Effect Of Grape Seed Proanthocyanidins Extracts On Synthesis And Expression Of Apolipoprotein A-â…  Under Condition Of High Glucose

Part one Inhibitory effect of grape seed proanthocyanidins extracts on the levels of apolipoprotein A-I of both plasma and aortic tissue in streptozocin induced diabetic ratsBackgroundMacroangiopathy is a major complication of both typeⅠand typeⅡdiabetes,and its main mechanism is not so clear.The pathologic changes of diabetic macroangiopathy can be from endothelial dysfunction to formation of atherosclerotic plaque,in which oxidative stress induced by chronic hyperglycemia may play a key role.Proanthocyanidins are naturally occurring polyphenolic compounds widely available in fruits,vegetables,nuts,seeds,flowers and bark.Grape seed proanthocyanidins extracts(GSPE),a combination of biologically active polyphenolic flavonoids including oligomeric proanthocyanidins,have been demonstrated to exert a novel spectrum of biological,pharmacological,therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress.Previous studies have proved that plasma apolipoprotein A-I(apoA-I),the major apolipoprotein of high density lipoprotein(HDL),by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells, is a protective factor of cardiovascular disease.And there is a negative relationship between the level of apoA-I and cardiovascular diseases.But neither the influence of GSPE on the levels of apoA-I of both plasma and aortic tissue in diabetic models nor the influence of fluctuation of levels of apoA-I on diabetic aortic angiopathy remained unknown.Our previous experiments showed that GSPE played a protective role in cardiomyocyte,nervous system,etc.In this study,we aimed to examine the effects of GSPE on the levels of apoA-I of plasma and aortic tissue in streptozocin (STZ) induced diabetic rats and to evaluate the relationship between GSPE and diabetic macro-angiopathy,which may be helpful to find a new drug target point.ObjectiveIn this study,we investigated the effect of grape seed proanthocyanidins extracts (GSPE),which have been proved to have anti-oxidative and anti-aging functions,on the levels of apolipoprotein A-I(apoA-I) of both plasma and aortic tissue in a rat model of diabetes.MethodsFifty-five rats were randomly divided into three groups:group C(control,n=15); group DM(diabetes,n=20);group DM+GSPE(GSPE administrated diabetes,n=20). GSPE(250 mg/kg body weight/d),were administrated to diabetic rats induced by STZ intravenous injection via tail veins,for 24 weeks.Plasma low density lipoprotein(LDL),high density lipoprotein(HDL)and apoA-I were determined in each group.Aftern all animals were sacrificed after 24 weeks,the aortas were excised about 1cm long from the end,fixed in 10%formaldehyde,and embedded in paraffin and cut into 4μm-thick sections for light microscopy.Then they were stained with hematoxylin-eosin under a light microscope at a magnification of 200×.Beside this, difference gel electrophoresis technique was used to explore differential expression protein after treatment with GSPE in diabetic rats.Western blot and immunofluorescence techniques were used to validate the protein expression levels of apoA-I in aortic tissue.ResultsIn untreated diabetic rats,the levels of LDL,HDL and apoA-I were higher than those in group C.With the treatment of GSPE,the levels of LDL,HDL and apoA-I decreased to normal values similar to those of control rats.In the diabetic rats, hypertrophy and disarray of endothelial cells,proliferation of smooth muscle cells were observed in aortic wall,and the internal elastic laminae were impaired.The lumina of aorta became smaller.After treatment of GSPE light microscopic findings were similar to those of the control rats.ApoA-I was found differential expressed in DIGE(DM group/C group:-1.68;DM+GSPE group/DM group:1.77).In the following Western blot and immunofluorescence experiment,the expression of apoA- I in aortic tissue was found to be the highest(p<0.05) in group DM,GSPE significantly reduced this expression(p<0.05).ConclusionsIn conclusion,GSPE can downregulate the expression of apoA-I in both plasma and aortic tissue,which may be contributed to the beneficial influences of GSPE on the diabetic aortic angiopathy.Part two Effects of Grape seed proanthocyanidins extracts on apolipoprotein A-I expression in HepG2 cells under the experimental conditions of high glucoseBackgroundApoA-I,which constitutes 70%of the apolipoprotein content of HDL,acts as acceptor for the transfer of phospholipids and free cholesterol from peripheral tissues and transports cholesterol in the liver and other tissues for excretion and steroidogenesis.In particular,apoA-I interacts with the ATP-binding cassette transporter A1 and accepts free cholesterol,and acts as a cofactor of the HDL-associated enzyme lecithin cholesterol acyl transferase(LCAT),thereby mediating the delivery of cholesterol ester to the liver.This process is important in reducing the accumulation of foam cells in the arterial walls.Furthermore apoA-I exerts antioxidant properties by removing lipid hydroperoxides from low-density lipoprotein(LDL),thereby reducing the oxidation of LDL and consequently the atherogenicity of these lipoproteins.Therefore,apoA-I also shows anti-inflammatory actions through the inhibition of the expression of adhesion molecules in endothelial cells and reducing the recruitment of monocytes in the arterial walls.It may be helpful to promote the expression of apoA-I to prevent the pathogenesis of diabetic angiopathy.ObjectiveProanthocyanidins are naturally occurring polyphenolic compounds widely available in fruits,vegetables,nuts,seeds,flowers and bark.Grape seed proanthocyanidins extracts(GSPE),a combination of biologically active polyphenolic flavonoids including oligomeric proanthocyanidins,have been demonstrated to exert a novel spectrum of biological,pharmacological,therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress.In this study,we investigated the effect of grape seed proanthocyanidins extracts(GSPE), which have been proved to have anti-oxidative and anti-aging functions,on the expression of apoA-I at both protein and gene levels of HepG2 cells in vitro under the experimental conditions of high glucose.MethodsCell viability was measured by SRB.The apoA-I mRNA expression was assayed by RT-PCR.The apoA-I protein level was assayed by Western blot.Firstly,HepG2 cells were incubated in 10%inactivated newborn calf serum in DMEM medium.Next, cells were incubated with high-sugar and sugar serum-free medium,and added different concentration of GSPE(2.5,5 and 10μg/ml) for more than 24 hours,and thereafter,investigated whether GSPE can promote more apoA-I expression in HepG2 cells under the experimental conditions of high glucose.Results1.In this experiment,HepG2 cells were incubated with high-sugar and sugar serum-free medium,and the HepG2 cells incubated with high-sugar medium produced less apoA-I no matter at protein or gene level.The difference was significant(p<0.05).2.When HepG2 cells were incubated with GSPE at concentration of 20μg/ml or above for about 4 hours,cell viability measured by SRB was lower than 50%. However,cell viability of HepG2 cells incubated with GSPE at concentration of 10μg/ml or below was higher than 70%for more than 24 hours.Therefore,we chose the HepG2 cells incubated with GSPE concentration of 2.5,5,10μg/ml to observe the effect of GSPE on the expression of apoA-I.3.After incubated with GSPE,the apoA-I expression of HepG2 cells were significantly elevated at both protein and gene levels compared to that of high sugar control(p<0.05).Moreover,this action of GSPE showed dose dependent,and the dose of 2.5μg/ml was optimal.Conclusions1.GSPE could inhibit HepG2 cell survival when HepG2 cells were incubated with high-sugar serum-free medium and GSPE at concentration of higher than 20μg/ml.2.In HepG2 cells,endogenous apoA-I was significantly suppressed following 24h of exposure to high concentrations of glucose.3.GSPE could promote expression of apoA-I dose dependently at both protein and gene levels when its concentration was lower than 10μg/ml.