Mechanism Of PI3K/AKT GSK-3? Signaling Pathway On Aortic Calcification In Type 2 Diabetic Rats

Background & Objective: Wnt/?-catenin signaling pathway is the most important signal pathway in bone formation.In vivo,inhibitors(LY294002 and TWS119)of PI3K/AKT GSK-3? signaling pathway were used to verify the relationship between Advanced glycation endproducts(AGEs)and PI3K/AKT GSK-3? signaling pathway in diabetic arterial calcification.Methods:(1)80-100 g(60 rats)of SD rats were fed in animal room.After one week,the rats were randomly divided into N group,DM group,DMSO group,LY294002 group,TWS119 group,12 rats in each group,and all the rat were given the different feeds.N group(12 rats)were fed with normal diet for 4 weeks,DM group,DMSO group,LY294002 group,TWS119 group(total 48 rats)were fed with high fat diet(HFD)+ nicotine for 4 weeks.After 4 weeks,fasting blood glucose(FBG)and body weight were measured(recorded as 0 days).After the data were measured,DM group,DMSO group,LY294002 group and TWS119 group(total of 48 rats)were injected intraperitoneally with streptozotocin(STZ)to construct the model of diabetic rats with vascular calcification.On the 3rd,5th,7th,14 th and 28 th day after intraperitoneal injection of STZ,random blood glucose was collected from rats.The concentration of rats blood glucose>16.6 mmol/L?3 in 5 times was judged to be successful in the model of diabetic rats.Some diabetic rats were unsuccessful which were given a small dose of STZ intraperitoneal injection.(2)The rats of type 2 diabetes mellitus combined with vascular calcification model was successfully modeled,N group rats were given 10ml/kg saline intraperitoneal injection;DM rats were also given 10ml/kg saline intraperitoneal injection;DMSO rats were given 10ml/kg DMSO intraperitoneal injection;LY294002 rats were treated with AKT inhibitor LY294002 intraperitoneally at 60mg/kg for 3 weeks.TWS119 rats were treated with GSK-3? inhibitor TWS119 according to 30mg/Kg of intraperitoneal injection for 3 weeks.(3)In SD rats,we injected PI3K/AKT GSK-3? signaling pathway(LY294002 and TWS119)intraperitoneally for a month.After 1 month,all the rats were sacrificed and immediately blood collection from the heart with 5ml empty needle.Immediately centrifuged the supernatant in the EP tubes,marked group and saved at-20 ?gerator temporary.Objective to investigate the expression of ?-catenin in the aorta and the osteogenic differentiation of the aorta.Detection the expression of related proteins and detection the expression of Wnt/?-catenin signaling-related signaling proteins.The related detection techniques were including Western Blot assay,immunohistochemistry,etc.Results:(1)Fatty rats(HFD)intramuscular injection and intraperitoneal injection of streptozotocin(STZ)could successfully construct diabetic rats with vascular calcification.(2)After the use of PI3K/AKT inhibitor LY294002 in vivo,it can reduce the calcium content,calcification and collagen expression in aorta,and decrease the elastic fiber and increase the completeness.(3)After the application of GSK-3? inhibitor TWS119 in vivo,the expression of ?-catenin protein was increased and the ? wnt signaling pathway was activated to up-regulate the expression of osteoblast-related proteins,including Runx2,OPG and BMP2.Conclusion:(1)PI3K/AKT GSK-3? signaling pathway is involved in the calcification of the aorta of type 2 diabetic rats and may play a role in the regulation of beta-catenin transcription.(2)The PI3K/AKT GSK-3?/?-catenin signaling pathway may be used as a new target for the treatment of calcification of diabetes mellitus in diabetic rats.