Cordycepin Prevents High Glucose-induced Apoptosis In Human Umbilical Vein Endothelial Cells

ObjectiveThe aim of this study was to investigate the protective effect of cordycepin on high glucose-induced human umbilical vein endothelial cells(HUVECs) and to provide the basis for the cordycepin treatment on vascular complications of diabetes. MethodsUnder aseptic condition, we dealt with umbilical cord by washing, digestion, collecting and centrifugation to isolate cells. The 3rd~5th generations were used in the test. HUVECs were identified by immunocytochemistry staining. They were divided into eight groups:(1) control group: 5.5mmol/L glucose;(2) high glucose group: 40mmol/L glucose;(3) resveratrol group: 40mmol/L glucose+10?mol/L resveratrol;(4) 1?mol/L cordycepin group: 40mmol/L glucose+1?mol/L cordycepin;(5) 10?mol/L cordycepin group: 40mmol/L glucose +10?mol/L cordycepin;(6) 20?mol/L cordycepin group: 40mmol/L glucose+20?mol/L cordycepin;(7) CLI-095 group: 40mmol/L glucose+20?mol/L cordycepin+1?mol/L CLI-095;(8) rapamycin group: 40mmol/L glucose+20?mol/L cordycepin+100nmol/L rapamycin. After 24~48h cultivation, Brd U 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. Results1. More than 95% of HUVECs were identified positive by immunocytochemistry staining.2. Compared with that of control group, absorbance(A) value of high glucose group was decreased significantly(P<0.05). Compared with that of high glucose group, A value of resveratrol group, 10?mol/L and 20?mol/L cordycepin groups were increased respectively(P<0.05). A value of 10?mol/L and 20?mol/L groups were higher than that of 1?mol/L group(P<0.05). Compared with that of 20?mol/L cordycepin group, A value of CLI-095 group and rapamycin group were further increased(P<0.05).3. Compared with that of control group, early apoptosis rate of high glucose group was increased significantly(P<0.05). Compared with that of high glucose group, early apoptosis rates of resveratrol group, 1?mol/L group, 10?mol/L group, 20?mol/L group were decreased respectively(P < 0.05). Apoptosis rates of 10?mol/L and 20?mol/L cordycepin groups were lower than that of 1?mol/L group(P<0.05). Compared with that of 20?mol/L cordycepin group, apoptosis of rapamycin group were further reduced(P<0.05) and that of CLI-095 group was not significantly changed(P>0.05).4. Compared with that in control group, m RNA expression of SIRT1 in high glucose group was decreased significantly(P<0.05). Compared with that in high glucose group, m RNA expression of SIRT1 in resveratrol group, 10?mol/L group, 20?mol/L group were increased respectively(P<0.05). SIRT1 m RNA expression in 20?mol/L groups was higher than that in 1?mol/L and 10?mol/L groups(P <0.05).Compared with that in 20?mol/L cordycepin group, SIRT1 m RNA expression in CLI-095 group and rapamycin group were further increased(P<0.05). Conclusions1. Cordycepin has a protective effect on endothelial cells in high glucose environment.2. Cordycepin can promote SIRT1 m RNA expression on HUVECs.3. Cordycepin activates SIRT1 expression independent of TLR4 and m TOR signaling pathway.4. Cotreatment of cordycepin with TLR4 signaling pathway inhibitor or m TOR signaling pathway inhibitor has synergistic effect on promoting cell proliferation and m RNA expression of SIRT1. Furthermore, cotreatment of cordycepin with m TOR signaling pathway inhibitor may synergistically suppress cell apoptosis.