The Mechanism By Which Klotho Protein Regulates Autophagy Via The AMPK/mTOR Signaling Pathway To Protect Vascular Endothelial Cells From H2O2-induced Damage Was Investigated

Objective Klotho protein was tested for its ability to protect umbilical vein endothelial cells(HUVECs)from H2O2-induced damage.Subsequent observations were made to better understand how klotho protein controls autophagy by regulating the AMPK/m TOR signaling pathway and enhances the protection of vascular endothelial cells from H2O2-induced damage.Methods Human umbilical vein endothelial cells(HUVECs)were cultivated,and hydrogen peroxide caused harm to the human umbilical vein endothelial cells(HUVECs)(H2O2).One blank control group,two H2O2 model groups(incubated with 200 umol/L H2O2 for 24 hours),and three Klotho protein groups(first incubated with 100 umol/L Klotho protein in the medium for 12 hours,then incubated with 200 umol/L H2O2 for 24 hours)were used in the experiment.4AMPK inhibitor Compound C group was incubated with 100 g/L of Klotho protein,10 mol/L of AMPK inhibitor Compound C,and 200 mol/L H2O2 for 12 hours.Using the MTT technique,the cell viability of the aforementioned groups was found.Using the dihydroethanediol technique,the reactive oxygen species(ROS)values in each group were calculated.Malondialdehyde(MDA)and superoxide dismutase(SOD)kits were used to gauge the amount of oxidative stress in each group’s cell culture media.By using immunofluorescence labeling,the alterations of microtubule associated protein 1 light chain 3(LC3II)were discovered.Autophagy-related proteins such as p62,Beclin-1,LC3II/LC3 I,proteins involved in the AMPK/m TOR signaling pathway,and apoptotic proteins were all detected using the Western blot method.Caspase3,Bax,Bcl-2.Flow cytometry was used to identify cell apoptosis.Results(1)HUVECs activity in the model group decreased in comparison to the control group,and the apoptosis rate increased(P<0.01);(2)ROS and MDA contents in cells significantly increased(P< 0.01),and SOD activity significantly decreased(P<0.01);(3)p-AMPK/AMPK increased,and the ratio of p-m TOR/m TOR decreased(P<0.01).(4)The expression of p62 reduced(P<0.05),the ratio of LC3II/LC3 I increased(P<0.05),and the autophagy-related protein beclin-1 increased(P<0.01).In comparison to the model group,(1)the Klotho protein group’s cell viability and survival rate increased(P<0.01);(2)its ROS,MDA,and SOD content reduced(P<0.01);its SOD activity increased(P<0.01);(3)its p-AMPK/AMPK ratio increased(P<0.01);and its ratio of p-m TOR to m TOR dropped(P<0.01);(4)The expression of p62 reduced(P<0.05),the ratio of LC3II/LC3 I increased(P<0.01),and the autophagy-related protein beclin-1increased(P<0.05).The results of the above indexes in AMPK inhibitor Compound C group were contrary to those in Klotho protein group.Conclusions Klotho protein activates the AMPK/m TOR signaling cascade and boosts autophagy to protect endothelial cells from H2O2-induced damage.