Study On The Effects Of Different Doses Of Resveratrol On High-fat Diet Mice With The Genes Of SIRT1, LXR-α, And ABCA1

IntroductionRecently, cardiovascular and cerebrovascular diseases have become major diseases of our residents. Dyslipidemia is elevation of serum cholesterol, triglyceride, or both, or a low high density lipoprotein level that contributes to the development of atherosclerosis. Epidemiological studies have associated high levels of total cholesterol (TC) and low-density lipoprotein (LDL) and reduced concentrations of high density lipoprotein (HDL) with the risk of atherosclerosis. Therefore, to know the lipid metabolism in cardiovascular disease has a positive application and scientific significance meaning. Resveratrol (RSV) is a polyphenlic flavonoid found in the grapes and skins of grapes that produce wine, raspberries, mulberries, blueberries and cranberries. In mouse and rat experiments, anti-cancer, anti-inflammatory, blood-sugar-lowering and other beneficial cardiovascular effects of resveratrol have been reported. Recently, reports indicate that resveratrol treatment produces beneficial effects in mammals are mainly mediated through SIRT1 (the mammalian homolog of Sir2).SIRT1 deacetylation function through participation in various metabolic activities, through a variety of histone and non histone proteins to perform different functions, including liver X receptor (LXRs) with cholesterol, triglycerides and glucose metabolism are closely related to the role of gene regulation for a class of nuclear receptors. Some cholesterol metabolites such as hydroxy steroids 24s,25-epoxy cholesterol is the activation of their endogenous ligand.Our present study was designed to elucidate the effects of resveratrol on the high-fat diet-induced dyslipidemia mice for 8 weeks, hypothesized that resveratrol treatment might activate SIRT1 in vivo. And SIRT1 though the LXRs to alleviate the dyslipidemia, delay the progression of atherosclerosis.Materials and methodsMale Kunming mice,8 weeks old, came from China Medical University (CMU) Laboratory Animal Center and were quarantined for 1 week. They were housed in cages (n=5/cage) kept at 22±3℃, with 50-60% relative humidity and controlled lighting that provided a 12h light-dark cycle. Free to drink water and a normal diet. Mice were randomly divided in two groups, a standard-diet group (n=10) and a high-fat diet group (n=40,10%fat). After 4 weeks feeding, we tested the total cholesterol of the venous blood from the canthus. The results of the TC carried out a significant difference between groups. Then, the high-fat-diet group was randomly divided into four groups (n=10/group), one with 0.5% Carboxymethyl cellulose solution (CMC), the other groups with resveratrol 5mg/(kg-bw-day),22.5mg/(kg-bw-day),45mg/(kg-bw·day) and 0.5% CMC for 8 weeks.Analytic ProceduresBlood samples were collected via orbital venous plexus from the anaesthetized mice for determination of serum lipids:using semi-automatic biochemical analyzer for determination of serum TC, triglyceride (TG), LDL-C, and HDL-C, TC and TG use a colorimetric assay according to manufacturer's instructions. LDL-C and HDL-C use a hydrogen peroxide clearance method to manufacturer's instructions.Serum and liver homogenate were prepared to measure the superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) by microplate reader. Xanthine oxidase (hydroxylamine method) determination of SOD activity, MDA with thiobarbituric acid (TBA) assay and GSH-Px use a colorimetric assay according to manufacturer's instructions.The mRNA expression on liver samples:according to TRIzol manual steps, to gain total RNA from approximately 1OOmg of liver. Then use 1μg total RNA, to carry out reverse transcriptase polymerase chain reaction (RT-PCR) reaction by Takara RNA PCR kit. then ran on 2% agarose gel electrophoresis and photographed under gel imaging camera analyzer. Compare with the ratio of (3-actin gene expression as relative content. Three kinds of primers were designed by Premier Primer 5, and evaluated by the Oligo6.0. Statistical analysisData were expressed as mean±standard deviation (mean±SD). Statistical analysis was performed by one-way ANOVA using SPSS 13.0 statistical software. P＜0.05 is considered as significant.Results1. The general condition of animals and body weightThe mice were generally in good condition, no abnormal growth and development. Body weight of mice before and after intervention, the low dose RSV had the most weight gain, however, no significant difference between the all the groups (P> 0.05).2. Liver appearance and pathologyLiver/body ratio were compared, the availability of high-fat group was higher than other groups (P＜0.05), RSV in each group had a slightly downward trend.In the liver appearance and shape, among the groups did not observe significant differences. High-fat group had a little of the increased weight and slightly greasy. Histological examination of liver sections by staining with hematoxylin-eosin revealed a loss of cellular integrity and some vacuoles and necrosis in the livers of both high-fat groups.3. The serum lipidsAfter 8 weeks treatment, the middle dose of RSV group'serum LDL-C level was significantly reduced compared with the high-fat diet group. And the low dose of RSV group'total cholesterol was reduced and others did not have any decreased after the treatment. To the level of TG, we didn't get any effective results yet. At the same time, the concentrations of HDL-C in the RSV group had a little Increasing tendency, but among the groups no statistical significance change.4. AntioxidantThe middle dose of RSV group was significantly reduced the serum MDA. And all the high-fat groups GSH-Px activity was lower than the standard diet group. But again the The middle dose of RSV group showed the role of elevated enzyme activity of glutathione.In the liver tissue, we got the same conclusion to the ability of resveratrol in the middle dose cleaning the MDA. But we saw the same effect of resveratrol in the serum to increase the enzyme activity of the GSH-Px. 5. The mRNA expression of SIRT1, LXR-a, and ABCA1We did not see a clear difference among the groups in the mRNA expression level. We tested the SIRT1, LXR-a, and ATP binding cassette transporter A1 (ABCA1) mRNA expression.Conclusions1. The dose of 22.5mg/(kg-bw·day) of RSV group can reduced the high-fat mice serum lipid levels of LDL-C and the dose of 5mg/(kg-bw·day) RSV group can reduce serum lipid levels of TC.2. The dose of 22.5mg/(kg-bw·day) RSV group can reduce the high-fat mice MDA levels and raise the enzyme activity of the GSH-Px.3. The effect of resveratrol on serum lipid levels of hyperlipidemic mice was not affected by affecting the liver in mice SIRT1, LXR-a, and ABCA1 mRNA expression. there may be other mechanisms.