The Direct Effect Of Grape Seed Procyanidins On Cultured Endothelial Cell And Its Effect On Injured Endothelial Cell By Advanced Glycation End Products

The first part The direct effect of grape seed procyanidins on culturedendothelial cellBackgroud: Atherosclerosis (AS) is one of the seriousest diseases which are bad for good health. AS can cause low angiokinesis, narrow lumens, thrombosis, and at last leads to many acute cardiovascular events. The disorder of structure and function of vascular endothelial cell, promoting factor of atherosclerosis, is associated with the production and development of atherosclerosis. Grape seed procyanidins (GPC) is a polyphenol found at high concentrations in grapes and red wine with reported anti-oxidant effect. It is mainly polymerized by monomers of catechin or epicatechin. Studies had reported that a number of antioxidants may have both anti-oxidant and pro-oxidant effects. In our experiment, human umbilical vein endothelial cells (HUVEC) were used, and we sought to investigate the effect of different dose of GPC on HUVEC for different times.Aim: To study the the effects of different GPC on cultured endothelial cell for different times, we will investigate from three part. (1) MTT (methyl thiazolyl tetrazolium,MTT) method was used to test cell proliferation; (2) Von Wille brand factor (vWF) was measured by enzyme linked immunosorbent assay(ELISA);(3) nitric acid reductase assay was used to test NO (nitric oxide, NO) content.Methods: Human umbilical vein vascular endothelial cells were cultured with GPC of different doses (10mg/l, 50mg/l, 100mg/1, 200mg/l, 400mg/l, 800mg/l). MTT method was used to test cell proliferation when cells were cultured for 12 h, 24 h and 48 h, respectively. Von Wille brand factor (vWF) was measured by enzyme linked immunosorbent assay(ELISA) and nitric oxide (NO) in culture medium by nitric acid reductase assay.Results: 1. When incubation for 12 h, as the GPC doses increased, the cell proliferations enhanced (P<0.01). The group of 200mg/l GPC had the strongest cell proliferation rate which was 162.47%. Though the cell proliferation rates of groups of 400mg/l GPC and 800mg/l GPC decreased, they were still higher than that of control. Incubation of HUVEC cells for 12h with increasing concentrations of GPC led to a dramatic reducing vWF level compared with the control(p<0.01). The NO content of each experimental group was higher that that of the control.2. When cells were cultured for 24 h, at 100mg/l level the cell proliferation was 1.39±0.03 which was the strongest compared with other groups. Incubation with increasing concentrations of GPC from 10mg/l to 100mg/l led to a dramatic reducing vWF level compared with the control.The vWF levels gradually increased compared with normal group when incubation with GPC of 200mg/l, 400mg/l and 800mg/l level. The reducing trend of NO content corresponded to the decreasing cell proliferation in experimental groups.3. When incubation for 48h, the cell proliferation was 1.38±0.03 at 10mg/l level which was the strongest compared with other groups. From 50mg/l GPC to 800mg/l GPC level, cell proliferations and NO content decreased gradually,but the vWF content increased significantly instead(p<0.05).Conclusion: GPC can increase proliferate activity of vascular endothelial cells at definite concentration and within certain time, but higher dose of GPC can injure endothelial cell and decrease NO secretion.The second part The protective effect of grape seed procyanidins oncultured endothelial cell injured by advanced glycation end productsBackgroud: Diabetes mellitus (DM) which is the ter-common disease second only is an acute and lifelong disease. As the research into DM goes to deepen, human have already realized that the nature of DM is vasculopathy. In 1999, Ammerican Heart Association (AHA) had drawn off that DM was a cardiovascular disease. At present, the important reason of diabetic dead is macroangiopathy. AS is the first death cause in diabetic. The incidence rate of AS in DM is 2-fold to 3-fold than in non-diabetic. AS in DM has earlier morbility, faster development, heavier pathogenetic condition and higher case fatality rate than non-diabetic. Prolonged hyperglycemia in diabetes leads to non-enzyme glycosylation pathway activation, and results in the production and accumulation of advanced glycation end products (AGEs) which is responsible for endothelial cell dysfunction and cellular damage. Interaction of AGEs with their receptor, RAGE, is very important in the occurrence and development of diabetic vessel complication. Procyanidins (PC), derived from plants, has been reported to possess a variety of potent properties including anti-oxidant, anti-inflammation, and radical-scavenging and so on. In the final of 1995, American free voting election, procyanidins was considered to be the most popular and fashionable plant amedica. Large amount of researches have been made to kinds of PC derived from different plants in the late forty years. The disorder of structure and function of vascular endothelial cell, promoting factor of atherosclerosis, is associated with the production and development of atherosclerosis. So, protecting vascular endothelial cell is the foundamental element for preventing and curing AS in DM. We have confirmed that GPC can inhibit anti-nonenzymatic glycation in diabetic rats, decrease AGEs accumulation in renal cortex. But the research into the protective effect of GPC on endothelial cells has been seldom reported. In our experiment, HUVEC were used. Cell proliferation, vWF, the receptor for AGEs (RAGE) and peroxisome proliferators-activated receptor γ (PPAR γ) expressions were measured respectively to investigate the protective effect of GPC on HUVEC and the possible mechanism.Aims: HUVEC were incubated with different dose of GPC and AGEs. To investigate the effect of GPC on HUVEC injured by AGEs and its possible mechanism, we observed from four parts. (1) MTT method was used to test cell proliferation; (2) Von vWF was measured by ELISA; (3) RAGE was measured by western blot analysis; (4) PPAR γwas measured by western blot analysis.Methods: AGEs-modified bovine serum albumin (AGEs-BSA) was prepared by incubating BSA with high concentration of glucose. The cultured HUVEC were divided into six groups: ①Normal group; ②BSA group: cells were incubated with 200mg/l BSA for 24 h; ③AGEs group: cells were treated with 200mg/l AGEs; ④GPC (low dose)+AGEs group: cells were pretreated with 10 mg/1 GPC for 4 h before 200mg/l AGEs treatment; ⑤GPC (middle dose)+AGEs group: cells were pretreated with 50mg/l GPC for 4 h before treated with 200mg/l AGEs; ⑥GPC (high dose)+AGEs group: cells were pretreated with 100mg/1 GPC before 200mg/l AGEs treatment. MTT method was used to detect the cell proliferation of HUVEC. Von Wille brand factor (vWF) was measured by enzyme linked immunosorbent assay (ELISA).And the receptor for AGEs (RAGE) and peroxisome proliferators-activated receptor γ (PPAR γ) expressions were measured by western blot analysis.Results: After treatment with AGEs, the proliferation of HUVEC was significantly reduced, and the proliferation rate was 90.53% of that of the normal, while the proliferations of cells pretreated with GPC of different concentration were 0.95-fold, 1.12-fold, and 1.23-fold than that of the normal. The vWF level in AGEs-treated HUVEC was higher than that of the normal (p<0.01). Pre-incubation with GPC of different concentrations could decrease the vWF level increased by AGEs in a dose-dependent manner. AGEs activated the expression of RAGE and inhibited PPAR γ expression in HUVEC, whereas GPC inhibited RAGE expression and activated PPAR γ expression in HUVEC simultaneously.Conclusion: AGEs inhibited HUVEC proliferation and injured HUVEC. GPC could protect HUVEC against damage induced by AGEs in a dose-dependent manner. It was possible that the mechanism of protective effect of GPC on the function of endothelial cell may relate to activation of PPAR γ expression and inhibition of RAGE expression.