Effect Of Total Flavonoids From Lycium Barbarum On Protecting From The Injury Of High Glucose Induced Oxidative Stress

Objective Using in-vitro assays to confirm flavonoids compounds from lycium barbarum’s protection from oxidative stress injury model of human umbilical vein endothelial cells(HUVEC) induced by high glucose(HG). Using in-vivo assays to research flavonoids compounds from lycium barbarum’s protection from injury model of diabetes mellitus(DM) rat induced by streptozocin(STZ). Doing some basic research on the molecular mechanism of the flavonoids compounds from lycium barbarum’s protection from the high glucose induced oxidative stress injury.Method1.Using cultured human umbilical vein endothelial cells and high glucose-induced human umbilical vein endothelial cells to build the oxidative stress damage model.This study was conducted as following: normal control group, high glucose injury group, TFLB protective groups(100, 200, 400 mg·L-1)and positive control group(vitamin C). Cell viability was measured by MTT assay. We have measured the change of levels and activity of lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD), the generating capacity of nitric oxide(NO)and Nitric oxide synthase.(NOS) in the diagnostic reagent kit. Using the enzyme-linked immunoassay(ELISA) method to measure the von willebrand factor(v WF),soluble thrombus regulatory protein(s TM) and soluble vascular endothelial cell protein C receptor(s EPCR) in order to observe the flavonoids from lycium barbarum’s protection from vascular endothelial cell injury caused by high glucose-induced human umbilical vein endothelial cells.2 Using the in-vivo assays to established diabetes mellitus rat model induced by Sreptozocin(STZ). First, randomly divided diabetic rats which are successfully established models into two groups: DM group and TFLB group. After separation, the total flavonoids from lycium barbarum were given to the rats by coloclysis for 4weeks. Rats from normal control group and diabetic model group were given the same amount of normal saline to fill the stomach by coloclysis and their fasting blood glucose levels were measure in every two weeks. After 4 weeks and measuring the fasting blood glucose in caudal vein blood, anesthetize the rats, the chest blood measure the change of blood lipids with biochemical kits and observe the regulation of TFLB on DM rats blood glucose and blood lipid. Use the enzyme-linked immunoassay(ELISA)to determine the change of von willebrand factor(v WF), soluble thrombus regulatory proteins(s TM), and soluble vascular endothelial cell protein C receptor(s EPCR). At last, excise a pancreas tissue and the aorta to do HE staining and immunohistochemical in order to observe the effect of TFLB on DM rat’s viscera organization ultrastructure and the improvement of the pathological physiology’s change.Result1. Using in-vitro assays to research flavonoids compounds from lycium barbarum’s protection from oxidative stress injury model of human umbilical vein endothelial cells(HUVEC)induced by high glucose(HG).(1) Successfully establish the high glucose induction of human umbilical vein endothelial cell injury model, and confirm that the molding concentration of high glucose is 40 mmol L-1 and the injury time lasts for 48 hours at last.(2) After TFLB protection, compared with the HG injured model group, the amount of release of MDA in the TFLB protective groups obviously decrease, the amount of release and activity of LDH decreases, SOD activity increases obviously, NO, NOS generation and the activity all increase obviously and the difference is significant(P <0.05, P < 0.01), and the medlar flavones shows dose dependent relationship in a certain concentration range.(3) After TFLB protection, compared with the HG injured model group, s TM in medlar flavone contents shows a declining trend. VWF and s EPCR content are on the decline, and the difference is significant(P < 0.05, P < 0.01).2. Using in-vivo assays to research flavonoids compounds from lycium barbarum’s protection from injury model of diabetes mellitus(DM)rat induced by streptozocin.(1) After TFLB protection, TFLB can delay the course of The rise of blood glucose,have obvious trend to decrease the level of blood-lipid.(2) After TFLB protection,compared with the DM model group, v WF and s TM and s EPCR deceases in every TFLB groups and the difference is significant(P < 0.05, P < 0.01).(3) After TFLB protection, compared with the DM model group, with the increasing of TFLB, islet number and number of secretory cells increase in rat, cell lineage relatively uniform neat, islet structure and form basic returns to normal, and relatively less of cells show vacuoles degeneration.(4) After TFLB protection, compared with the DM model group, the aortic intima in in TFLB content in low group thickens obviously with mountain-like protrusions.There are vascular morphology changes and uneven changes of thickness with partly visible patches. Outer membrane has mild damage, endothelial cells have proliferated and still not neat. The aortic intima in in TFLB content in high group thickens mildly endothelial cells have proliferated mildly and arrgmge neat and outer membrane has no obvious damageConclusion1. After the protection of TFLB from injury model of HUVEC induced by HG, the activity of antioxidant enzymes(SOD) in HUVEC was improved,the generating capacity and the activity of NO and NOS was increased, the generating capacity of MDA and LDH was decreased, the content of v WF、s TM 、s EPCR of vascular endothelial function was reduced and achieved the protection of vascular endothelial cells at last. The preliminary mechanism may be related to flavonoids from Lycium barbarum fight free radicals. The fight free radicals can protect endothelial cells, and then reduce the lipid oxidation caused by HG.2. After the protection of TFLB from DM model induced by STZ, the islet function DM rat was protected obviously, insulin resistance was improved, the increasing speed of blood glucose was delayed, the disorder of lipid metabolism was adjusted,and then the progressing of DM was delayed, the injury of vascular endothelial cell by high blood glucose was reduced and achieved the cardiovascular protection.