The Mechanism Of The Amelioration Of D-fagomine On High Glucose-induced Oxidative Damage In HUVECs

Endothelial cells line the inner surface attached to various vascular cells,and are the morphological basis for blood vessel formation.The overproduction of ROS in endothelial cells cause oxidative stress,and mitochondrial is the main site of ROS production,its structure and functions play an important role in maintaining the normal physiological function of endothelial cells.In addition,the study found that the activation of AMPK can significantly improve the expression of SIRT1 and PGC-1α,improve endothelial cells damage.Thus,the early and long-term interventions using dietary supplement of natural plant extracts and other bioactive substances to active AMPK,inhibit oxidative stress and protect endothelial cells function are an effective measure for the prevention or treatment of vascular complications of diabetes.D-fagomine is a polyhydroxy azasugars alkaloid and it’s an analogue of DNJ.Studies have reported that DNJ can improve high glucose-induced endothelial cells damage by reduce the production of ROS,but the specfic methanism was not clear.Buckwheat is widely accepted as a hypoglycemic substance,and as the only food source for D-fagomine,if the D-fagomine has similar function as DNJ,then we can supply D-fagomine to take an effect and explore the relationship bteween functional substances and function in buckwheat more deeply.Firstly,we determined the content of D-fagomine in different parts of tartary buckwheat plants during different harvest periods and 18 different varieties of buckwheat grains using HPLC.Secondly,we used D-fagomine standard as the research object,high glucose stimulation to mimic diabetic environment.Western blot and Assay Kits were used to study the effect of D-fagomine on oxidation damage in HUVECs.Furthermore,the molecular target and the mechanism of D-fagomine on high glucose-induced oxidative damage in HUVECs were studied.The main results of this study were as follows:(1)The content of D-fagomine in different parts of tartary buckwheat plants during four growth periods and 18 different varieties of buckwheat seeds were determined by HPLC-FLD.The results showed that different parts of tartary buckwheat plants contain D-fagomine,the highest relative content of D-fagomine was in grain stage and the content D-fagomine in mature grains of buckwheat was higher than other parts.The determination of the content of D-fagomine in tested 18 different varieties of buckwheat grains showed that the content of D-fagomine in common buckwheat was higher than that of tartary buckwheat.(2)To study the effect of D-fagomine on high glucose-induced oxidative damage in HUVECs.The test results showed that D-fagomine significantly decreased the production of ROS induced by high glucose,protected the mitochondrial function,and improved the activity of antioxidant enzymes(P<0.05)in HUVECs.In addition,D-fagomine significantly increased the phosphorylation level of AMPK via LKB1pathway(P<0.05).Research showed that D-fagomine improved oxidative stress in HUVECs by protecting mitochondrial function,reducing the production of ROS and increasing the activity of antioxidant enzymes.(3)To reveal the molecular mechanism of D-fagomine on high glucose-induced oxidative damage in HUVECs.The test results showed that the expression levels of SIRT1 and PGC-1α proteins were significantly up-regulated by D-fagomine(P<0.05).The using of AMPK and SIRT1 inhibitors showed that D-fagomine modulated the activity of antioxidant enzymes in mitochondrial by AMPK/SIRT1/PGC-1α pathway,thereby improved the high glucose-induced oxidative damage in HUVECs.The above results showed that D-fagomine was distributed in different parts of tartary buckwheat plants,and the content of D-fagomine in buckwheat grain was the highest,and the content of D-fagomine in common buckwheat grains was higher than that of tartary buckwheat grains.D-fagomine protected mitochondrial function,improved the activity of antioxidant enzymes,thereby improved the high glucose-induced oxidative damage in HUVECs by LKB1/AMPK/SIRT1/PGC-1α/antioxidant enzymes pathway.The results are helpful to our understanding of the traditional medicinal and edible food resources,to make clear the relationship between functional material and functional activity in buckwheat,and comprehensive utilization of resources guidance.