Role And Mechanism Of SIRT1 In Angiogenesis Of Diabetic Wounds

BackgroundChronic wound is a common complication of diabetes mellitus,which seriously affects the quality of life and safety of patients.As the high incidence and recurrence,the diabetic wounds treatment has caused a heavy burden on social medical resources and economy.Chronic diabetic ulcer has become the main cause of death and disability in diabetic patients.Literature reports that of those patients with DFUs,25% subsequently experience a lower extremity amputation,with the mortality rate from amputation approaching 50% five-year post-amputation.The cause of the diabetic wound are not fully understood,and there are limited therapeutic agents and methods to repaire these wound.Therefore,it is still an urgent need to explore the mechanism of diabetic wound healing and find new therapeutic agents.Dysfunction of angiogenesis is an important cause of diabetic wound healing,and it is an important target for improving diabetic wound healing.Endothelial dysfunction is an important feature of diabetes mellitus,which is closely related to the occurrence and development of diabetic vascular complications.The main manifestation in wound healing is the weakening response to hypoxia and injury-induced angiogenesis.Oxidative stress is the main cause and central link of endothelial dysfunction caused by diabetes mellitus.Therefore,protecting endothelial cells from oxidative stress and promoting angiogenesis is one of the effective ways to promote wound healing of diabetes.SIRT1 is an important transcription factor that maintains cell homeostasis and resists environmental stress.It regulates cell functions such as DNA damage repair,inflammatory response,oxidative stress and apoptosis.A large number of studies have shown that SIRT1 has protective effect on tissue damage caused by diabetes mellitus and its complications by regulating the above cellular functions.It has great potential in the prevention and treatment of diabetes mellitus and its complications.However,the role of SIRT1 in diabetic wounds is still unclear.Therefore,we hypothesize that SIRT1 can improve wound healing efficiency of diabetes mellitus by protecting endothelial cells from oxidative stress injury and enhancing wound angiogenesis.ObjectiveTo explore the effect of SIRT1 on the function of diabetic wounds endothelial cells and further reveal the mechanism of angiogenesis dysfunction in diabetic wounds,so as to explore therapeutic agents and theoretical basis for the repair of diabetic wounds.Methods and materials1.Collecting skin tissue from patients with clinical diabetes mellitus or acute trauma patients(without history of diabetes),using conventional molecular biology methods such as qRT-PCR and Western Blot to detect the expression of SIRT1 in wound tissues,using 8-oxo-dG staining to assess the level of oxidative stress in wound margin tissue.2.Construct the STZ-induced diabetic mouse wound model,and set up the PBS treatment group and SRT1720 treatment group,using the normal mouse wound model as a control.The SIRT1 expression in intact skin and wound tissue mice on the 7th day post operation was detected by qRT-PCR and Western Blot.On the 1st,3rd,7th and 14 th postoperative day,the diffence of wound healing of three groups was observed to investigate the effect of SIRT1 on diabetic wound healing.The changes of wound perfusion in each group on the 1st,3rd,and 7th day of the day were detected by laser Doppler flow imaging.The wound tissue from diabetic mice model group and the diabetic model + SRT1720 treatment group were collected on the 7th day after operation,and CD34 antibody staining was used to evaluate the angiogenesis in wounds tissue;the effect of SIRT1 on wound angiogenesis in diabetic mice was investigated.3.Construct cell model with HUVECs and set up control group(low glucose culture),hyperglycemia group(high glucose culture)and hyperglycemia + SRT1720 treatment group;use conventional molecular biology methods such as qRT-PCR and Western Blot to detect SIRT1 expression levels of HUVEC in high glucose culture group and low glucose culture group.The effects of SIRT1 on migration,proliferation and angiogenesis ability of HUVECs in high glucose environment were studied by scratch test,Transwell test,Ki67 immunofluorescence staining assay and tube formation experiment.The expression of SIRT1 in HUVEC cells was interfered by siRNA transfection technique.The effects of SIRT1 on migration,proliferation and angiogenesis of HUVECs were studied by Transwell assay,Ki67 immunofluorescence staining assay and tube formation assay.4.Construct cell model with HUVECs and set up control group(low glucose culture),hyperglycemia group(high glucose culture)and hyperglycemia + SRT1720 treatment group.and use ROS detection kit to detect the level of ROS production in each group.To investigate the activity and expression levels of key molecules in the AKT/NRF2 signaling pathway,and explore the possible mechanism of SIRT1 protection of endothelial cells under high glucose conditions.Results1.Compared with the wound tissue from patients with acute trauma,the mRNA and protein expression of SIRT1 in diabetic wound tissue was significantly decreased(P < 0.05,P < 0.001);The wound tissue from patients with acute trauma showed a lower level of 8-oxo-dG expression,while diabetic wound tissue showed higher levels of 8-oxo-dG expression.2.The expression of SIRT1 in intact skin of diabetic mice was lower than that of normal mice(P < 0.001,P < 0.01);The expression of SIRT1 in wound tissue of diabetic mice on the 7th day post injury was lower than that of normal mice(P < 0.001,P < 0.01).The evaluation results of wound healing rate showed that the wound healing rate of diabetic mice was lower than that of normal mice on the 3rd,7th and 14 th day after injury(P < 0.05,P < 0.01,P < 0.01);The wound healing rate of diabetic mice plus SRT 1720 treatment group was higher than that of diabetic mice on the 7th and 14 th day after injury(P < 0.01,P < 0.01).Laser Doppler results showed that the blood perfusion of wound surface in diabetic mice was lower than that in normal mice on the day of wound construction and the 1st,3rd and 7th day after injury(P < 0.01,P < 0.01,P < 0.01,P < 0.01),while the blood perfusion of wound surface in diabetic mice + SRT1720 group was higher than that in diabetic mice on the 3rd and 7th day after injury(P < 0.05,P < 0.05).CD34 immunohistochemical staining showed that the vascular density of wounds in diabetic mice treated with SRT 1720 was significantly higher than that in diabetic mice on the 7th day after injury(P < 0.01),and the arrangement of neovascularization was regular and the lumen was smooth.3.Compared with the low-glucose group,the SIRT1 expression in HUVECs was significantly decreased in the high-glucose group(P < 0.01,P < 0.001).Scratch test results showed that the scratch healing rate of HUVECs in high glucose culture group was lower than that in low glucose culture group at all observation points(24,48 and 72 hours after scratch)(P < 0.01,P < 0.01,P < 0.01),while the scratch healing rate of HUVECs in high glucose culture + SRT 1720 group was higher than that in high sugar culture group(P < 0.05,P < 0.05,P < 0.05).Transwell cell culture experiment showed that the number of HUVECs in high glucose culture group was significantly lower than that in low glucose culture group 24 hours later(P < 0.01),while the number of HUVECs in high glucose culture + SRT 1720 treatment group was significantly higher than that in high glucose culture group 24 hours later(P < 0.01).Ki67 immunofluorescence staining showed that the expression level of HUVECs Ki67 in high glucose culture group was significantly lower than that in low glucose culture group(P < 0.01),while the expression level of HUVECs Ki67 in high glucose culture + SRT1720 treatment group was significantly higher than that in high glucose culture group(P < 0.01).The results of tube formation experiment showed that the total length of lumen and number of branch nodes of HUVECs in high glucose culture group were significantly lower than those in low glucose culture group(P < 0.001,P < 0.001)while the total length and number of branch nodes of HUVECs in high glucose culture + SRT 1720 group were significantly higher than those in high glucose culture group(P < 0.01,P < 0.01).The results of siRNA interference experiment showed that the number of HUVECs perforating cells,Ki67 expression,total lumen length and number of branch nodes in SIRT1 interference sequence transfection group 24 hours later were lower than those in invalid sequence transfection group(P < 0.01,P < 0.001,P < 0.01,P < 0.001).4.The level of ROS production of HUVECs in high glucose culture group was significantly higher than that in low glucose culture group(P < 0.01),while the levels of AKT phosphorylation,NRF2 and NQO1 expression were lower than those in low glucose culture group(P < 0.05,P < 0.001,P < 0.001);the level of ROS production of HUVECs in high glucose culture + SRT1720 treatment group was significantly lower than that in high glucose culture group(P < 0.05),while the levels of AKT phosphorylation were higher than those in high glucose culture group(P < 0.01).The expression levels of NRF2 2 and NQO1 were higher than those in high glucose culture group(P < 0.05,P < 0.01)Conclusion1.The results showed that low level of oxidative stress in acute traumatic tissue was the requirement of tissue repair,and severe oxidative stress in diabetic wound tissue might be one of the reasons that destroyed the healing process of diabetic wound,and the inhibition of SIRT1 expression in diabetic wound tissue might be related to the difficulty of wound healing.2.SIRT1 activation can effectively promote wound healing in diabetic mice,which may be related to its active regulation of wound angiogenesis.3.SIRT1 is an indispensable transcription factor for HUVECs to maintain good migration,proliferation and differentiation.High glucose stress inhibits the expression of SIRT1 in HUVECs,which is one of the mechanisms leading to impaired migration,proliferation and differentiation of HUVECs.SIRT1 activation can effectively improve the impaired endothelial cell function under high glucose stress.4.The increase of ROS production in HUVECs induced by high glucose may be related to the inhibition of AKT phosphorylation,the decrease of NRF2 and its downstream gene NQO1 expression.SIRT1 activation may increase AKT phosphorylation,up-regulate the expression of NRF2 and NQO1,and inhibit the excessive production of ROS in endothelial cells under high glucose culture.