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INTRODUCTION
Chronic ethanol intake is a serious social problem since it increases social expenses in terms of health and family problems. Recently, a great increase in the number of Koreans who chronically consume ethanol has been associated with such problems. One health problem with ethanol intake is dysregulated hepatic lipid metabolism. For example, patients abusing alcohol are observed to have triglyceride accumulation in their hepatocytes, a critical process in disease progression leading to late stage hepatitis and cirrhosis [1,2,3]. Abnormal lipid metabolism includes fat accumulation in the liver caused by an imbalance between the degradation and synthesis of fatty acids. Development of hepatic steatosis is therefore initially seen in deleterious effects of excessive alcohol intake [4,5,6]. In addition, hypertriglyceridemia (increased blood triglyceride levels) caused by elevated very low-density lipoprotein and chylomicron levels in the blood [7], and hypercholesterolemia caused by enhanced cholesterol biosynthesis and decreased bile acid excretion [8] were observed in chronic alcohol consumption patients.
DWP208, a sodium succinate form of ZYM-201 (Fig. 1), is a saponin derivative obtained from a ziyu-glycoside of Sanguisorba officinalis L. (Rosaceae). This compound has been reported to be an orally available drug. Orally administered DWP208 dose-dependently inhibited in vivo tissue factor (TF) activity with an ED50 of 1.7 mg/kg [9]. Furthermore, ZYM-201 diminished both in vitro and in vivo tumor necrosis factor (TNF)-production with an IC50 and ED50 of 69.4 M and 87.4 mg/kg, respectively [9], suggesting that DWP208 may have therapeutic activity against tissue TF- and TNF-mediated diseases such as septic shock and against hyperlipidemic conditions. Indeed, this compound strongly attenuated the hyperlipidemia induced by diabetic and high fat diet conditions as did other saponin components from Sapindus rarak DC, floratheasaponins, and Eugenia jambolana [10]. However, because a major cause of hyperlipidemia is chronic alcohol intake, we investigated the curative activity of DWP208 in a rat model of chronic alcohol consumption.
METHODS
Materials
DWP208, a sodium succinate form of ZYM-201 (ziyuglycoside II methylester) [9], was synthesized by adding succinic anhydride and sodium-2-ethylhexanoic acid. The yield of DWP208 was 83% and its chromatographic purity was greater than 95.3% according to HPLC analysis. Fenofibrate, cholesterol, (3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT), succinic anhydride, and sodium-2-ethylhexanoic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Absolute alcohol was purchased from Fluka (Buchs, Switzerland). All other chemicals and reagents used were of pharmaceutical grade. Fetal bovine serum and RPMI1640 were obtained from GIBCO (Grand Island, NY, USA). RAW264.7 cells were purchased from ATCC (Rockville, MD, USA).
Animals
Male Sprague-Dawley (SD) rats weighing 150-160 g were purchased from Daehan Biolink (Daejeon, Korea) and were given ad libitum access to a commercial diet (Samyang, Korea) and water. They were housed in a temperature-controlled room at 20℃ with lighting on for 12 h. Humidity was maintained between 50 and 70%. Our study was approved by the Animal Ethics Committee of Kangwon National University (Chuncheon, Korea) and was performed in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the Korea National Institute of Health.
Induction of hyperlipidemic conditions
To induce hyperlipidemic conditions, 10% (w/w) aqueous ethanol was chronically treated for 8 weeks as reported previously [11]. At the end of the treatment, using these rats, DWP208 (0 to 10 mg/kg) or fenofibrate (5 mg/kg) were orally administered once a day for 1 week. According to previous methods [12], animals were anesthetized and blood was drawn by cardiac puncture 2 h after the final drug administration. Serum was obtained by centrifugation of blood at 3,000 rpm for 15 min.
Biochemical analysis of serum
Serum was separated by centrifugation at 1,000 g for 15 min. The levels of total cholesterol, total triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) were determined using an automatic analyzer (Hitachi 7020, Japan) [13]. Determination of hydroxyl radicals was done by a method previously reported [14]. Lipase activity in serum was also determined by a method previously published [15]. Lipid peroxidation levels in liver and serum were measured using an assay for thiobarbituric acid reactive substances (TBARS) [16]. SOD activity was assayed in the serum and liver using a technique that involves inhibition of pyrogallol auto oxidation at pH 8.0 [17].
Biochemical analysis of liver samples
Liver samples for enzymatic analyses were prepared according to the methods of a previous report [18]. Each liver sample (1 g) was homogenized (10%, w/v) in four volumes of 0.1 M potassium phosphate buffer (pH 7.5), then centrifuged at 600 g for 10 min, 10,000 g for 20 min, and 105,000 g for 60 min to prepare the microsomal fraction. The microsomal fraction was used for the assay of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase. Total lipids, total cholesterol, and triglyceride in the liver homogenate prepared with 0.9% NaCl were enzymatically determined using assay kits (Asan Pharmacy, Seoul, Korea). Protein concentrations were determined by the Bradford method [19] using BSA as the standard.
Statistical analysis
Student's t-test and a one-way ANOVA were used to determine the statistical significance of differences between values for the various experimental and control groups. Data are expressed as mean±standard error (SEM) and the results are taken from at least four independent experiments performed in 10 rats. p<0.05 was considered to be statistically different unless otherwise indicated.
RESULTS AND DISCUSSION
Since we found that DWP208, a water-soluble salt form of ZYM-201, is able to normalize the up-regulated levels of hyperlipidemic parameters in an animal model of streptozotocin-induced diabetes that was simultaneously fed a high fat diet [20], we examined the effects of DWP208 on alcohol-induced hyperlipidemia. Chronic alcohol addiction is more prevalent in the Korean population and causes serious effects such as liver damage, leading to the generation of fatty liver, cirrhosis, and liver cancer. Although getting the patient to abstain from alcohol drinking would be ideal, alcohol-induced liver damage and subsequent malfunction of liver metabolism, causing the generation of various vascular diseases, such as atherosclerosis and stroke should be restored. To date, no drug has been reported to be a strong candidate for treating these symptoms of chronic alcohol intake. In the current study, we investigated the possibility that DWP208 can be used as a remedy to ameliorate alcohol-induced liver dysfunction in an animal model of alcohol-induced hyperlipidemia.
During chronic alcohol (10%) treatment, it has been shown that liver function and lipid metabolism are altered. Thus, alcohol treated individuals display striking hyperlipidemic symptoms such as (i) up-regulated levels of LDL, VLDL, atherosclerosis index (AI), serum triglyceride and total cholesterol, hepatic total lipid, cholesterol, and triglyceride levels, and serum lipoproteins and HMG-CoA reductase contents (Fig. 2, 3, 4, 5, Table 1), and (ii) down-regulated levels of body weight, HDL, and SOD activity (Table 2, Fig. 2A, and Fig. 5C). The alcohol-induced up-regulation and down-regulation of these parameters were previously reported [21,22], indicating that our experimental plan was feasible.
Also, DWP208 was able to ameliorate the hyperlipidemia induced by a high fat diet or streptozotocin [20]. Furthermore, DWP208 protected against the hyperlipidemia induced by chronic alcohol intake. The most striking activity of DWP208 was its effects on LDL levels. Alcohol-induced up-regulation of LDL was completely restored by DWP208 treatment at 3 to 10 mg/kg (Fig. 2A). Reduced HDL levels were also restored and enhanced VLDL level were normalized. Consequently, AI value increased in alcohol treatment was strongly recovered in DWP208 treatment groups (3 and 10 mg/kg), as standard compound fenofibrate did (Fig. 2B). DWP208 also attenuated the up-regulated triglyceride and total cholesterol levels in serum (Fig. 3A, Table 1).
To see if the regulation of serum metabolism of lipid molecules by DWP208 was due to modulation of serum lipase levels, the activity of this enzyme was tested. However, chronic alcohol treatment failed to significantly increase the serum activity of lipase (Fig. 3B); indeed, there was a slight decrease. DWP208 did not alter serum lipase activity, while fenofibrate increased it up to 2 fold, suggesting that DWP208-mediated regulation of serum parameters is not serum lipase dependent. In fact, hepatic levels of total lipid (Fig. 4A), total cholesterol (Fig. 4B), and triglycerides (Fig. 4C) upregulated by 10% alcohol treatment were apparently restored by DWP208 (Fig. 5), suggesting that altered hepatic lipid metabolism can be targeted by DWP208. However, HMG-CoA reductase, a critical enzyme for cholesterol biosynthesis [23], did not appear to be a target enzyme involved in DWP208-mediated amelioration of hyperlipidemic conditions. Thus, DWP208 at doses up to 10 mg/kg did not suppress the activity of this enzyme, although the enhancement of HMG-CoA reductase activity was very marginal in alcohol treatment group (Table 3), suggesting that other metabolic enzymes or pathological events could be the target of DWP208.
Since finding a direct target of DWP208 is not easy, we focused on exploring the effect of this compound on the generation of major pathological molecules. Numerous literature reports have commonly stressed the critical role of radicals generated during alcohol intake in the malfunction of hepatic lipid metabolism [5,24]. We found that DWP208 suppresses radical generation under streptozotocin treatment during high fat diet conditions [20]. Indeed, Fig. 5 strongly supported an anti-oxidative role of DWP208 in alcohol-treated conditions. Thus, this compound dose-dependently suppressed lipid peroxide (Fig. 5A) content and serum hydroxyl radical levels (Fig. 5B). Furthermore, the activity of SOD, an anti-oxidative enzyme against superoxide and hydroperoxide [25], was restored to normal levels after alcohol treatment had clearly reduced it (Fig. 5C). This suggested that the anti-oxidative property of DWP208 may be a critical factor in its anti-hyperlipidemic effects.
In general, it has been known that most saponin components are not strong anti-oxidants due to lack of chemical features [26]. Instead, some saponins such as ginsenoside Rb1 and gypenosides indirectly protect against cell damage induced by radicals by suppressing NF-B [27] and increasing glutathione levels [28]. We also failed to find direct neutralizing activity of DWP208 against SNP-induced radical generation [20]. Therefore, our results and previous reports suggest that DWP208 is protective by indirectly increasing cellular anti-oxidant systems. Since various biological activities of saponins - such as acute hepatic failure induced by lipopolysaccharide and D-galactosamine in mice [29], gastric ulcer induced by HCl/EtOH [30], and oxidative injury of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium ion (MPP(+) [28] - are derived from their neutralizing activity against oxidative stress, DWP208 seems to have similar protective activities against these pathological symptoms. Further tests of the pharmacological actions of DWP208 in such models are now warranted.
In summary, we found that orally administered DWP208 can reverse the hyperlipidemia generated by chronic intake of alcohol. This compound normalized both up-regulated hyperlipidemic parameters such as LDL, VLDL, AI, triglyceride, total cholesterol, and down-regulated hyperlipidemia parameters such as absolute body weight, SOD activity, and HDL in serum and liver. According to our data, the ameliorative activity of DWP208 seems to be due to its indirect anti-oxidative activity by which lipid peroxide and hydroxyl radical levels are reduced and the activity of SOD is increased. Considering that chronic alcohol abuse is a serious social problem and is increasing particularly fast in Far East Asian countries, the complications of alcohol consumption, including hyperlipidemia and atherosclerosis, will be greatly increased. Our data strongly suggest that DWP208 can be used as a remedy against alcohol-induced hyperlipidemia.
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