Roles Of SIRT1 On The Damage Of High Glucose Cultured Human Umbilical Vein Endothelial Cells

Objective:SIRT1 has been shown recently to lower plasma glucose levels,improve insulinsensitivity and atheroslerosis.To investigate the possible roles of SIRT1 in diabeticmacrovascular diseases,high glucose cultured primary human umbilical veinendothelial cells(HUVEC) is used.The underlying mechanisms are also investigated.Methods:Part 1,the expression of SIRT1 in high glucose culured HUVEC cells wereinvestigated,cells were divided into control group (5.5 mmol/L glucose),highglucose group (33.3 mmol/L glucose),hyperosmotic control group (27.8 mmol/Lmannitol+5.5 mmol/L glucose) The expression of SIRT1,eNOS,E-selectin,endothelin-1 mRNA were analyzed using RT-PCR;and the expression of SIRT1protein using Western blot.Supernatent of cells were collected for the analysis of thecontents of NO using nitric acid reductase.Part 2,the effects of SIRT1 activatorresveratrol on the function of high glucose culured HUVEC cells were investigated.Cells were divided into control group,high glucose group(48hours),1μmol/Lresveratrol pretreatment (24hours)group plus high glucose group.Part 3,the effects ofresveratrol on the functions of high glucose culured HUVEC after SIRT1"silence"were investigated.Cells were divided into control group,high glucose group,1μmol/L resveratrol pretreatment (24hours)group plus high glucose group,SIRT1siRNA or LipofectamineTM 2000 combined with resveratrol and high glucose group.For the latter two groups,cells were treated with 16.7 nmol/L SIRT1 siRNA (or not)in the assistance of Lipofectamine 2000 in order to interfere the expression of SIRT1.Part 4,cells were divided into control group,normal glucose group,high glucosegroup,1μmol/L resveratrol pretreatment (24 hours)group plus high glucose group.After 48 hours,cells in the latter three groups were stimulated by 100 nmol/L insulinfor 30 min.Nitric acid reductase was used to analyze the NO contents in supernatent.Results:(1) Compared with normal control cells,high glucose downregulated the levels ofSIRT1 both at the level of mRNA and protein(P＜0.05),and downregulated thelevels of eNOS mRNA (P＜0.01) and the secretion of NO (P＜0.01),butupregulated the levels of E-selectin (P＜0.05) and endothelin-1 (P＜0.05);the levels of SIRT1,eNOS,E-selectin,endothelin-1 and NO ofmannitol group did not change.(2) Compared with high glucose groups,1μmol/L resveratrol pretreatment for 24hours significantly increased the expression of SIRT1 mRNA(P＜0.01) and protein(P=0.01) of high glucose cultured HUVEC.Resveratrol also increased the level ofeNOS mRNA (P＜0.05),the secretion of NO (P＜0.01),but decreased the level ofE-selectin (P＜0.05),and the level ofendothelin-1 (P＜0.05).(3) After"knock out"the expression of SIRT1 of HUVEC,the up-regulating effectsof eNOS,NO and the down-regulating effects of E-selectin induced by resveratroldisappeared.But the down-regulating effects of resveratrol on the expression ofendothelin-1 did not change.The levels of SIRT1,eNOS,NO and endothelin-1 inLipofectamine 2000 group were not different from those in resveratrol alonetreatment group.(4) 100 nmol/L insulin treatment for 30 rains significantly stimulated the secretion ofNO in HUVEC.But high glucose impires the insulin roles on endothelial cells.Whenpretreatment endothelial cells with resveratrol significantly promote the stimulatingeffects of insulin on the secretion of NO in endothelial cells(P＜0.01).Conclusions:(1) SIRT1 is down-regulated when endothelial cells is cultured at high level ofglucose,SIRT1 activitor can improve the dilation of endothelial cell and downregulate the expression of E-selectin in endothelial cells cultured in high glucose,andfunctions as an endothelial cell protector,and therefore may have the great promise inthe prevention or therapy of diabetic macrovascular diseases.(2) Resveratrol can protect the endothelial cells from high glucose induced damageby stimulating the expression of eNOS and the secretion of NO and by inhibiting theexpression of E-selectin in SIRT1 dependent manner.Morover,resveratrol can inhibitthe expression of endothelin-1 in SIRT1 non-dependent manner in endothelial cellscultured in high glucose.Resveratrol may also have the potential to improve theinsulin resistance of endothelial cells induced by high glucose.In conclusion,resveratrol has the great prospects in the protection and therapy of diabeticmacrovascular diseases and atherosclerosis.