A Study Of The Antioxidative Effect Of Curcumin And Its Analogue L3 And Their Influence On Diabetes And Vascular Complication In Mice

PartⅠObjective: To explore the effects of curcumin and its analogue L3 on human umbilical vein endothelial cell injury induced by oxidative stress in vitro and to investigate their possible regulatory mechanisms.Methods: Peroxide hydrogen (H2O2) was used to induce human umbilical vein endothelial cells (HUVECs) injury. The cell survival rate was evaluated with MTT assay to explore the protective effect of curcumin and its analogue L3 on vascular endothelial cells injury. The activities of cytosolic superoxide dismutase (SOD),glutathione S transferase (GST), glutathione peroxidase (GSH-PX) and catalase (CAT) and the levels of cytosolic glutathione (GSH) and malondialdehyde (MDA) were determined in H2O2-treated HUVECs. The fluorescence agent DCF-DA was used to detect intracellular generation of reactive oxygen species (ROS), and DAF-FM DA to detect intracellular generation of nitric oxide (NO). Level of extracellular nitric oxide (NO) was measured by nitrate reductase method. Protein expression of endothelial nitric oxide synthase (eNOS) in H2O2-treated HUVECs was detected by Western blotting analysis. Activities and expressions of caspase 3 and caspase 9 were determined by spectrophotometric and Western blotting assay. The fluorescence agent rhodamine 123 (RH 123) was used to detect mitochondrial membrane potential (MMP). Phosphorylation of ERK1/2, translocation of nuclear factor-kappa B (NF-кB) and protein expression of p53 were investigated by Western blotting analysis.Results: Thirty six-hour treatment with 200μM H2O2 significantly decreased the viability of HUVEC cells, which was accompanied with apparent apoptotic features including the mitochondrial depolarization and increased activities and expressions of caspase 3 and caspase 9. In addition, it is observed that H2O2 increased the amount of malondialdenhyde (MDA),the activity of glutathione S transferase (GST) and the generation of reactive oxygen species (ROS), and decreased the levels of glutathione (GSH) and intra/extracellular nitric oxide (NO), the activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-PX) and catalase (CAT) and the expression of endothelial nitric oxide synthase (eNOS) in HUVEC cells. Co-incubation with 12.5-50μM L3 resulted in a significant increase of cell viability in a concentration-dependent manner, while 12.5-25μM curcumin didn't exhibit any protective effect. Also, L3 resulted in a significant recovery from H2O2-induced cell apoptosis and decreased other H2O2-induced damage in a concentration-dependent manner, which might be achieved via decreasing of p53 expression and NF-кB translocation and increasing of ERK1/2 phosphorylation.Conclusion: L3 could attenuate H2O2-induced injury in HUVECs in a concentration-dependent manner and showed protective effect on vascular endothelial cells. The probable mechanisms closely associated with its protective effect might include repressing ROS generation, increasing the activities of antioxidant enzymes, improving redox status, upregulating eNOS expression, promoting NO synthesis, maintaining mitochondrial membrane potential and inhibiting cell apoptosis in H2O2-exposed HUVECs.PartⅡObjective: To evaluate the effects of curcumin and its analogue L3 on streptozotocin and high cholesterol diet-induced diabetes and hyperlipidemia in mice and their possible protection of multiple organ involvement under diabetic condition.Methods: Male ICR mice were injected intraperitoneally streptozotocin solution (50mg/kg body weight)once every other day for a total of five times to induce diabetes and then were fed either only high cholesterol diet or additional intragastrically administrated curcumin or L3 of different doses. Each half of the mice were sacrificed at the end of four and sixteen weeks, the levels of plasma glucose, glucose tolerance, glycated hemoglobin, insulin,plasma and liver lipids (TG, TC, HDL-C and LDL-C), glutathione (GSH) and malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) , glutathione S transferase (GST), glutathione peroxidase (GSH-PX) and catalase (CAT) were detected. The fluorescence agent DCF-DA was used to detect reactive oxygen species (ROS) generation in pancreas, and DAF-FM DA to detect nitric oxide (NO) generation in aortic arch. Level of plasma nitric oxide (NO) was measured by nitrate reductase method. Protein expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in aortic arch was detected by immunohistochemistry analysis. Histopathological sections of the liver and aortic arch were examined.Results: After a total of five STZ injections diabetic status was induced successfully in mice. Plasma and liver lipids increased significantly four or sixteen weeks after the mice were fed high cholesterol diet. Oxidative stress parameters and activities of antioxidant enzymes altered significantly. The generation of ROS was elevated in pancreas. The level of NO was depressed significantly both in aortic arch and in plasma. Expression of LOX-1 increased significantly in aortic arch. Apparent steatosis and artherosclerosis were observed in liver and aortic arch histopathological sections. The glucose and fatty metabolisms were improved after treatment with high-dose curcumin and medium/high-dose L3. Oxidative stress parameters and activities of antioxidant enzymes were also improved. The generation of ROS was attenuated significantly in pancreas. The level of NO increased significantly both in aortic arch and in plasma. Expression of LOX-1 was downregulated significantly in aortic arch. Steatosis and artherosclerosis were attenuated in liver and aortic arch histopathological sections.Conclusion: Curcumin and its analogue L3 appear to be beneficial in improving diabetic status and in protecting multiple organ involvement in STZ and HCD-induced diabete and hyperlipidemia in mice.