Intermedin Protects Against Vascular Endothelial Cells Injury Induced By Hypoxia/Reoxygenation By Inhibiting ROS

Objective:The rarefaction and destruction of the peripapillary capillaries（PTC）can cause ischemia and hypoxia of the kidneys,leading to the transition from acute kidney injury（AKI）to chronic kidney disease（CKD）.The damage of endothelial cells induced by oxidative stress is the root cause of PTC damage.Intermedin（IMD）can inhibit the local oxidative stress response of kidney tissue,but its mechanism of protecting endothelial cells is still unclear.Endothelial nitric oxide synthetase（eNOS）decoupling is an important source of reactive oxygen species（ROS）.what’s more,our preliminary experiments show that IMD can inhibit eNOS decoupling,and the mechanism is worthy of further study.In this experiment,the effects of IMD on human umbilical vein endothelial cells（HUVECs）under hypoxia/reoxygenation（H/R）were studied to clarify whether IMD can inhibit eNOS decoupling by stabilizing the guanosine triphosphatecyclohydrolase-1（GTPCH-1）-tetrahydrobiopterin（BH₄）axis,so as to reduce ROS production and protect vascular endothelial cells.Methods:HUVECs were cultured in a three gas incubator(hypoxia conditions:92%N₂+5%CO₂+3%O_2;reoxygenation conditions:5%CO_2,37°C)to establish an H/R model.HUVECs were randomly divided into four groups:control group,H/R model group,H/R+IMD group,H/R+IMD+GTPCH-1 inhibito 2,4-diamino-6-hydroxypyrimidine（DAHP）group.H/R model was established according to the hypoxia 18h and reoxygenation 6h obtained in the pre-experiment.DHE-ROS detection method and DAF-FM-DA method are used to detect the effect of IMD on ROS and NO levels in each group.Western blot was used to detect the effect of IMD on the expression of eNOS dimer/monomer and GTPCH-1 protein in each group.High-performance liquid chromatography（HPLC）was used to detect the effect of IMD on the content of BH₄.Results:Compared with the control group,the level of ROS in the H/R model group was significantly increased,indicating that hypoxia-reoxygenation induced oxidative stress.After IMD intervention was given on the basis of H/R model,ROS level decreased significantly,which proved that IMD can inhibit ROS production.Compared with the control group,the NO level in the H/R group decreased significantly,and NO increased after IMD administration,which proved that IMD could inhibit the decrease of NO after H/R injury and improve endothelial dysfunction.After H/R,the expression of eNOS dimer protein decreased,and the expression of monomer increased.After IMD intervention,the expression of eNOS dimer increased compared with H/R group,indicating that IMD can inhibit eNOS decoupling.Compared with the control group,GTPCH-1 protein expression decreased and BH₄ content decreased significantly after H/R,After IMD intervention,GTPCH-1 expression increased and BH₄ content increased too.DAHP could eliminate the effect of IMD on BH₄ and GTPCH-1,which proved that IMD inhibited eNOS decoupling through GTPCH-1-BH₄ axis.Conclusion:（1）IMD reduced the production of ROS and protected the vascular endothelial cells by inhibiting the uncoupling of eNOS induced by H/R.（2）IMD inhibits eNOS uncoupling and reduces ROS by stabilizing GTPCH-1—BH₄axis.