Mechanism Of Liraglutide On Proliferation, Apoptosis And Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells

Objective:1. To investigate the effects of liraglutide(Lira) on proliferation, apoptosis of human umbilical cord mesenchymal stem cells(h UC-MSCs) in vitro and to compare the effects of liraglutide and glucagon like peptide-1(GLP-1). 2. To investigate the effects of liraglutide on proliferation, apoptosis of h UC-MSCs in vitro and to explore the relationship with the PI3K/Akt pathway. 3. To investigate the effect of liraglutide on differentiation of h UC-MSCs in vitro.Meathods:1. HUC-MSCs were seeded in 25cm2 culture flask, and then changed the medium every 2-3days. When the cell confluency reached 60-70%, the experimental groups were divided into the control group, GLP-1 groups and Lira groups. The GLP-1 and Lira groups were divided into three concentration of 10-9, 10-8, 10-7mol/L by the 1:10 proportional respectively. After cultured with GLP-1 or liraglutide for 24 h or 48 h, compare Liraglutide and GLP-1 with proliferation measured by CCK-8 assay and poptosis rate assessed using Annexin V‐FITC/PI flow cytometry. 2. HUC-MSCs were seeded in 25cm2 culture flask. When the cell confluency reached 60-70%,the experimental groups was divided into the control group, Lira group, LY group and Lira+LY group.Lira group were incubated with liraglutide for 24 h.LY group were incubated with LY294002 for 24 h. Before incubated with liraglutide for 24 h Lira+LY group were incubated with 20μmol/L LY294002 for 2h.To detect the concentration of cyclic ydenosine monophosphate(c AMP) by ELISA and detect the expression of p-Akt, Akt protein by western blot technology.3. P5 cells of h UC-MSCs were induced into IPCs by high glucose, nicotinamide and liraglutide for three protocols in vitro. Firstly, the cells were cultured in HG-DEME medium containing 25mmol/L high glucose for 10 days; Secondly, the cells were cultured in LG-DEME medium with 10 mmol/L nicotinamide for 7 days.Finally the cells were cultured in LG-DEME medium with 10-8mol/L liraglutide for 7 days.The morphology of cells was observed by inverted microscope.The induced cells were confirmed by dithizone(DTZ) staining. While the insulin level that was cultured with or without glucose was measured by ELISA. The expression of PDX-1 and Insulin protein in each induced phage cells were detected by western blot.Results:1. After cultured with GLP-1 or Lira for 24 h,The result showed that the proliferation GLP-1 10-9mol/L,10-8mol/L,10-7mol/L and Lira 10-8mol/L,10-7mol/L concentration groups were higher than the control group(P<0.05), the apoptosis rate were lower than the control group(P<0.05). Group comparison of GLP-1 concentration groups showed that the differences of proliferation was not statistically significant(P>0.05); The apoptosis rate of GLP-1 10-8mol/L and 10-7mol/L concentration groups were lower than the10-9mol/L concentration group(P<0.05); The apoptosis rate of GLP-1 10-7mol/L concentration group were lower than the GLP-1 10-8mol/L concentration group, but the difference was not statistically significant(P>0.05). Group comparison of Lira concentration groups showed that the proliferation and apoptosis rate were statistically significant(P<0.05); Then the highest growth rate and lowest apoptosis rate was Lira 10-7mol/L concentration group.After 48 h,the GLP-1 10-9mol/L,10-8mol/L and Lira 10-9mol/L concentration groups compared with the respective control group,the difference was not statistically significant(P>0.05); The proliferation of GLP-1 10-7mol/L and Lira 10-8mol/L,10-7mol/L concentration groups were higher than the respective control group(P<0.05); The apoptosis rate of different GLP-1 and Lira concentration groups were lower than the respective control group(P<0.05); Group comparison of GLP-1 concentration groups showed that the GLP-1 10-8mol/L group compared with the GLP-1 10-9mol/L,the difference was not statistically significant(P>0.05);the proliferation of GLP-1 10-7mol/L concentration groups were higher than the GLP-1 10-9mol/L concentration group(P<0.05); the GLP-1 10-7mol/L group compared with the GLP-1 10-8mol/L,the difference was not statistically significant(P>0.05);The proliferation of Lira 10-8mol/L and 10-7mol/L concentration groups were higher than the Lira 10-9mol/L concentration group(P<0.05); Compared with Lira 10-8mol/L concentration group, the proliferation of Lira 10-7mol/L concentration group increased, but the difference was not statistically significant(P>0.05). Group comparison of GLP-1 and Lira concentration groups showed that the apoptosis rate was statistically significant(P<0.05). The proliferative activity of the same concentration group at different time points,the proliferative activity of cultured with Lira for 48 h compare was lower than the proliferative activity of cultured with Lira for 24h(P <0.05). 2. After the intervention with h UC-MSCs for 24 h, compared with the control group, the concentration of c AMP in Lira group was increased significantly(P<0.05),LY group significantly decreased(P<0.05),Lira+LY group decreased,but the differences was not statistically significant(P>0.05); Compared with the Lira group, the concentration of c AMP in LY group and Lira+LY group was decreased significantly(P<0.05).Compared with LY group, the concentration of c AMP in Lira+LY group was increased significantly(P<0.05). The results showed that,after the intervention with h UC-MSCs for 24 h, compared with the control group,the expression of P-AKT protein in Lira group was increased(P<0.05), but the LY group was decreased(P<0.05).While compared with LY group, the expression of P-AKT protein in Lira+LY group was increased. 3.The morphological features of the cells were observed under inverted microscope.The morphology of h UC-MSCs was spindled before induction. After the first induction phase,the cells clustered. After the second phase, the h UC-MSCs organized into spheroid structures while the h UC-MSCs began to form suspended clusters. Finally, after the third phase,there were more suspended clusters than before. DTZ-stained cellular clusters appeared after the second phase and became more apparent after the third phase. There was little insulin in supernatants fluid in before induction and released gradually increased after induction. The insulin didn’t increased when added glucose or not in before induction and first phase.(P>0.05). In contrast, the cells had a markedly response to the high glucose at the end of the second and third phase(P<0.05). The expression of PDX-1 and Insulin protein were negative before induction,but after induction the expression of PDX-1 and Insulin protein was increased gradually.Conclusions:1. Liraglutide and GLP-1 could promotes proliferation and inhibit the apoptosis of h UC-MSCs in a dose-dependent manner, while the mechanism may be activation of the PI3K/Akt signaling pathway. 2. Liraglutide could increase c AMP concentration of h UC-MSCs, and block the inhibitory effect on the c AMP concentration of LY294002 in h UC-MSCs. 3. HUC-MSCs could be induced into IPCs by high glucose, nicotinamide and liraglutide in vitro.