The Effect Of Dapagliflozin On Autophagy And Apoptosis In Diabetic Rat Macroangiopathy

Objective: To investigate the effect of dapagliflozin(Dapa)on autophagy and apoptosis of diabetic rat macroangiopathy and the relevant mechanisms.Methods: Male SD rats fed with high glucose and high fat and induced by STZ were used to establish an experimental diabetic macroangiopathy rat model;SD rats without STZ injection were the normal control group(n = 12)and fed with a general diet.The prepared models of the rats were randomly divided into diabetes group(n = 12),low-dose treatment group(n = 12),and high-dose treatment group(n=12).The low-dose treatment group was intragastrically administrated with dapagliflozin 3 mg/kg.d.The high dose treatment group was given 5 mg/kg.d dapagliflozin in the same way.The normal control group and diabetic group were given the same volume of sterilized water instead of dapagliflozin.After 12 weeks,blood was collected via eyepit to measure the levels of serum total cholesterol(TCH),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)levels.Thereafter,the aortic root tissue was taken by hematoxylin-eosin(HE)staining to observe pathological changes.Real-time quantitative PCR(RT-qPCR)was used to detect the expression levels of Beclin-1,p62,Bax,and Bcl-2 mRNA in thoracic aorta tissue.Besides,protein expression levels of beclin-1,p62,LC3,Bax and bcl-2 in thoracic aorta were analyzed by western blot(WB).Results: 1.Blood glucose,blood lipid,and body weight in each group:Compared with the normal control group,blood glucose,TG,TCH,LDL-C,and HDL-C in the diabetic rats were significantly increased(P<0.01);Compared with the diabetes group,the blood glucose,TG,TCH,and LDL-C in the high-dose treatment group and the low-dose treatment group were both significantly reduced(P<0.01),and HDL-C made a slight difference.In comparision to the low-dose treatment group,the high-dose treatment group had lower blood glucose(P<0.01),and there was no difference in the levels of TG,TCH,LDL-C and HDL-C.Compared with the control group,the weight of the the diabetic group was lower than that of the control group.The intervention of Dapagliflozin had no effect on the body weight of the rats in the low-dose treatment group and the high-dose treatment group(P>0.05).2.HE staining of thoracic aorta of rats in each group: The aortic intima of the normal control group was intact and flat,and the endothelial cells were flat against the inner elastic plate.They were flat,smooth muscle cells were arranged in parallel,long spindle shape,uniform nucleus size,and uniform cytoplasmic stain.The elastic fibers are intact and not broken.In the diabetic group,the intimal thickening of the aorta was uneven,and the endothelial cells were partially shed.The median membrane was thickened of the,the number of smooth muscle cells increased,the arrangement was disordered,and the number of layers was increased;The pathological changes of aorta in the high and low dose treatment group were improved by comparison with the diabetes group.3.RT qPCR of thoracic aorta in each group: RT-qPCR of thoracic aorta:Compared with the normal control group,the result of the diabetic group presented a totally difference,more specifically,the expression of Bax mRNA(P<0.01)and Beclin-1 mRNA(P<0.05)was increased,while the Bcl-2 and P62 mRNA expression decreased(P<0.01).In addition,in comparision with the diabetic group,in the low-dose treatment group,the levels of Bax and Beclin-1 mRNA in the low-dose treatment group were decreased(P<0.01),the levels of Bcl-2 mRNA(P<0.05)and p62 mRNA(P<0.01)were increased.There was no significant difference in the expression levels of Bax,Beclin-1,Bcl-2 and P62 mRNA between the low-dose treatment group and the high-dose treatment group.4.Thoracic aorta WB of rats in each group: Comparison between the control group and the diabetes group,Beclin-1 protein content increased(P<0.05),Bax protein content significantly increased(P<0.01),p62 protein content significantly decreased(P<0.01),Bcl-2 protein content decreased(P<0.05),and LC3?/LC3? increased in the diabetes group(P<0.05).Compared with diabetes group,Beclin-1 protein content significantly decreased(P<0.01),Bax protein content decreased(P<0.05),Bcl-2 and p62 protein contents increased(P<0.05),and LC3?/LC3? decreased in the low-dose treatment group(P<0.05),while Bax and Beclin-1 protein contents significantly reduced(P<0.01),Bcl-2 and p62 protein contents increased(P<0.01),and LC3?/LC3? decreased(P<0.05)in the high-dose treatment group.Compared with the low-dose treatment group,Beclin-1 Protein content reduced significantly(P<0.01),Bax protein content decreased(P<0.05),Bcl-2and p62 protein contents increased(P<0.01),and LC3?/LC3? had no significant difference in the high-dose treatment group(P<0.05).Conclusions: 1.Dapagliflozin can improve the metabolism of glucose and lipid,and affect autophagy and apoptosis in diabetic rat macroangiopathy.2.Dapagliflozin can alleviate diabetic rat macroangiopathy,which may be related to the inhibition of autophagy and the decrease of apoptosis in diabetic rat macroangiopathy.