| Research purposes:To test the effects of Exendin-4 on bone mineral density,strength and morphology in diabetic mice,and to explore the prevention and treatment effect of Exendin-4 on diabetic bone loss and its possible mechanism.Experimental method:Thirty 12-week-old male ICR mice were fed adaptively for one week and randomly divided into experimental group(20 mice)and control group(10 mice).The experimental group was fed with high glucose and high fat diet for 8 weeks,and then intraperitoneal injection with streptozotocin(STZ,75mg/kg)to establish the animal model of diabetic.Then they were randomly divided into diabetic group(DM group)and Exendin-4 drug treatment group(Ex-4 group,8μg/(kg·d))for 9weeks.Mice fed with normal diet were used as control group(NC group).The body weight and random blood glucose level of mice were measured weekly.Bone mineral density test,biomechanical properties test,tissue sections Masson-Goldener trichrome staining,and bone histometric analysis were performed.One mouse in each group was selected,and BMSCs were isolated and cultured according to the whole bone marrow adherent method.BMSCs were cultured to the third generation,and divided into the following three groups: 1)blank control group(NC group),2)diabetic group(DM group),3)Exendin-4 treatment group(Ex-4 group;Exendin-4,1μM).The differentiation and mineralization of BMSCs into osteoblasts induced by Exendin-4 were observed by alizarin red staining and alkaline phosphatase activity(ALP).Western blot was used to observe the effect of Exendin-4 on osteogenic differentiation and the regulatory effect of Exendin-4 on Wnt/β-Catenin pathway.Result:1.After STZ modeling,the random blood glucose of mice in the DM group increased rapidly at the 8th week and reached a relatively stable level after 12 weeks.Blood glucose levels in the Ex-4 group peaked at week 12 and then continued to decline,with significantly lower blood glucose levels than those in the DM group.Exendin-4 can reduce the blood glucose level in diabetic mice.2.The BMD,maximum pressure,flexural stiffness,compression stiffness and elastic modulus of the right tibia of mice in the DM group were significantly lower than those in the NC group,and Exendin-4 treatment could significantly improve the above parameters.3.Bone histomorphological analysis indicated that the number,thickness,girth,area and volume of bone trabeculae in DM group were significantly decreased compared with normal mice,while the space between bone trabeculae was significantly increased.Exendin-4 treatment could significantly improve the bone morphology of diabetic mice4.Alizarin red staining indicated that the formation rate of calcified nodules in DM group was significantly lower than that in NC group.Compared with the DM group,the formation rate of calcified nodules was significantly increased in the Ex-4group.Alkaline phosphatase activity assay indicated that compared with the NC group,the ALP activity in the DM group was significantly decreased during osteogenesis,while the ALP activity in the DM group was significantly increased after the treatment with Exendin-4.Exendin-4 intervention can improve the osteogenic differentiation of bone marrow mesenchymal stem cells in DM group.5.Western blot showed that Wnt3 a,β-Catenin and Runx2 protein expressions were significantly down-regulated in the DM group compared with the NC group(P<0.05).Exendin-4 treatment significantly increased the expression of Wnt3 a,β-Catenin and Runx2(P<0.05).Conclusion:Exendin-4 can promote the osteogenic differentiation of BMSCs in mice by up-regulating the protein expression of important molecules in the Wnt/β-Catenin signaling pathway,increase the bone density and bone strength of mice,improve the bone tissue morphology,and play an important role in the prevention and treatment of bone loss in diabetic mice. |
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