Related Apoptosis-reducing Signal Pathways In Human Umbilical Vein Endothelial Cells Through Upregulation Of SIRT1 By Cordycepin

Objective The aim of this study was to investigate whether the ERK?m TOR?AKT and TLR4 signaling pathways play a role on the upregulation of SIRT1 by cordycepin and the protective effect of cordycepin on high glucose-induced apoptosis in human umbilical vein endothelial cells(HUVECs)and to clarify the protective mechanism of cordycepin on endothelial cells.Methods The 2rd~3th generations of HUVECs in logarithmic growth phase were selected for the experiment.And four pathway inhibitors were used in the test.All cells were divided into fourteen groups:(1)control group;(2)high glucose group;(3)resveratrol group;(4)10?M cordycepin group;(5)20?M cordycepin group;(6)CLI-095(TLR4inhibitor)group;(7)20?M cordycepin + CLI-095 group;(8)rapamycin(m TOR inhibitor)group;(9)10?M cordycepin + rapamycin group;(10)20?M cordycepin+rapamycin group;(11)AZD5363(AKT inhibitor)group;(12)20?M cordycepin +AZD5363 group;(13)U0126(ERK inhibitor)group;(14)20?M cordycepin + U0126 group.Brd U-ELISA method was used to detect cell proliferation and annexin V-FITC/PI double staining flow cytometry was used to detect cell apoptosis.Real-time quantitative PCR was used to detect m RNA expression of SIRT1.Western blot was used to detect protein expression of SIRT1.Results1.Cordycepin intervention experiment: Compared with that in control group,expression of SIRT1 and absorbance value in high glucose group were decreased significantly(P < 0.05),early apoptosis rate were increased significantly(P < 0.05).Compared with that in high glucose group,m RNA expression of SIRT1 and absorbance value in 20?M cordycepin groups were increased significantly(P < 0.05),early apoptosis rate were decreased significantly(P < 0.05).Compared with that in 10 ?M cordycepin group,m RNA expression of SIRT1 in 20 ?M cordycepin group were increased significantly(P<0.05),early apoptosis rate were decreased significantly(P<0.05).2.m TOR signal pathway: Compared with that in rapamycin group,m RNA and protein expression of SIRT1 in 20?M cordycepin + rapamycin groups were significantly increased(P < 0.05).Compared with that in cordycepin group,protein expression of SIRT1 in 20?M cordycepin + rapamycin groups were further increased(P<0.05).Compared with that in high glucose group,absorbance values in rapamycin group were significantly increased,early apoptosis rates were significantly decreased(P<0.05).Compared with that in 10?mol/L cordycepin group and in rapamycin group,absorbance values of 10?M cordycepin + rapamycin groups were significantly increased respectively(P<0.05).3.ERK signal pathway: Compared with that in high glucose group,m RNA expression of SIRT1 in U0126 groups were unchanged(P>0.05).Compared with that in high glucose group,m RNA expression of SIRT1 in 20?M cordycepin group were significantly increased(P < 0.05).Compared with that in 20?M cordycepin group,m RNA expression of SIRT1 in 20?M cordycepin + U0126 group were significantly decreased(P<0.05).Compared with that in high glucose group,absorbance values and early apoptosis rates in U0126 group were unchanged(P>0.05).Compared with that in high glucose group,absorbance values in 20?M cordycepin group were significantly increased,early apoptosis rates were significantly decreased(P<0.05).Compared with that in 20?mol/L cordycepin group,absorbance values in 20 ?M cordycepin + U0126 group were significantly decreased,early apoptosis rates were significantly increased(P<0.01).4.AKT signal pathway: Compared with that in high glucose group,m RNA expression of SIRT1 in AZD5363 group were significantly increased(P < 0.05).Compared with that in 20?mol/L cordycepin group,m RNA expression of SIRT1 in 20?M cordycepin + AZD5363 group were unchanged(P>0.05).5.TLR4 signal pathway: Compared with that in 20?mol/L cordycepin group,protein expression of SIRT1 in 20?M cordycepin + CLI-095 group were unchanged(P>0.05).Conclusions1.Cordycepin activated SIRT1 expression through the ERK signaling pathway,but independent of m TOR,AKT and TLR4 signaling pathway.2.Cotreatment of cordycepin with m TOR inhibitor had synergistic effect on SIRT1 expression.3.Activation of SIRT1 had a protective effect on endothelial cells in high glucose environment.4.Treatment of cordycepin may suppress cell apoptosis and promote cell proliferation through the upregulation of SIRT1.