, 30, and 50 mg/kg per day YWPC increased signifi- zymography. The results demonstrated that YWPC at 10, cantly in comparison with the control group, which was 30, and 50 mg/kg per day significantly inhibited MMP-2, enhanced more in the rosuvastatin group when compared 9 activity and protein expression when compared to the to the control group and YWPC groups (Fig. 5). control group. Rosuvastatin, used as a positive control, also significantly inhibited MMP-2 and MMP-9 activity Discussion

Many epidemiological studies have demonstrated that moderate consumption of polyphenol-rich foods, such as fruits and vegetables, tea, and red wine, is associated with a reduction in overall mortality, especially the reduced risk of coronary heart disease (CHD). Yellow wine is also a rich source of polyphenolic compounds. However the regulative effects of yellow wine on CHD have not been completely evaluated yet. Therefore, we planned this study to examine the potential beneficial effects of YWPC from yellow wine in LDLR−/− mice fed a high-fat diet. The dose level of YWPC to rats Fig. 2. Atherosclerosis lesion areas of the aorta artery in the 5 is comparable to human exposure, and the effect of groups. Data are expressed as the mean ± S.D. #P < 0.01 versus control; *P < 0.01 versus rosuvs, &P < 0.01 versus YWPC (10 mg/kg rosuvastatin was tested for comparison. per day). Rosuvs, rosuvastatin. n = 3 per group. Studies have found that a high level of plasma choles-

Fig. 3. Effects of rosuvastatin and 10, 30, and 50 mg/kg per day YWPC on MMP-2 (A) and MMP-9 (B) protein production in the aorta isolated from LDLR−/− mice treated with high fat diet after 14 weeks. They were analyzed by western blotting and normalized with the housekeeping gene b-actin. Data are expressed as the mean ± S.D. #P < 0.01 vs. control, *P < 0.05 and **P < 0.01 vs. rosuvs, &P < 0.05 vs. YWPC (10 mg/kg per day). Rosuvs, rosuvastatin. YWPC Improve Atherosclerosis 137

Fig. 4. Effects of rosuvastatin and 10, 30, and 50 mg/kg per day YWPC on MMP-2 (A) and MMP-9 (B) activity. Protein extracts from the aortic arch of LDLR−/− mice after 14 weeks on high-fat diet. They were examined by gelatin zymography assay. Data are expressed as the mean ± S.D. #P < 0.01 vs. control, *P < 0.05 and **P < 0.01 vs. rosuvs, &P < 0.05 vs. YWPC (10 mg/kg per day). Rosuvs, rosuvastatin.

Fig. 5. Effects of rosuvastatin and 10, 30, and 50 mg/kg per day YWPC on TIMP-1 (A) and TIMP-2 (B) protein production in the aorta isolated from LDLR−/− mice treated with high-fat diet after 14 weeks. They were analyzed by western blotting and normalized with the housekeeping gene b-actin. Data are expressed as the mean ± S.D. #P < 0.01 vs. control, *P < 0.05 and **P < 0.01 vs. rosuvs, &P < 0.05 vs. YWPC (10 mg/kg per day). Rosuvs, rosuvastatin. terol is a risk factor contributing to the incidence and reached a plateau. The lipid effects cannot be attributed severity of CHD. The present study showed that dietary to differences in food or energy intake. It was consistent YWPC at levels of 10, 30, and 50 mg/kg per day reduced with other investigations that showed a hypocholesterol- circulating total cholesterol and LDL cholesterol levels emic effect in rats fed diets containing phenolic com- as well as the rosuvastatin in LDLR−/− rats compared pounds derived from red wine or tea (26, 27). The mecha- with that in the control group. There was no statistically nism by which polyphenols decrease the cholesterol significant difference between YWPC at 30 and 50 mg/kg level may be through their interaction with cholesterol per day. We speculated that a maximum beneficial carriers and transporters present across the brush border effect of YWPC on blood lipids was gained at a dose membrane, which inhibits the absorption of cholesterol of 30 mg/kg per day and then the lipid-lowering effect and enhances fecal excretion of cholesterol and bile 138 X Zhai et al acid (27, 28). In our study, YWPC and rosuvastatin TIMPs are a family of specific endogenous inhibitors tended to increase the serum HDL-C level, although the of MMPs whose activity correlates with decreased MMP effect was not significant. HDL plays an important activity in atherosclerotic plaques and plays a key role in anti-atherogenesis role by mediating reverse cholesterol maintaining the balance between the particular units of transport (RCT) to promote cholesterol efflux from ECM (40). There are currently 4 known TIMPs — TIMP macrophages and transporting the cholesterol to the liver 1 to TIMP 4. Each TIMP member operates with different (29). Several studies have shown that both polyphenols inhibition efficiencies against the different MMPs. It and rosuvastatin could enhance HDL-mediated choles- has been demonstrated that TIMP-1 binds and slows terol efflux via up-regulation of ABCA1 protein expres- activation of the latent form of MMP-9, whereas TIMP-2 sion and reduce atherosclerosis (29 – 31). binds and regulates activation of MMP-2 (41). In order In addition, increasing evidence demonstrated that to investigate the mechanism underlying inhibition of the dietary polyphenols had the capacity to reduce LDL MMP secretion, we mainly detected the expressions of susceptibility to oxidation which could operate to benefit TIMP-1 and TIMP-2 protein. The results of western atherosclerosis (32). The activity of polyphenols for blot analysis showed that with respect to the control protecting LDL from oxidation could be attributable, group, the protein expression of TIMP-1 and TIMP-2 at least in part, to both binding to the LDL particle was significantly higher in both the YWPC and rosuvas- against metal-catalyzed oxidation and scavenging tatin groups. The plausible mechanism mediating this property of free radicals (33), whereas the reduction beneficial effect of rosuvastatin may be through statin- of oxLDL by rosuvastatin is due to the anti-oxidative induced increase in nitric oxide bioavailability, as endo- properties, which are correlated with the reducing thelial nitric oxide synthase gene transfer has been equivalent donating property or direct hydroxyl radical- demonstrated to decrease MMP activities and increase scavenging activity (34). Thus, additional research is TIMP-2 secretion (42), whereas YWPC inhibition against necessary to determine the inhibitory effect of YWPC the transcription and expression of MMP-9 gene may be on LDL oxidation. attributed to the downregulation of activator protein-1 The development of atherosclerotic lesions is a chronic (AP-1) and nuclear factor-nB (NF-nB), which could also inflammatory process that is characterized by excessive up regulate the expression of TIMP (43). This result is in vascular remodeling with accumulation of cells and agreement with an earlier study in which polyphenols lipids within the intimal layer of the pathological artery increase the expression of TIMP-1 and TIMP-2 (44). (35). ECM is the main composition which constitutes Therefore, the prevention of MMP activation by YWPC blood vessel wall. Remodeling of ECM, including its is likely to be mediated by the increase of TIMP level, degradation and synthesis, is in dynamic balance in the thus weakening the ECM degradation that occurs in organism. MMPs are a family of proteolytic enzymes atherosclerotic arteries. In the present study, we found a that participate in the degradation of components of the reduction of the plaque lesion area in animals treated ECM, resulting in atherosclerotic plaque formation and with YWPC and rosuvastatin. Based on the studies vulnerability (36). Studies in mice have suggested that above, it is possible to conclude that YWPC decreases different members of the MMP family may exert different the activity of MMP-9 and MMP-2 protein and increase effects on atherogenesis. In particular, MMP-2 and MMP-9 the expression of TIMP-1 and TIMP-2 to alleviate have been identified as major proteinases degrading atherosclerotic lesion. most collagens in atherosclerotic plaque lesions during On the other hand, MMP and TIMP also act as key atherosclerotic lesion formation and instability, leading fibrogenic factors, which are important for hepatic fibro- to an increased risk of cardiovascular events (36, 37). sis progression (45). The development of hepatic fibrosis Therefore, the inhibition of MMP-2 and MMP-9 is can be prevented by polyphenols via a modulation of considered to be an important target for therapeutic MMP-2 activity (46). Furthermore, polyphenols con- intervention. Previous findings have pointed out that sumption has been shown to be inversely correlated polyphenols prevent effectively the expression of MMP- with liver damage (47) and markers of liver function 9 in endothelial cells and the Ang II–induced expression such as alanine aminotransferase (ALT) and aspartate of MMP-2 in vivo (38, 39). In our investigation, we transaminase (AST) (48). However, Galati et al. (49) demonstrated that the activities and expressions of found that administration of tea polyphenols resulted in both MMP-2 and MMP-9 were reduced by YWPC and a significantly increased level of plasma ALT level in rosuvastatin. These data suggested that YWPC have a mice, indicative of liver injury. Liver is an important distinct effect on MMP-2 and MMP-9 activities and organ participating in phenolics metabolism. We are expressions that may be related with a limited athero­ planing future studies to identify the potential biological sclerotic effect. effects of YWPC on biomarkers of liver function. YWPC Improve Atherosclerosis 139

Diet is a cornerstone in the prevention of cardio­ the Science and Technology Plan Project of Zhejiang Province vascular disease. In general, the effects of red wine (No. 2012C33040), technology projects for medicine and health of polyphenols have been conducted at doses ranging Zhejiang Province (WKJ2011-2-018), and by the Zhejiang Provincial program for the cultivation of High level Innovative Health Talents between 20 and 40 mg/kg for 1 – 4 week in vivo. In our (No. 2012241). experiments, 3 different concentrations of YWPC were used for 14 weeks. The results showed that all 3 doses of References YWPC are adequate to produce sufficient circulating concentrations of compounds which are able to relieve 1 Renaud S, de Lorgeril M. Wine, alcohol, platelets, and the French atherosclerosis. At present, statin is the main way for the paradox for coronary heart disease. 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