Bone Marrow Mesenchymal Stem Cells Promotes The Recovery Of AD Cell Model Via Platelet Derived Growth Factor-BB Regulating PI3K/AKT Signaling Pathway

Objective:In our previous study,Bone marrow mesenchymal stem cells(BMSCs)transplantation ameliorated the cognitive function of the rats Alzheimer’s disease(AD)model.The platelet derived growth factor-BB(PDGF-BB)was found increased significantly in the hippocampus of rats,after BMSCs transplantation.In this study,we will further explore and elucidate the role of PDGF-BB in BMSCs treatment of AD by cell experiments in vitro.Methods:Firstly,the HIV lentivirus was used to construct an APP695 over expressed lentivirus recombinant to transfect the human neuroblastoma cell line(SH-SY5Y),and then the stable cell line was screened by purinomycin(SH-SY5Y-APP695).After BMSCs and SH-SY5Y-APP695 were co-cultured for 72h through Millicell Hanging Cell Culture Inserts,the morphological changes of SH-SY5Y-APP695 were observed by inverted phase contrast microscope.The cell viability was measured by MTT method.The expression level of Neuronal Class Ⅲ β-Tubulin(Tuj1)was detected by using Immunofluorescence stain.Enzyme linked immunosorbent assay(Elisa)was employed to examine the protein level of β-Amyloid 1-42(Aβ1-42)and Platelet derived growth factor(PDGF-BB).PDGF-BB,Phosphatidylinositol 3-kinase(PI3K),phosphorylated-PI3K(p-PI3K),protein kinase B(AKT),phosphorylated-AKT(p-AKT)was detected by Quantitative real time polymerase chain reaction(Q-PCR)and Western blot respectively.Moreover,the expression of PDGF-BB was interfered by small interfering RNA(siRNA)method,and LY294002,a specific PI3K inhibitor was used to suppress the PI3K signaling pathway,with aim to explore the intensive mechanism involved in BMSCs’ effect on pathology and viability of AD cell model exerted by PDGF-BB and PI3K interaction.Results:1.BMSCs increased the mRNA level of PDGF-BB and improved the cell viability of the Aβ1-42 administrated SH-SY5Y cell.The MTT test showed that the cell viability of Aβ1-42 treated SH-SY5Y cells significantly decreased compared with the sham group(P=0.000<0.01).Compared with the Aβ1-42 group,the cell viability of A Aβ1-42+BMSC group markedly increased(P=0.007<0.01).The results of Q-PCR showed that the level of PDGF-BB mRNA in the Aβ1-42 group was lower than that of the sham group,but there was no statistical significance(P=0.0911>0.05).The PDGF-BB mRNA level in Aβ1-42+BMSC group increased significantly compared with Aβ1-42 group(P=0.001<0.01).2.The over expression of APP695 induced the morphological changes of SH-SY5Y cells,increased the Aβ1-42 expression and decreased the cell viability.The morphological detection revealed that the surface of the cells was rough and the cell protuberance became shorter in the APP group,when compared with the NC group.The results of MTT showed that the APP group presented a lower cell vitality than that of the NC group(P=0.007<0.01);The Q-PCR and Elisa results showed that both the mRNA(P=0.000<0.01)and protein levels of Aβ1-42 in APP group were significantly increased compared with the NC group(P=0.012<0.05).3.BMSCs promoted the cell viability,improved the mRNA and protein levels of PDGF-BB,reduced the level of A P 1-42,and increased the mRNA level of PI3K and AKT in the AD cell model.MTT results indicated that cell viability of APP+BMSC group was significantly increased than that of APP group(P=0.000<0.05).Q-PCR and Elisa results showed that PDGF-BB mRNA(P=0.000<0.05)and protein(P=0.000<0.05)levels in APP+BMSCs group was greatly increased respectively,compared with the APP group.4.After PDGF-BB and PI3K signaling pathway was interfered,the beneficial effects of BMSCs co-culturing about the amelioration of the cell viability and the Aβ1-42 clearance were abolished.The expression level of PI3K and AKT and synapse related protein GAP43 and SYP was substantially reduced.After PDGF-BB was interfered,the morphology of the AD cells in the APP+sh-PDGF-BB+BMSC group was obviously changed,the surface of the cells was rough,the cell protruding became shorter,the MTT result revealed that the cell vitality significantly decreased(P=0.043<0.05)compared with the APP+HSV+BMSC group.In the APP+sh-PDGF-BB+BMSC group,the mRNA(P=0.000<0.05)and the protein level(P=0.000<0.05)of PDGF-BB substantially decreased,the mRNA(P=0.000<0.05)and protein level(P=0.000<0.05)of Aβ1-42 both markedly increased,the mRNA levels of the key molecules of the signaling pathway PI3K/AKT,PI3K(P=0.000<0.05)when compared with the APP+HSV+BMSC group.The p-AKT(P=0.000<0.05),p-PI3K(P=0.000<0.05),GAP43 and SYP protein level greatly decreased respectively,when compared with the APP+HSV+BMSC group.After the inhibition of the PI3K signal pathway,the cell morphology in APP+BMSC+LY294002 group changed obviously,the cell protruding became shorter,relative to the APP +DMSO+BMSC group.The MTT results indicated that the cell vitality significantly decreased(P=0.000<0.05),the mRNA level(P=0.000<0.05)and protein level(P=0.000<0.05)of Aβ1-42 significantly increased.The mRNA and protein levels of PI3K(P=0.000<0.05),AKT(P=0.000<0.05)and p-PI3K(P=0.000<0.05),p-AKT(P=0.000<0.05),GAP43(P=0.000<0.05)and SYP(P=0.000<0.05)all significantly decreased,when compared with the APP+DMSO+BMSC group.Conclusion:In the co-culturing of BMSC and SH-SY5Y-APP695 condition,PDGF-BB significantly reduced the deposition of Aβ1-42 by activating the phosphorylation of PI3K/AKT signaling pathway,and enhanced the protein level of GAP43 and SYP,thus improved the cell viability of the SH-SY5Y-APP695 cell line of AD cell model,ultimately promoted the repair of cell damage in SH-SY5Y-APP695 cell line,a common AD in vitro cell model.