Effects And Mechanism Of Recombinant Human Brain Natriuretic Peptide On Lower Limb Ischemia In Diabetic Mice

BackgroundVasculopathy is one of the most comMon chronic complications of diabetes,and it is also an important cause of disability and death in patients with diabetes.One of the main pathogenesis of diabetic angiopathy is the damage of vascular endothelial cells caused by hyperglycemia.Due to the complex pathogenesis of extensive and diffuse diabetic vascular lesions,many treatment have limitations.It is meaningful to study on the mechanism of action of bioactive substances on vascular lesions in diabetic patients.Brain natriuretic peptide(BNP)is produced by ventricular myocytes,can mediate a series of effects including diuresis,natriuresis,hemangiectasis,inhibition of sympathetic nervous system and renin-angiotensin-aldosterone system,reduction of myocardial cell apoptosis,etc.Recently,several clinical researches indicated that BNP may be related to glucose metabolism,also it may be closely related to the occurrence of diabetic angiopathies and the change of lower extremity blood flow.However,there are few reports about the effects of BNP on diabetic angiopathy and its role in vascular endothelial cells exposed to high concentration of glucose.ObjectiveTo investigate the effects of recombinant human brain natriuretic peptide on stimulating HUVECs autophagy induced by high concentration of glucose and inhibiting apoptosis with the establishment of in vitro human umbilical vein endothelial cells autophagy model induced by high concentration of glucose and in vivo diabetic lower limb ischemia mice model.Also to explore the mechanism of rhBNP autophagy regulation effects on diabetic lower limb ischemic lesions.MethodsDiabetic mice model was established by intraperitoneal injection of streptozocin(STZ),while lower limb ischemic model was induced by ligation of femoral arteries.Mice were randomly divided into model group and rhBNP treatment group,mice in model group were given intraperitoneal injection of 0.3-0.4 ml saline per day,while 0.3-0.4 ml rhBNP were injected to mice in rhBNP group instead.Regular observations were performed to record the necrosis rate of mice lower extremities.Laser Doppler was used to evaluate the blood flow of mice lower extremities before modeling and on the 0th,3rd,7th,14th,and 21st days after operation.Eight mice from both model group and rhBNP group were sacrificed at 3rd day after operation,and their lower limb gastrocnemius muscles were taken.Related protein expression of LC3,ERK,AKT,Bcl-2 were detected using Western blot.Ten mice from both model group and rhBNP treatment group were sacrificed at 21st day after operation,and their lower limb gastrocnemius muscles were taken.HE staining,TUNEL kit and CD31,SMA imMunofluorescence staining were used to observe inflamMation infiltration,tissue necrosis,apoptosis and angiogenesis.Western blot was used to detect the expression of vascular growth factor HGF and VEGF in ischemic lower limbs.In vitro HUVECs apoptotic models induced by high concentration of glucose were separately treated with rhBNP,autophagy inhibitor 3-methyl adenine(3-MA)and autophagy enhancer Rapamycin(RAPA),in order to investigate the effect of rhBNP on autophagy,apoptosis and related signaling pathways of HUVECs induced by high concentration of glucose.ResultsThe levels of HGF,LC3,and p-ERK in mice of rhBNP group were significantly higher than those in the model group,and the cases of severed limbs and gangrene were significantly less than those in the model group.The results of laser Doppler detection showed that the improvement of lower limb ischemia in the rhBNP group was significantly better than that of the model group.Compared with the model group,TUNEL staining results indicated that apoptosis of endothelial cells in rhBNP group was decreased.The CD31 and SMA staining showed increased angiogenesis and increased expression of HGF in rhBNP group.The above changes indicate that rhBNP has a significant effect in improving lower limb ischemia in diabetic mice.In vitro,when HUVECs exposed to high concentration of glucose,cell proliferation was inhibited,apoptosis was increased,LC3-II/I levels were decreased,activity of ERK pathway was decreased,and levels of HGF were decreased significantly.These changes suggest that inhibition of ERK protein expression may inhibit autophagy and promote apoptosis and further inhibit the activity of HUVECs after high concentration of glucose induction.rhBNP intervention significantly enhanced HUVECs cell viability and proliferation,increased the level of LC3-II/I,decreased the level of Bax,caspase-3 apoptotic protein,increased the protein expression of ERK,and increased the level of HGF.It was suggested that rhBNP may enhance the activity of HUVECs by increasing protein expression of ERK and promoting autophagy.ConclusionsThe exposure of high concentration of glucose might inhibit the proliferation of HUVECs,inhibit autophagy,promote apoptosis,and inhibit ERK signaling pathway.rhBNP has the potential to promote angiogenesis and improve lower limb ischemia in diabetic mice.The mechanism may be related to the stimulating of ERK/LC3 signaling pathway to increase autophagy and reduce apoptosis.