| Objectives: 1.To establish a rat model of diabetes mellitus and observe the effects of high mobility group box 1(HMGB1),LC3-II(Beclin-1)and LC3-II(Beclin-GFP),mTOR(Mammalian target of rapamycin),PI3K(Phosphatide 3-kinases)and pAkt(Phosphorylated protein kinase B);2.To investigate the effects of sitagliptin on autophagy and PI3 K / AKT / mTOR signaling pathway in diabetic rats with macrovascular complications.Methods:Fifty-five male Sprague Dawley(SD)rats of 5 weeks old(weighing about 180g-200g)without specific pathogen-grade(SPF)were randomly divided into normal control group(n = 10)and Model group(n = 48).The rats in the normal group were fed with normal feed.And the other model group was fed with high fat high sugar diet and all were free to drink water.After 4 weeks,the level of blood insulin(FINS),fasting blood glucose(FBG),triglyceride(TG),cholesterol(TC)and low density lipoprotein(LDL)were tested after 12 hours of fasting,the model group was intraperitoneally injected with STZ at a dose of 35 mg / kg.After 72 hours,tail vein blood of rats was randomly selected to be diabetic rats(BS)≥ 16.7 mmol / L for three consecutive times not modeled successfully).Rats that did not model success were excluded from the study.The rats in group A were fed with normal diet.The rats in model group were divided into five groups randomly divided into five groups according to the principle of randomization: group B(n = 10),sitagliptin 1.0 mg / kg.d(group C),n = 9),sitagliptin 5.0 mg / kg.d(group D,n = 9),sitagliptin 1.0 mg / kg.d + PI3 K inhibitor LY294002 10 mg / kg / d(group E,n= 9),sitagliptin 5.0 mg/ kg.d + PI3 K inhibitor LY294002 10 mg / kg / d(group F,n= 9).The rats in group A and group B were fed with normal saline only,the rats in groups C and D were treated with sitagliptin in the corresponding doses,and the rats in groups E and F were treated with a gavage of sitagliptin correspondingly,and were subcutaneously injected with PI3 K inhibitor LY294002 10 mg / kg / d(continuous injection for 2 weeks).All groups were given drugs at 8:00 a.m.and a total of 16 weeks intervention was performed.At the end of the 16 th week,blood was collected from each group to determine biochemical indexes such as FBG,TG.At the end of the 16 th weekend after model establishment,the rats were sacrificed after body weighing.5-6ml blood samples were drawn from the apex to detect the biochemical markers such as FBG,FINS,TG,TC and LDL-C.The free aortic arch was connected to the thoracic aorta The blood vessels were divided into three sections.The first section was used for HE staining and oil red O staining.The second section was used to measure the area of plaque and collagen fibers,macrophages and smooth muscle cells by immunohistochemistry Phagocytosis,and phagocytosis.Another group was used to detect HMGB1(high mobility group box 1),phosphatidylinositol 3-kinase class III(PI3K),phosphorylated protein kinase B(pAkt),mammalian target of rapamycin,Beclin-1 protein expression and qRT-PCR detection of HMGB1 expression.Results:1.At the end of the 4th week(the first week after successful modeling),the body weight and biochemical indexes of the rats in each group were significantly higher than those in the normal control group(P <0.05).The levels of insulin,blood glucose,HOMA-IR,TG,TC and LDL were significantly higher than those in the normal group(P <0.05).2.Compared with the control group A,the FBS,TG,TC and LDL-C of the model group were higher than those of the control group A,compared with the model group B,The above indexes in group C,D,E and F were significantly lower(P <0.05),the difference between group C and group D was statistically significant(P <0.05)There was no significant difference between group D and group F(P> 0.05).3.Compared with group A,the HMGB1,LC3-II,Beclin-1,mTOR,PI3 K and p Akt in thoracic aorta of sitagliptin intervention group at 16weeks: Beclin-1 expression was significantly lower than that of B group(P <0.05).Compared with B group,the expression of Beclin-1 in C,D,E and F groups was significantly increased(P <0.05)Compared with group A,the expression of mTOR,PI3 K and pAkt in diabetic rats increased significantly(P <0.05),and the expression of these indexes was lower in group D and C(the difference was statistically significant(P <0.05).The expressions of mTOR,PI3 K and pAkt in group E,group C and group F were significantly lower than those in group D,and the expressions of LC3-II and Beclin-1 were significantly increased(P <0.05).4.Sitagliptin intervention at 16 weeks by HE staining and oil red O staining results,B group showed vascular intimal defect,endothelial cells occasionally shedding,smooth muscle cells showed significant proliferation,disordered arrangement,elastic fiber disorder,showing a large number of foam cells.Compared with group B,the area of aortic plaque in group C,D,E and F was significantly lower,and the area of aortic plaque was significantly lower in group E and C,F and D compared with group D.Conclusions:1.The expression of HMGB1,LC3-II and Beclin-1 in the thoracic aorta of STZ-induced diabetic rats decreased significantly and the expression of mTOR increased,suggesting that the autophagy activity was inhibited;2.Sitagliptin may protect against diabetic macroangiopathy by up-regulating the expression of HMGB1,inhibiting the PI3 K / Akt / mTOR signaling pathway and promoting autophagy. |
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