Effects Of GLP-1(7-36a) And Its Metabolite (9-36a) On Oxidative Stress Injury Of Human Umbilical Vein Endothelial Cells Through CYP450-EETs Pathway And Its Mechanism

Objective:With the largest population in the world,China also has the largest number of diabetes mellitus（DM）patients,and the prevalence of diabetes is still increasing rapidly,accompanied by the continuous occurrence and development of complications.Among them,vascular complications are the primary cause of disability and death of diabetes,to improve and delay vascular complications is an urgent problem.Among many drugs for the treatment of diabetes,glucagon-like peptide-1（GLP-1）has been gradually written into major guidelines for its high glucose-lowering efficiency without causing hypoglycemia and cardiovascular benefits.The CYP450-EETs metabolic pathway plays an important role in the treatment of vascular injury.To explore the possible mechanism of action between GLP-1（7-36a）and its metabolites（9-36a）and CYP450-EETs,and to provide more ideas for the prevention and treatment of clinical diabetes vascular complications.Methods:Experiment 1:Human umbilical vein endothelial cells cultured in vitro were divided into NC group,H₂O₂group,H₂O₂+7-36a group,and H₂O₂+9-36a group.100 nmol/L GLP-1（7-36a）and 100 nmol/L GLP-1（9-36a）were pretreated for 1h,and then 600μmol/L H₂O₂was added for 3h.Cell proliferation level was detected by the Cell Counting Kit-8（CCK-8）,reactive oxygen species（ROS）level was detected by the Reactive Oxygen Detection Kit,and Western blot（WB）method to detect the expression of glucose regulated protein 78（GRP78）and cytochrome P450 2J2（CYP2J2）signaling pathway proteins.Experiment 2:Human umbilical vein endothelial cells cultured in vitro were divided into NC group,H₂O₂group,H₂O₂+7-36a group,H₂O₂+9-36a group,H₂O₂+7-36a+Danazol group,and H₂O₂+9-36a+Danazol group.100 nmol/L GLP-1（7-36a）and 100nmol/L（9-36a）were pretreated for 1h,and 1μmol/L Danazol was added for 3h,followed by 600μmol/L H₂O_2.CCK-8 was used to detect cell proliferation level,ROS level was detected by reactive oxygen species kit,and GRP78 and CYP2J2 protein expression was detected by WB method.Results:（1）H₂O₂can induce oxidative stress in HUVECs,and GRP78 protein expression is significantly increased,with statistical significance（P<0.05）.（2） GLP-1（7-36a）and（9-36a）pretreatment of HUVECs could improve oxidative stress.Compared with H₂O₂treatment group,GRP78 protein expression was significantly decreased,while CYP2J2 protein expression was significantly increased,with statistical significance（P<0.05）.（3）Added with Danazol,a CYP2J2 inhibitor,Compared with GLP-1（7-36a）and its metabolite（9-36a）pretreatment group,the oxidative stress level of H₂O₂treated HUVECs was significantly increased,the expression of GRP78 protein was increased,and the expression of CYP2J2 protein was decreased,with statistical significance（P<0.05）.Conclusions:GLP-1（7-36a）and its metabolite（9-36a）improve H₂O₂induced oxidative stress,possibly through the CYP450-EETs signaling pathway.