| Objective:Investigating the effects of combined therapy of liraglutide and umbilical cord mesenchymal stem cells on the glucose metabolism and islet beta cell apoptosis,then exploring the relation with the ASK1/JNK/BAX singnal pathway.Method:1.According to the random number table method,90 male SD rats were divided into normal control group(NC group,n=8)and model group(n= 80).The rats of normal control group were fed with conventional feed,and the rats of model group were fed with high fat and high glucose diet.After consuming the diet for 8 weeks,rats in model group were intraperitoneally injected with 30 mg/kg of STZ.Three days later,FPG of the rats were measured,the rats with FPG≥11.1 mmol/L three times were considered as type 2 diabetic mellitus rats.The modeling rate is 72%.Then the T2 DM rats were randomly selected and divided into the following four groups: type 2 diabetic mellitus model group(T2DM,n=10),hUC-MSCs treatment group(T2DM+hUC-MSCs,n=10),liraglutide treatment group(T2DM+LIRA,n=10)and hUC-MSCs combined with liraglutide treatment group(T2DM+LIRA+hUC-MSCs,n=10).Each group were given the appropriate intervention 8 weeks respectively.Among them,umbilical cord mesenchymal stem cells intervention group were treated with intravenous infusion of hUC-MSCs by vena caudalis at the first week and fifth week,the cell number were 1×106;umbilical cord mesenchymal stem cells negative group were treated with intravenous infusion of 100μl nutrient solution without umbilical cordmesenchymal stem cells by vena caudalis at the first week and fifth week,liraglutide positive intervention group were treated with subcutaneous injection of liraglutide twice a day,the dose of liraglutide was 200μg/kg;liraglutide negative control group were treated with subcutaneous injection of corresponding doses of saline twice a day.2.In the process of experiment,the general conditions of rats were recorded,fasting blood glucose were tested every week;eight weeks after intervention,glycosylated Hemoglobin(HbA1c)and intraperitoneal glucose tolerance test(IPGTT)were detected;C-peptide,Glucagon-like peptide-1 and glucagon were measured by ELISA.Pancreatic pathological changes were stained with HE,immunohistochemical method was used to detect the expression of insulin and glucagon,qRT-PCR was used to detect the mRNA of ASK1,JNK,Bax,and Bcl-2 in pancreas.Results:1.After treatment for 8 weeks,compared with the NC group,the body weight of T2 DM group were significantly higher than that of NC group(P<0.05),there was no significant difference in T2DM+LIRA and T2DM+LIRA+hUC-MSCs group.(P>0.05).Compared with T2 DM group,the body weight of three treatment groups were significantly decreased(P<0.05).Compared with T2DM+hUC-MSCs treatment group,the body weight of T2DM+LIRA and T2DM+LIRA+hUC-MSCs group were significantly lower than that of T2DM+hUC-MSCs group(P<0.05),there was no significant difference between the T2DM+LIRA group and T2DM+LIRA+hUC-MSCs group(P>0.05).Compared with NC group,the FPG and HbA1 c of T2 DM group were significantly increased.Compared with T2 DM group,the FPG and HbA1 c of three treatment groups were significantly decreased(P<0.05).Compared with T2DM+hUC-MSCs group,the FPG and HbA1 c of T2DM+LIRA group and T2DM+LIRA+hUC-MSCs group were significantly lower than those in T2DM+hUC-MSCs group(P<0.05).Compared with T2DM+LIRA group,the FPG and HbA1 c of T2DM+LIRA+hUC-MSCs group were also decreased(P<0.05).The results showed that the glucose tolerance of T2 DM group was significantly lower than that ofNC group,and the blood glucose level of 0 min was increased,the blood glucose reached the peak at 30 min,and keep hyperglycemia for 120 min,the difference was statistically significant(P<0.05).Compared with T2 DM group,the glucose tolerance of the three treatment groups was significantly improved,the blood glucose were decreased at all time points in three treatment groups(P<0.05).Compared with T2DM+hUC-MSCs group,the blood glucose were decreased at all time points in T2DM+LIRA group(P<0.05),the blood glucose was decreased at 0min,90 min and120 min points in T2DM+LIRA+hUC-MSCs group(P<0.05).Compared with T2DM+LIRA group,the blood glucose was decreased at 120 min points in T2DM+LIRA+hUC-MSCs group(P<0.05).2.After treatment for 8 weeks,the levels of serum C-p,GLP-1 and Glu in rats were measured.Compared with NC group,the levels of serum C-p and GLP-1 in T2 DM group were significantly decreased,but the levels of serum GLu increased(P<0.05).Compared with T2 DM group,serum C-p were increased in T2 DM + hUC-MSCs group(P<0.05),but there was no significant difference in GLP-1 and GLu levels(P>0.05),the levels of serum C-p and GLP-1 were significantly increased in T2DM+LIRA group and T2DM+LIRA+hUC-MSCs group,but the levels of serum GLu decreased(P<0.05).Compared with T2DM+hUC-MSCs group,the levels of serum C-p and GLP-1 in T2DM+LIRA group and T2DM+LIRA+hUC-MSCs group were significantly increased,while the GLu were decreased.(P<0.05).Compared with T2DM+LIRA group,serum C-p and GLP-1 in T2DM+LIRA+hUC-MSCs group were also significantly increased,while the GLu were also significantly decreased(P<0.05).3.After the treatment for 8 weeks,pancreatic pathological changes were stained with HE and observed under light microscope.In NC group,the islets of rats were intact,the number of islet cells were large,the cells were well arranged and the cytoplasm was abundant;compare with NC group,the islets of rats in T2 DM group were disorder and significantly reduce;compared with T2 DM group,islet structure and islet cells in three treatment groups were significangtly ameliorated.Among them,pancreatichistopathological changes in T2DM+LIRA+hUC-MSCs group were improved better than the other two treatment groups.4.After the treatment for 8 weeks,the expression of insulin and glucagon in islet tissue were measured.Compared with NC group,the ratio of insulin positive area in islet tissue was significantly decreased in T2 DM group while the ratio of glucagon positive area in islet tissue was significantly increased(P<0.05);Compared with T2 DM group,the ratio of insulin positive area in islet tissue was significantly increased in the three treatment groups while the ratio of glucagon positive area in islet tissue significantly decreased(P<0.05);Compared with T2DM+hUC-MSCs group,the ratio of insulin positive area in islet tissue was increased in T2DM+LIRA group and T2DM+LIRA+hUC-MSCs group while the ratio of glucagon positive area in islet tissue significantly decreased(P<0.05).Compared with T2DM+LIRA group,the ratio of insulin positive area in T2DM+LIRA+hUC-MSCs were higher than T2DM+LIRA group while the ratio of glucagon positive area in islet tissue were lower than T2 DM + LIRA group(P<0.05).5.After the treatment for 8 weeks,the expression of ASK1,JNK,BAX and Bcl-2mRNA in pancreatic tissue of rats were measured.Compared with NC group,the expression of ASK1,JNK and Bax mRNA in pancreatic tissue of T2 DM group were significantly increased,while Bcl-2 mRNA were significantly decreased(P<0.05).Compared with T2 DM group,the expression of ASK1,JNK and Bax mRNA in the pancreatic tissue of the three treatment groups were significantly decreased,while the expression of Bcl-2 mRNA was increased(P<0.05).Compared with T2DM+hUC-MSCs group,the expression of ASK1,JNK and Bax mRNA in T2DM+LIRA group and T2DM+LIRA+hUC-MSCs group were decreased,while the Bcl-2 mRNA were increased(P<0.05).Compared with T2DM+LIRA group,the expression of JNK and Bax mRNA in pancreatic tissue of T2DM+LIRA+hUC-MSCs group were decreased while the expression of Bcl-2 mRNA were increased(P<0.05),the expression of ASK1 mRNA was not statistically significant(P> 0.05).6.Correlation between FBG and pancreatic tissue ASK1 mRNA,JNK mRNA,apoptosis gene Bax and anti-apoptotic gene Bcl-2 in rats:The FBG was positively correlated with the relative expression of ASK1 m RNA(r=0.837,P<0.01),JNK mRNA r=0.849,P<0.01),Bax mRNA(r=0.894,P<0.01)in pancreas,but negatively correlated with the relative expression of Bcl-2 m RNA(r=-0.812,P<0.01).7.Correlation between pancreatic insulin positive area and pancreatic tissue ASK1,JNK,apoptosis gene Bax and anti-apoptotic gene Bcl-2 in rats: The positive area of pancreatic insulin was negatively correlated with the relative expression of ASK1mRNA(r=-0.861,P<0.01),JNK mRNA(r=-0.904,P<0.01),Bax m RNA(r=-0.920,P<0.01)in pancreas,but positively correlated with the relative expression of Bcl-2 mRNA(r =0.838,P<0.01).Conclusion:1.Liraglutide or umbilical cord mesenchymal stem cells single and the combination of the two intervention could improve glucose metabolism and inhibit pancreatic β-cell apoptosis in type 2 diabetes mellitus rats which were induced by gving A high-fat diet and low-dose streptozotocin intraperitoneal injection;2.Compared with a single therapy of liraglutide or umbilical cord mesenchymal stem cells,the combination of the two intervention could improve glucose metabolism and inhibit pancreatic β-cell apoptosis better in type 2 diabetes mellitus model rats;3.Liraglutide combined with umbilical cord mesenchymal stem cell could improve glucose metabolism and injibit islet β cell apoptosis in a ASK1/JNK/BAX pathway-dependent manner. |
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