| Now,it is widely recognized that obesity, especially abdominal obesity is closely associate with occurrence, progression and diagnosis of metabolic syndrome. Lipid metabolism disorder is a main risk for metabolic syndrome, cardiovascular disease and diabetes. Excessive circulating free fatty acids and fat deposition in non-adipose tissue increase the risk of tissues injury and insulin resistance. Inflammatory cytokines secreted by adipose tissues,such as IL-6, TNF-α, can cause a state of inflammatory response, increase levels of reactive oxidative species(ROS), which may induce a variety of cardiovascular diseases, such as atherosclerosis. Therefore, to improve dyslipidemia and decrease fat deposition will contribute to alleviate the conditions of diseases and reduce the occurrence of death.Sirtl is a NAD-dependent histone deacetylase as a member of Sirtuins protein family. An increasing number of studies have shown that Sirtl plays an important role in regulating nutritional and metabolic balance and increasing cell survival under stress.Therefore,it is proposed that Sirtuins family will be a potential targets for the therapy of metabolic syndrome.Resveratrol(RES) is a natural compound attributed to non-flavonoid polyphenol discovered earliestly with a function of activating Sirtl activity. In many aging-associated diseases, such as cardiovascular disease and diabetes,it shows a good effect. Studies indicated that the joint action of statin and resveratrol can not only enhance the hypolipidemic efficacy, but also increase myocardial protection.Berberine(BBR) is a natural compound attributed to isoquinoline alkaloids with a hypolipidemic efficacy.The hypolipidemic mechanism of berberine is different with statins, which is to upregulate LDLR expression in liver cells. Studies indicated that the combination of berberine with statin can enhance the hypolipidemic efficacy.In addition, berberine also has an antioxidant effect,but the mechanism is not clear.In this study, the lipid-lowering efficacy of combination of berberine with resveratrol and the antioxidant role of berberine are explored. The primary study of their mechanism also be explored. The goal is to provide a guidance to improve the clinical effectiveness of lipid-lowering of berberine and reduce the incidence of adverse reactions.Meanwhile, the primary study of Sirtl protein expression in D-galactose-induced aging mice also be investigated. We selected a low dose of berberine combined with resveratrol to study the synergy of Berberine and Resveratrol in hyperlipemia hamster model.The results showed:based on the detection of total cholesterol, Triglyceride, and LDL-cholesterol, administration of low-dose of berberine (25mg/kg)or resveratrol(20mg/kg) alone showed little hypolipidemic effect, while it showed a significant hypolipidemic effect in the combination group(p<0.05).More over, the combination of low-dose of berberine with resveratrol showed a synergetic effect on lipid-lowering.Through 3T3-L1 adipocyte models,we evaluated the impact of berberine and resveratrol on adipogenesis. The results showed that Berberine and resveratrol had a capability to reduce the accumulation of lipid droplets in adipocyte with a dose-depedent relatioship.12μmol/L or 20μmol/L berberine combined with 25μmol/L resveratrol showed a certain synergetic effect.In order to study the synergetic mechanism of Berberine combined with resveratrol, we hypothesis that resveratrol can enhance the efficacy of LDLR expression regulated by berberine. By indirect fluorescent labeling technology and flow cytometer assay, the mean fluorescent intense in combination of 12μmol/Lberberine and 10μmol/L resveratrol group increased by 8%compared with berberine group,which implied that resveratrol potentialize the effect of berberine on LDLR expression. Meanwhile, the results of Western blot were consistent with that of flow cytometry. Secondly, using fluorescence properties of berberine,we detected the amount of berberine in cells by flow cytometry to evaluate the influence of resveratrol on amount of berberine in cells.It was showed:After pre-treatment of 10μmol/L resveratrol for 2h,the amount of berberine in liver cells significantly increased by 16%(p<0.05), compared with the control group.Meanwhile, we studied the effect of berberine on expression level of sirtl protein, and the linkage between Sirtl and LDLR were also investigated. The results showed that 1.It's the first time to discover that Berberine can up-regulate the expression of Sirtl protein in vitro and in vivo.The expression levels of protein and mRNA abundance of Sirtl were significantly increased by Western blot and RT-PCR.2.the expression level of Sirtl and LDLR protein are positive correlation. After berberine treatment, an increase of Sirtl and LDLR protein expression can be detected by Western blot, and a decrease of Sirtl and LDLR protein expression can be detected by Western blot after a Sirtl specific inhibitor Sirtinol.Based on the effect of berberine on expression level of Sirtl protein, we speculated Berberine can enhance the capability of cells to resist to oxidative stress damage due to upregulation of Sirtl protein. Therefore, we designed some experiments to evaluate the effect of Berberine on enhancing cell resistance to oxidative stress, including the premature aging of young embryo lung diploid fibroblasts (HPF) induced by H2O2, cell apoptosis induced by H2O2 and generation of oxygen free radicals in liver cells induced by H2O2. Experimental results showed that 1. Berberine and resveratrol can significantly reduce the generation of senescence-associated-β-galactosidase in young embryo lung diploid cells induced by H2O2, and increase the resistance of cells to sublethal H2O2-induced cell senescence-like change and survival of cells.2.20μmol/L Berberine can enhance cell resistance to lethal H2O2-induced cell apoptosis and maintain cell survival. The decrease of PARP fragment and caspase-3 activation fragment were detected by Western blot, but expression level of sirtl protein did not decline.3. 20μmol/Lberberine and lOμmol/Lresveratrol can protect the expression of Sirtl protein from decline induced by lethal H2O2 4.12μmol/L Berberine and 10μmol/L resveratrol can reduce generation of intracellular reactive oxygen species induced by sublethal H2O2Through establishment of aging model induced by D-galactose, we investigated the expression levels of Sirtl protein in aging model.We established the aging mice model induced by different doses of D-galactose. Aging Model was evaluated using the index of body weight, appearance, biochemicals assay in blood.The results showed:In D-galactose treatment group and control group, body weight did not have significant differences. However, in its appearance, we observed that the D-galactose treatment group showed many of yellow hair and white hair, reduction of spontaneous activity, compared with the control group. In D-galactose treatment group, serum superoxide dismutase (SOD) content were significantly reduced (p<0.05), malondialdehyde (MDA) content were significantly increased (p<0.05) compared to control group. In the liver tissue of aging animal, we detected a decrease of Sirtl and Hsp70 protein.In this study, the results showed:1.It is the first time to discover that the combination of berberine with resveratrol can enhance the hypolipidemic effect of berberine.and there is a synegetic effect of resveratrol and berberine on decreasing lipid accumulation in adipocyte.It is primary showed that resveratrol can increase the accumulation of berberine in liver cells and sequently increase the amount of LDLR protein expression could be associated with the efficacy of combination.2.It is the first time to discover that berberine can up-regulate Sirtl expression in vitro and in vivo. It is primary shoed that Berberine can decrease premature aging of cells induced by sublethal H2O2 the apoptosis of cells induced by lethal H2O2 and enhance survivral of cells under oxidative stress. Upregulating Sirtl expression and reduce the production of reactive oxidative species induced by H2O2 could be associated with the effect of berberine on improving the resistance of cells to oxidative stress injury.3.Our primary study in D-galactose-induced aging mice indicated that expression levels of Sirtl protein decreased in liver tissue. |
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