Protective Effect Of Intermedin On Vascular Endothelial Cells Damage Induced By Hypoxia/reoxygenation And Its Mechanisms

Objective:Delaying the transformation of acute kidney injury?AKI?to chronic kidney disease?CKD?is a hot topic in the field of renal diseases.The decrease of peritubular capillary?PTC?caused by vascular endothelial cell injury plays an important role in the transformation from AKI to CKD,while reactive oxygen species?ROS?is an important mechanism that causes vascular endothelial cell damage.Our previous studies have shown that intermedin?IMD?can reduce acute and chronic renal injury by inhibiting oxidative stress,but the effect on vascular endothelial cells is still unclear.In this experiment,the endothelial cell hypoxia/reoxygenation?H/R?model was established to observe the effect of IMD on vascular endothelial cell injury,and the mechanism of protecting endothelial cells was explored by measuring ROS.Methods:Human umbilical vein endothelial cells?HUVECs?were used as experimental objects and cultured in vitro,and H/R model was established in vitro through a three-gas incubator.They were divided into control group,H/R model group,H/R+IMD group,H/R+N-acetyl-L-cysteine?NAC?group,and H/R+IMD+hydrogen peroxide?H₂O₂?group.Observe the morphology of endothelial cells with an inverted microscope,detect the cell survival rate with MTT colorimetric method,and observe the effect of IMD on the survival rate of endothelial cells.The expression of caspase-3 active fragment was detected by Western blot,and the effect of IMD on endothelial cell apoptosis was observed.DAF-FM-DA method was used to determine the fluorescence intensity of nitric oxide?NO?;ELISA method was used to detect endothelin-1?ET-1?,monocyte chemoattractant protein-1?MCP-1?and vascular cell adhesion molecules-1?VCAM-1?,observe the effect of IMD on endothelial cell function;ROS fluorescence intensity was detected by DHE-ROS method to evaluate the effect of IMD on ROS content.Results:HUVECs with normal morphology are arranged in a typical paving stone pattern under microscope.The best time for endothelial cells H/R model is 18h of hypoxia and 6h of reoxygenation.Compared with the control group,the survival rate of endothelial cells in the H/R model group decreased,suggesting that endothelial cells can be damaged after H/R.After H/R+IMD,the survival rate of endothelial cells increased,and there was no significant difference from the results of NAC intervention,indicating that IMD can protect endothelial cells,and its effect is consistent with the level of known antioxidant NAC.It was found that the protective effect of IMD on endothelia cells disappeared by co-treatment of IMD and strong oxidant H₂O₂,indicating that the protection of endothelial cells by IMD may be related to antioxidant effects.The expression of caspase-3 active fragment after H/R is activated,suggesting that H/R can cause apoptosis of vascular endothelial cells.Compared with the model group,the expression level of caspase-3activity fragment decreased significantly after IMD and NAC intervention,and there was no statistical significance between the two groups,indicating that IMD can inhibit the apoptosis of endothelial cells,its anti-apoptotic effect was similar to that of NAC pretreatment.After treated with IMD and H₂O₂,the anti-apoptotic effect of IMD was lost,it is speculated that IMD exerts anti-apoptotic effect through antioxidant.The NO content of HUVECs decreased and the content of ET-1 increased after H/R,which also induced the up-regulation of MCP-1 and VCAM-1 expression,suggesting that endothelial cell injury can cause vasoconstriction,and increase expression of inflammatory factors.After intervention with IMD and NAC,the NO level increased,while the expression of ET-1,MCP-1 and VCAM-1 decreased,and there was no significant difference between the two groups,indicating that IMD can inhibit vasoconstriction,inflammation and improve endothelial dysfunction,which the results were the same as NAC.After co-treatment of IMD and H₂O₂,H₂O₂ can eliminate the above-mentioned IMD effects,indicating that IMD inhibits vasoconstriction and inflammation is related to antioxidant effects.The ROS content in the model group was significantly higher than that in the control group,suggesting that endothelial injury is related to oxidative stress.Compared with the H/R model group,the expression of ROS decreased after IMD and NAC intervention,and there was no significance between the two groups,indicating that IMD can inhibit the production of ROS,and its effect of inhibiting oxidative stress is consistent with that of NAC.Compared with H/R+IMD group,ROS content increased after co-treatment with IMD and H₂O₂,which confirmed that IMD reduced ROS production by inhibiting oxidative stress.Conclusion:?1?IMD has protective effect on endothelial cell injury induced by H/R;?2?The protective effect of IMD on endothelial cells is achieved by inhibiting ROS.