Protective Effects Of Nobiletin On High Glucose-induced Injury In Cultured Human Umbilical Vein Endothelial Cells

Background:In recent years,the incidence of diabetes mellitus is increasing rapidly all over the world,and cardiovascular complications have become the main cause of death in diabetic patients. The mechanisms of diabetic complications are complex,in which endothelial dysfunction plays an important role. Therefore,how to protect the endothelial function becomes a new target for the therapy of cardiovascular complications. Nobiletin is a flavonoid compound extracted from orange peel, which has been reported to possess anti-thrombosis,anti-fungal,anti-inflammatory and anti-allegic poverty. However,there is no study about whether Nobiletin could protect against high glucose induced dysfunction of human umbilical vein endothelial cells.This study was designed to investigate the protective effect of Nobiletin on the injury of human umbilical vein endothelial cells induced by high glucose in vitro,and to explore its underlying mechanism.Methods:Human umbilical vein endothelial cells (HUVECs) were supplied by the central laboratory of Shandong Provincial Hospital. HUVECs were incubated at 37℃in a humidified atmosphere of 95% air-5% carbon dioxide in RPMI-1640 medium,which is supplemented with 10% FBS. HUVECs in good conditions were used for experiments.In experiments, HUVECs cultured in RPMI-1640 medium served as normal control groups(concentration of glucose:5.5mmol/L). HUVECs were incubated with high glucose (30mmol/L,HG group) for 48 h. To observe the effect of nobiletin on high glucose-mediated cell injuries, HUVECs were pretreated with nobiletin (2.5,5,10,20 and 40μmol/L; A group, B group, C group, D group, E group, respectively) for 18 h before incubation with high glucose (30mmol/L). HUVECs injuries were evaluated by cell morphological assessment,cell vitality and the activity of nitric oxide (NO) and superoxide dismutase (SOD) in supernatant.Results:1.Morphological observation of HUVECs:HUVECs in the control group, morphologically, can be seen small spindle,circular,the majority of cells agglomerans under the inverted microscope and showed cobblestone-like arrangement.They had a clear border,rich cytoplasm and small particles. While compared with the control group, HUVECs that exposed to high glucose (30 mmol/L) showed chromatin condensed, little cytoplasm with unclear borders and many cell fragments emerged. HUVECs cultured with high concentration nobiletin (10,20,40μmol/L)were almostly the same as those in the control group. While low concentration nobiletin(2.5,5μmol/L) were almostly the same as those exposed to high glucose.2.The cell vitality,the activity of SOD and content of NOCompared with the control group, the cell vitality(OD optimum),the SOD content and secrete of nitric oxide(NO)was decreased obviously in HUVECs that exposed to high glucose (30 mmol/L)(P<0.01). Meanwhile,in nobiletin group(10,20,40μmol/L),compairing with high glucose group(30mmol/L),the cell vitality(OD optimum),the SOD content and the secrete of nitric oxide(NO)was advancing obviously(P<0.01).while in other nobiletin group (2.5,5μmol/L).comparing with high glucose group(30mmol/L), the cell vitality(OD optimum),the SOD content and secrete of nitric oxide(NO) have no significant difference.Conclusions:1. High glucose can induced the cell viability descending and active oxygen produced increasing and the antioxidant capacity descending, and caused HUVEC injury. 2. The nobiletin can protect the injury of HUVEC induced by high glucose in vitro and this protective effect is dose dependent. The mechanism may be associated with scavenging oxidative free radicals and increasing the secreat of NO.