Dissertation > Medicine, health > Pharmacy > Drug basic science > Medicinal Chemistry

Effect of Grape Seed Proanthocyanidins Extracts on Synthesis and Expression of Apolipoprotein A-Ⅰ under Condition of High Glucose

Author DiZuo
Tutor GaoHaiQing
School Shandong University
Course Internal Medicine
Keywords Grape seed proanthocyanidins Diabetic vascular disease Aortic Apolipoprotein A-I Rats HepG2 cells
CLC R914
Type PhD thesis
Year 2009
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The first part of grape seed proanthocyanidins inhibit diabetic rat aorta apolipoprotein AI expression background speculated that the World Health Organization in 2025 adults with diabetes will reach 300 million. # 2 in terms of the number of diabetic patients, China ranked India. Diabetes is a major health, social and economic problems facing the elderly. Diabetic vascular disease is a common chronic complications of diabetes patients with diabetes to death, one of the leading causes of disability, the pathological change process begins with endothelial dysfunction may progress to atherosclerotic plaque formation, the pathogenesis is not clear However, the oxidative stress induced by chronic hyperglycemia may be one of the important reasons. Grape seed proanthocyanidins (grape seed proanthocyanidinsextracts, GSPE) is a mixture of polyphenols extracted from grape seeds proanthocyanidins content of more than 95%. Animal experiments showed that, GSPE has anti-oxidation, anti-atherosclerosis and cardiovascular protection. And, GSPE concurrent vascular lesions of streptozotocin (streptozotocin, STZ)-induced diabetic rats, myocardial disease, kidney disease, and neuropathy have a protective effect. As we all know, apolipoprotein AI (apolipoprotein AI, apoA-I) is the main protein component of high-density lipoprotein (high density lipoprotein, HDL). Studies have shown that plasma HDL content with coronary artery disease were negatively correlated, that is, the higher the HDL levels, lower incidence of coronary artery disease. ApoA-I clear the coronary protective effect. Monitoring GSPE apoA-I levels and therefore bring effect to explore the mechanisms of the GSPE vascular protection will help to identify drug targets and to provide a theoretical basis for the study of new prevention of complications of diabetes drugs. The purpose of a large number of studies have confirmed that grape seed proanthocyanidins (GSPE) has powerful antioxidant and anti-aging. The experimental observation of grape seed proanthocyanidins apolipoprotein AI (apoA-I) in the plasma and aortic tissue of streptozotocin (STZ)-induced diabetic rats. Methods 55 male Wistar rats were randomly divided into a control group (C group, n = 15), diabetes mellitus (DM group, n = 20) and diabetes GSPE treatment group (DM GSPE group, n = 20). Diabetes group and diabetes GSPE treated rats disposable intravenous injection of 0.1% STZ 55 mg / kg, the control group was injected with the same dose of citrate buffer citrate buffer. 72h after the injection, take the rat tail blood serum glucose, blood glucose ≥ 16.7mmol / L as a successful model of diabetes standards. Daily DM group given saline DM GSPE group GSPE (250mg/kg) was administered daily gavage. Continuous observation for 24 weeks. Determination of animals in each group plasma low-density lipoprotein (LDL), high density lipoprotein (HDL) and apolipoprotein AI (apoA-I) levels. The animals were sacrificed after 24 weeks, The thoracic aorta was separated quickly, and phosphate buffer solution with ice thorough cleaning to remove blood components, then immediately placed in liquid nitrogen and cryopreserved. Thoracic aorta cut into small pieces about 1cm from the roots, and fixed in 10% formaldehyde, embedded in paraffin, cut into 4μm thick slices, line hematoxylin - eosin staining observed at 200 times the light microscope. Diabetic rat aorta differentially expressed proteins by fluorescence difference gel electrophoresis observed after treatment with GSPE, and the application of mass spectrometry analysis and identification of the protein spots. The difference in the application of the technical verification of the Western blot and immunofluorescence identified by GSPE treatment protein apoA-I expression in diabetic rat aorta. Results diabetic rats plasma LDL, HDL and apoA-I water the average higher than the normal control group, after treatment with GSPE LDL, HDL and apoA-I levels to near normal. Diabetic group rat aortic endothelial cell hypertrophy, structural disorder, and smooth muscle cell proliferation, destruction of internal elastic panels, smaller lumen after GSPE treatment, none of the above changes the vascular structure close to the control group. Fluorescence difference gel electrophoresis and mass spectrometry analysis showed differential expression in diabetic rat aorta apoA-I GSPE treatment (DM group / group C: -1.68; DM GSPE group / DM group: 1.77). After Western blot and immunofluorescence experiments verify the differentially expressed proteins, and found that the highest levels of apoA-I in aortic tissue of diabetic rats (p <0.05), GSPE, it was able to effectively reduce this expression (p <0.05 ). Conclusion 1.GSPE help ease the macrovascular complications of diabetes can be adjusted; 2.GSPE apoA-I in the expression level of the aortic tissue, and the regulation may achieve one of the mechanisms of the protective effect on vascular; 3.apoA- I express the number of modified change may be one of the diabetic rat aortic disease pathogenesis, but still needs further experimental verification. The second part of grape seed proanthocyanidin extracts of HepG2 cells the high sugar under the conditions of the experimental expression of apolipoprotein AI affect background apolipoprotein AI (apoA-I) is one of the main component of high-density lipoprotein (HDL), total HDL protein content of about 50%, mediated by a number of important physiological functions. It can be combined with the blood vessel wall of the ATP binding transporter Al (ABCAl), free cholesterol induced outwardly from the vascular endothelium of the macrophages or other epidermal tissue is induced to a reverse cholesterol transport (RCT) in the initial stage, then can activate the binding to HDL, lecithin cholesterol acyl transferase (LCAT), and the free cholesterol esterification, and then from the surface of HDL transferred to the kernel, and then transported to the liver, is the crucial role of factor of reverse cholesterol transport in vivo. Recent studies have shown that it also has other biochemical effects, including antioxidant, anti-inflammatory, anti-endotoxin. Therefore, to promote expression of apoA-I will help to ease the process of atherosclerosis. The purpose grape seed proanthocyanidins (GSPE) natural antioxidants extracted from grape seeds. In this study, in vitro culture of human hepatoma cell line HepG2 as the research object, whether from the protein and gene level observed GSPE induced HepG2 apoA-I expression. First inactivated newborn calf serum DMEM medium with 10% conventional cultured HepG2 cells and passaged then serum-free medium with high sugar and low sugar, add different concentrations (2.5, 5 and 10μg/ml) that GSPE holds substantial promise for the treatment of DN, and GSPE HepG2 cells incubated for 24 h, the SRB assay during GSPE on the proliferation of HepG2 cells, Western Blot determination of apoA-I protein levels in cells, RT-PCR determination of changes in apoA-ImRNA. Observe the GSPE whether to promote the conditions of the experimental high sugar high expression of apoA-I in HepG2 cells. Results of the experiments with high sugar and low-sugar medium and incubated HepG2 cells and found that, regardless of apoA-I from HepG2 cells incubated in high glucose medium proteins or genes water were significantly lower than those with low glucose medium (p <0.05). HepG2 cells were co-incubated add GSPE with serum-free high glucose medium SRB detect cell survival found that GSPE holds substantial promise for the treatment of DN, and GSPE concentrations above 20μg/ml and incubated for four hours, that appear obvious apoptosis of HepG2 cells; in the The GSPE concentrations below 10μg/ml groups, jointly incubated for 24 hours no significant apoptosis, HepG2 cells, the cell survival is above 70%, Therefore, the experiment eventually established 2.5,5,10 μg / ml GSPE intervention group as observed object. . GSPE After incubation, both from the gene or protein level, HepG2 cells express higher than those of pure high-sugar incubated apoA-I, the difference was statistically significant (p <0.05), and this type of role GSPE concentration, 2.5 μg / ml, a promoting effect as the best. Conclusion 1.20μg/ml concentration GSPE significantly inhibited in vitro incubation with serum high glucose medium HepG2 cell survival; experimental high glucose can inhibit the expression of apoA-I protein and gene level; 3. high glucose 10μg/ml concentration GSPE from protein and gene level to promote the expression of apoA-I, and the role GSPE concentration, when the the GSPE concentration of 2.5μg/ml role as the best.

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