Beneficial Effects Of Low-intensity Continuous Ultrasound On Ischemia-induced Angiogenesis In Type 2 Diabetic Mice

Objective:Recently the morbidity of type 2 diabetes mellitus has increased sharply,peripheral artery disease is a major vascular complication of diabetes mellitus.Therapeutic post-ischemic angiogenesis can improve clinical symptoms of patients.However,there still lack of effective treatments.A growing body of research shows that low-intensity continuous therapeutic ultrasound(TUS)could repaire damaged tissue and accelerate vascularization of ischemic tissues.However,it is little known that the effect of low-intensity continuous therapeutic ultrasound treatment on ischemic-induced angiogenesis in type 2 diabetic individuals.The aim of this investigation was to observe the effect of low-intensity continuous therapeutic ultrasound on ischemia-induced angiogenesis in type 2 diabetic mouse model and high glucose-induced dysfunction in human umbilical vein endothelial cells(HUVECs),and detect the underlying molecular biological mechanism,provide new perspectives for the clinical treatment of type 2 diabetic vasculopathy.Methods:1.To study the effect of low-intensity continuous therapeutic ultrasound on ischemia-induced angiogenesis in type 2 diabetic mice.The mouse model of type 2diabetic was induced by a high-fat diet(HFD)for 8 weeks,followed by a single intraperitoneal injection of Streptozotocin(100 mg/kg),hindlimb ischemia was induced by unilateral femoral artery ligation.The treatment of low-intensity continuous therapeutic ultrasound(TUS,1MHz,0.3W/cm~2,9min/day,for a continuous 14 days after surgery)was given according to the experiment grouping design,infrared thermal imaging analysis was employd to evaluate blood perfusion of hindlimb,the score of hindlimb ischemia was assessed according to grading standard,capillary density of ischemic tissue was detected by immunohistochemical staining,endothelial apoptosis and oxidative stress were measured by immunofluorescence staining,mitochondrial function was analyzed by Real-time Polymerase Chain Reaction,protein expression of Akt,p-Akt,e NOS,p-e NOS,VEGF,Bcl-2,Bax,AIF,Cytochrome c,Caspase3 and c-Caspase3 in ischemic muscles were detected by Western Blot.2.To evaluate the effect of low-intensity continuous therapeutic ultrasound on high glucose-induced angiogenesis function in HUVECs.The expression of Akt,p-Akt,e NOS,p-e NOS and VEGF in HUVECs were determined by Western Blot,the cell proliferation activity,cell migration ability and tube formation ability were evaluated by CCK-8 assay,Transwell test and Matrigel test respectively.3.To evaluate the effect of low-intensity continuous therapeutic ultrasound on high glucose-induced apoptosis in HUVECs.TUNEL assay was employed to detect cell apoptosis,the level of ROS in HUVECs was analyzed by DCHF-DA ROS kit,mitochondrial ROS level was measured by using Mito SOX Red Mitochondrial Superoxide Indicator,mitochondrial membrane potential was determined by using JC-1,the protein expression of Bcl-2,Bax,AIF,Cytochrome c,Caspase3 and c-Caspase3 were measured by western Blot.Results:1.Low-intensity continuous therapeutic ultrasound can promote ischemia-induced angiogenesis in type 2 diabetic mice.Compared with the normal mice,increased ischemia score of hindlimb,decreased blood perfusion,reduced capillary density,elevated oxidative stress levels,impaired mitochondrial function,decreased proangiogenic factors,up-regulated apoptotic proteins as well as down-regulated anti-apoptotic proteins were captured in diabetic mice.Low-intensity continuous therapeutic ultrasound not only rescued the toxic effects of diabetes but also improved these indicators in normal group.2.Low-intensity continuous therapeutic ultrasound can modify high glucose-induced angiogenesis function in HUVECs,which may be related to restoration of PI3K-Akt-e NOS signal pathway.Compared with the normal group,PI3K-Akt-e NOS signal pathway was impaired in HUVECs incubated with high glucose.However,the low-intensity continuous therapeutic ultrasound can robust rescue the impaired pathway,followed by elevated VEGF expression,improved cell proliferation activity,enhanced cell transmembrane migration ability and tubule like structure formation ability.However,the protective effects of low-intensity continuous therapeutic ultrasound can be abrogated by L-NAME or LY294002.3.Low-intensity continuous therapeutic ultrasound can suppress high glucose-induced apoptosis in HUVECs via ameliorating mitochondrial function.Compared with the normal group,high glucose can dramtically accelerate HUVECs apoptosis,up-regulate apoptotic factors expression,down-regulate antiapoptotic protein level,increase the total ROS and mitochondrial ROS in HUVECs,reduce the mitochondrial membrane potential.Low-intensity continuous therapeutic ultrasound not only restored the damage effect of high glucose but also improved these indicators in normal group.Conclusion:Low-intensity continuous therapeutic ultrasound can augment angiogenesis and blood perfusion in type 2 diabetic mice.The mechanism may be related to the restoration of PI3K-Akt-e NOS signal pathway and improvement of mitochondrial function in HUVECs.