Adropin Improves Endothelial Cell Damage Induced By AngiotensinⅡ By Reducing Oxidative Stress

Objective:To study the effect and mechanism of Adropin protein(adropin)in improving endothelial cell damage induced by angiotensin-II(ang II)by reducing oxidative stress.Methods:1.Cell cultureSelecte human umbilical vein endothelial cells(HUVECs)in good condition and moderate density from the resuscitated cell lines,cultivate cells for 48 hours after passage,and divide cells into 6 groups,namely control group,Adropin group,and Ang II group,Ang II+Adropin 10ng/ml group,Ang II+Adropin 100ng/ml group,Ang II+Adropin 1000ng/ml group,except for the control group and Ang II group,all the other groups were pretreated with different concentrations of Adropin for 30 minutes.The group was treated with Ang II at a concentration of 1μmol/l for 24 h and waited for follow-up experiments.The control group was treated with 10% FBS medium as a control.2.TUNEL kit to detect level of cell apoptosisAfter the above-mentioned experimental method,take out the umbilical vein endothelial cells in the petri dish,aspirate the medium,add 50μl TUNEL detection solution to the sample,mount the slide with anti-fluorescence quenching mounting solution,and observe under a fluorescence microscope.The apoptosis rate was determined by comparing the average fluorescence intensity of the cells in the treatment group and the cells in the untreated group.3.CCK-8 to detect cell viabilitySet up the control group according to the above experimental methods,insert the passaged cells into a 96-well plate,add the corresponding drug treatment to each well according to the experimental needs,add 10 microliters of CCK-8 solution to each well after the end,and measure the absorbance of each well with a microplate reader.Compare the average absorbance of treated and untreated cells to determine cell viability.4.Detection of oxidative stress indicators4.1 ROS release determinationThe experimental method is as above.The reactive oxygen detection kit uses the fluorescent probe DCFH-DA for reactive oxygen detection.After processing according to the experimental method,it is directly observed with a laser confocal microscope to detect the intensity of fluorescence before and after stimulation in real time.The ROS release content was determined by comparing the average fluorescence intensity of cells in the treatment group and the cells in the untreated group.4.2 Determination of MDA levelThe experimental method is as above.The operation is performed in accordance with the instructions of the malondialdehyde(MDA)kit.The absorbance value of each well is measured by a microplate reader,and the intracellular MDA level is determined by comparing the average absorbance of the treated group and the untreated group.4.3 Determination of SOD levelThe experimental method is as above,and the operation is performed according to the instructions of the superoxide dismutase(SOD)kit,and the absorbance value of each well is measured by a microplate reader.The intracellular SOD level was determined by comparing the average absorbance of the treated group and the untreated group.4.4 Determination of NO levelThe experimental method is as above.According to the instructions of the NO determination kit,operate as follows,and measure the absorbance of each well with a microplate reader.The cell NO level was determined by comparing the average absorbance of the treated group and the untreated group.5.RT-q PCR detect the m RNA levels of Intercellular cell adhesion molecule(ICAM-1)and Vascular cell adhesion molecule(VCAM-1).6.Western Blot6.1 Detect the protein expression levels of GAPDH,Adropin,ICAM-1,p-e NOS,total-e NOS in umbilical vein endothelial cells damaged by Ang II;6.2 Detection of Adropin treatment of Ang II damaged endothelial cells GAPDH,ICAM-1,VCAM-1 protein,and the expression level of pathway proteins PI3 K,p-AKT,total-AKT,p-e NOS,total-e NOS Result:1.The Ang II group vs.the control group,the Ang II group(1μmol/l)has significant increase in cell apoptosis(P<0.001);the Ang II+Adropin group vs.Ang II group compares the Ang II+Adropin group with significant decrease in cell apoptosis(P<0.01),And decreased in a concentration-dependent manner with Adropin.2.The Ang II+Adropin(1000ng/ml)group also significantly relieved the Ang II group(1μmol/l)from inhibiting the viability of umbilical vein endothelial cells(P<0.05).3.Oxidative stress:3.1Treatment of umbilical vein endothelial cells in the Ang II+Adropin group can reduce the generation of ROS induced by Ang II(P<0.01),in a dose-dependent manner.3.2 Ang II+Adropin(1000ng/ml)VS Ang II group(1μmol/l)group,the intracellular MDA activity was also significantly inhibited(P<0.01).3.3 Compare with the control group,the treatment of umbilical vein endothelial cells with Ang II(1μmol/l)resulted in a decrease in the production of superoxide dismutase SOD and vasodilation factor NO(P<0.01),while Adropin(1000ng/ml)reversed Ang II Inhibition of SOD and NO production(P<0.05)4.RT-q PCRIntracellular ICAM-1,VCAM-1m RNA: Ang II group vs control group,Ang II group expression increased significantly(P<0.01,P<0.0001);Ang II+Adropin(1000ng/ml)vs Ang II group,Ang II+Adropin significantly decreased(P<0.01,P<0.0001).5.Western Blot1)The protein expression of Adropin,ICAM-1,VCAM-1,P-e NOS: Ang II concentration group vs.control group,the Adropin protein expression level of Ang II group was significantly decreased(P<0.0001),and the protein expression level of Adropin in Ang II(1μmol/l)Obvious statistical significance;Ang II concentration group vs.control group,ICAM-1 and VCAM-1 increased significantly in Ang II group(P<0.01),and P-e NOS decreased in a concentration-dependent manner(P<0.01).In the Ang II+Adropin(1000ng/ml)group vs.Ang II group,the ICAM-1 and VCAM-1 of Ang II+Adropin were significantly inhibited compared with the Ang II group(P<0.01,P<0.05).2)Expression of PI3 K,P-AKT,AKT,P-e NOS,e NOS proteinAng II(1μmol/l)group VS Control group,Ang II’s PI3 K,P-AKT,P-e NOS protein expression was significantly inhibited(P<0.01,P<0.001 P<0.0001),but Ang II+Adropin(1000ng/ml)Group vs Ang II group,the inhibitory effects of PI3 K,P-AKT,and P-e NOS in the Ang II+Adropin group were significantly alleviated(P<0.05,P<0.05,P<0.01).There was no significant difference in the expression of total-AKT and total-e NOS protein in each group(P>0.05).Conclusion:1.Ang II can induce human umbilical vein endothelial cells apoptosis and up-regulation of inflammatory adhesion molecules ICAM-1 and VCAM-1,leading to damage of endothelial cells;Adropin significantly inhibits endothelial damage induced by Ang II;2.Adropin significantly reduces expression of the ang II-induced oxidative stress ROS and MDA in human umbilical vein endothelial cells,and significantly relieves the inhibitory effect of Ang II on SOD and NO in human umbilical vein endothelial cells.3.Adropin improves the oxidative stress induced by ang II by activating the PI3K-AKT-e NOS pathway.