Experimental Study On The Effects Of Liraglutide Combined With Insulin On Fracture Healing In Streptozotocin-induced Diabetic Rats

Part One Experimental study on fracture healing in streptozotocin(STZ)-induced diabetic ratsObjective:Establishing the type 2 diabetes mellitus model,which used an 8-week high-fat diet(HFD)combined with intraperitoneal low-dose(35 mg/kg)STZ.Femur fracture healing in this model were evaluated by X-ray,micro-CT,and histomorphological and immunohistochemical staining.Methods:1.Twenty male 2-month-old Sprague Dawley rats were raised in a barrier laboratory environment.Six rats were in the simple fracture group(control fracture,CF group)and the remaining rats were prepared as type 2 diabetes fracture models(diabetes mellitus and fracture,DF group).2.During the observation period,blood glucose of all rats were determined weekly.Six weeks after establishing the fracture,specimens were taken for micro-CT examination of the bone mineral density(BMD),trabecular separation(TB.SP),and trabecular number(TB.N).X-ray examination was used to evaluate the fracture healing status,and safranine O/fast green staining was applied to analyze the proportion of bone structure to total callus.Immunohistochemical staining was performed to analyze beta-catenin expression in the callus.Results:1.Body weight and blood glucose.Following intraperitoneal STZ injection,the rats appeared spiritually malaise and displayed withered hair,polydipsia,polyuria,weight loss,and other symptoms.Approximately one week after establishment of the closed femoral fracture,the rats’ activity increased,the claudication status had essentially resolved,and the surgical incision had healed smoothly.Before conducting the modeling experiment,there was no significant difference in blood glucose levels among the rats.After 8 weeks of feeding with the HFD,the non-fasting blood glucose level was increased compared with the control group.After intraperitoneal STZ injection,the blood glucose levels were statistically significantly increased(P<0.05).2.X-ray analysis:The resultant score of the DF group was lower than that of the CF group,but this difference was not significant between the two groups(P>0.05).3.Micro-CT test results:Micro-CT software analysis results showed that at 6 weeks after the femoral fracture,the BMD,BV/TV,and TB.N in the callus of the DF group were statistically significantly lower than those in the CF group,while the TB.SP in the DF group was statistically significantly higher than that in the CF group(P<0.05).4.Results of safranine O-fast green staining:At 6 weeks after the femoral fracture,fusiform callus connections were established in both the CF and DF groups,and the red-stained chondrocytes were essentially absent and replaced by woven bone.The bone tissue area to total callus area was significantly lower in the DF group than that of the CF group(P<0.05).5.Results of immunohistochemical staining for beta-catenin:The IOD method was applied to analyze the immunohistochemical beta-catenin staining intensity.The expression intensity in the callus of beta-catenin in the DF group was statistically significantly lower than that in the CF group(P<0.05).Part Two Effect of liraglutide combined with insulin on fracture healing in STZ-induced diabetic ratsObjective:To establish a type 2 diabetes mellitus rats’ model with closed femoral fracture,and insulin and/or liraglutide were injected subcutaneously.We aimed to investigate the effects of a combination of liraglutide and insulin on fracture healing in a rat model of diabetes and the mechanisms involved.Methods:1.A total of 30 healthy male 2-month-old Sprague Dawley rats were raised in a barrier laboratory environment.The animals were divided into five groups for the experiment:simple fracture group(CF group,n=6),diabetic fracture group(DF group,n=6),diabetic fracture,insulin treated(DFI group,n=6),diabetic fracture,liraglutide treated(DFL group,n=6),diabetic fracture,insulin and liraglutide treated(DFIL group,n=6).2.Blood glucose and body weight were determined weekly in all rats.Six weeks after establishing the femoral fracture,specimens were taken for micro-CT examination,X-ray examination,Safranine O-fast green staining,TRAP staining,ALP staining and the immunohistochemical staining of type I collagen(COL I)and osteocalcin(OCN)in the callus.Results:1.General state of the animals:Before the experiment,there were no significant differences in the body weights and blood glucose of the rats.After 8 weeks of feeding with a HFD,the weights and blood glucose of these rats increased and were significantly higher compared with the CF group(P<0.05).Following intraperitoneal STZ injection,the rats appeared spiritually malaise,and displayed withered hair,polydipsia,polyuria,weight loss,and other symptoms.After intraperitoneal STZ injection,the blood glucose level increased significantly.The fasting blood glucose of rats in the DFI,DFL and DFIL groups was significantly decreased.The blood glucose level of the rats in the DFIL group was significantly lower compared with the DFI group(P<0.05).After intraperitoneal STZ injection,the body weights of the rats were decreased.The body weight began to increase approximately 3 weeks after STZ injection,and subsequently increased gradually.The weight of rats in the CF group increased gradually.By the end of the experiment,the weight of rats in the CF group was statistically significantly higher than that in the other groups(P<0.05).Approximately one week after the establishment of a closed femoral fracture,the rats’ activity increased,the claudication status had essentially resolved,and the surgical incision healed smoothly.2.Results of the micro-CT analysis of bone callus microstructure:At 6 weeks after establishing a femoral fracture model,the callus BMD,BV/TV,and TB.N in the DF group were significantly lower than those in the CF group(P<0.05).The BMD,BV/TV,and TB.N in the DFI,DFL,and DFIL groups were significantly higher than those in the DF group,while the TB.SP was significantly lower than that in the DF group(P<0.05).The TB.N in the DFIL group was significantly higher than that in the DFI group(P<0.05).On the three-dimensional reconstruction images and sagittal section view of the bone morphology,it could be observed that the fracture types were horizontal or short oblique.In the CF group,the bone trabeculae were sparse in the callus,and no significant continuous bone trabeculae had formed.3.X-ray score results:X-ray photography showed that the fractures in each group displayed fusiform callus formation at 6 weeks after the fracture.The callus in the DFI,DFL,and DFIL groups was relatively dense,and the fracture line was blurred or even absent.The fracture line in some rats in the DF group remained clear.However,there were no significant differences in the X-ray scores among the groups.4.Staining results of safranine O-fast green and for alkaline phosphatase and osteoclast TRAP of the callus site:The area of bone tissue to total callus in the CF group was significantly higher than that of the other groups(P<0.05).There were no significant differences in the bone tissue area to total callus area among the DF,DFI,and DFL groups.The bone tissue area to total callus area in the DFIL group was significantly higher than in the DFI group(P<0.05).The ALP staining intensity in the callus of rats in the DF group was significantly lower than that of the other groups(P<0.05).The ALP staining intensity in the callus of the DFIL group was significantly higher than that of the DFI group(P<0.05).The TRAP staining intensity in the DFL,DFI,and DFIL groups were significantly lower than that in the DF group(P<0.05),and the TRAP staining intensity in the DFIL group was significantly lower than that in the DFI group(P<0.05).5.Immunohistochemical staining results for COL Ⅰ and OCN in the femoral callus:The expression intensities of OCN and COL Ⅰ in the callus of rats in the DFI,DFL,and DFIL groups were significantly higher than that in the DF group(P<0.05).The OCN and COL Ⅰ expression intensity in the callus of rats in the DFIL group were higher than that in the DFI group(P<0.05).Part Three Study on the effect of liraglutide on MC3T3-E1 cell differentiation into osteoblasts under the action of high glucoseObjective:To observe the effects of different D-glucose concentrations on MC3T3-E1 cell differentiation in vitro.The effects of different liraglutide doses on MC3T3-E1 cell differentiation and its protective effect on high glucose-stimulated MC3T3-E1 cells were observed.Methods:1.MC3T3-E1 cells purchased from the cell bank of Shanghai Chinese Academy of Sciences were used for in vitro experiments.After cell culture and passage,the specific groups were as follows:Different D-glucose concentrations:Control group:cells cultured in α-MEM basic medium for 24 hours;D-glucose groups:cells cultured in α-MEM basic medium and induced with D-glucose at a final concentration of 5.5,11.2,or 22.4 mM for 24 hours.Different concentrations of liraglutide:Control group:cells cultured in α-MEM basic medium for 24 hours;liraglutide groups:cells cultured inα-MEM basic medium and induced with liraglutide at a final concentration of 10-5,10-4,or 10-3 mM for 24 hours.Treatment and administration group:Control group:cells cultured in α-MEM basic medium for 24 hours;Group L:cells cultured in α-MEM basic medium and induced with liraglutide at a final concentration of 10-3 mM for 24 hours;Group G:cells cultured in α-MEM basic culture medium induced with D-glucose at a final concentration of 22.4 mM for 24 hours;Group G+L:cells cultured in α-medium and induced with liraglutide at a final concentration of 10-3 mM for 1 hour followed by D-glucose at a final concentration of 22.4 mM for 24 hours.2.Alizarin red staining was performed to evaluate the area of calcified nodules in cells,and Image-Pro Plus software was used to observe and calculate the cell red IOD value.An immunoblotting method was used to determine BMP2,SP7,OCN,COL Ⅰ,β-catenin,and α-tubulin.Image-Pro Plus software was used to observe and calculate the grayscale values.Results were compared with the α-tubulin expression level.Results:1.Alizarin red staining:MC3T3-E1 cells were pretreated with liraglutide and induced by D-glucose.The results of alizarin red staining showed that compared with the control group,only a small number of red-stained osteoblasts differentiated and formed in the D-glucose stimulation group,and no mineralized nodules were observed.Compared with the D-glucose-induced group,in the D-glucose and liraglutide group,increased osteoblast differentiation and mineralized nodules were observed.The IOD value was calculated and the difference was statistically significant by analysis of variance(ANOVA)(P<0.05).2.Stimulation of MC3T3-E1 cells with different glucose concentrations:Compared with the control group,the protein expression level was significantly decreased in a dose-dependent manner,and the difference was significant by ANOVA(P<0.05).3.Different concentrations of liraglutide stimulated MC3T3-E1 cells:Compared with the control group,the BMP2,SP7,OCN,COL Ⅰ,and β-atenin protein levels were significantly upregulated in a dose-dependent manner by liraglutide,and the differences were significant by ANOVA(P<0.05).4.After pretreatment with liraglutide,MC3T3-E1 cells were stimulated with D-glucose:Compared with the control group,the D-glucose induced group showed significantly decreased BMP2,SP7,OCN,COL I,andβ-catenin protein levels.Compared with the D-glucose induction group,the BMP2,SP7,OCN,COL Ⅰ,and β-catenin protein expression levels of the liraglutide and D-glucose groups were all statistically significant upregulated by ANOVA(P<0.05).Conclusions:1.A HFD combined with a small-dose STZ intraperitoneal injection could successfully establish a diabetes type 2 rat model,and bone histomorphology analysis confirmed that the diabetic rats displayed delayed fracture healing.Furthermore,β-catenin in the callus was significantly reduced,suggesting that this may be associated with delayed fracture healing.2.At the observed time point and drug intervention dose,the fracture healing in diabetic rats was delayed.Insulin or liraglutide alone could improve the fracture-healing process.The combination of liraglutide and insulin displayed better blood glucose control and thus a greater ability to promote fracture healing in STZ-induced diabetic rats than with either alone.3.D-glucose stimulation inhibited osteoblast differentiation and mineralization,and attenuated osteoblast markers BMP2,SP7,COL I,OCN,and β-catenin.Liraglutide reduced osteoblast damage caused by D-glucose stimulation,promoted osteoblast differentiation and mineralization,and increased osteoblast markers BMP2,SP7,COL Ⅰ,OCN,and β-catenin.