Study On Protection Of Mesenchymal Stem Cells-conditioned Medium Against HD-induced Apoptosis In VSC 4.1 Cells And

Objective: The purpose of this study was to investigate whether mesenchymal stem cell supernatant(MSC-CM)attenuates HD-induced VSC4.1 cell apoptosis by NGF / Akt / Bad dependency.Materials and Methods: VSC 4.1 cells,a cell line of dorsal motor neurons were treated with HD alone or 5,10 and 15% MSC-CM.VSC4.1 cells were divided into some groups: Control group,HD group,HD+MSC-CM group? HD+NGF group?HD+MSC-CM/NGF+inhibitor(ab16161,antibody against NGF),k252a(Trk A inhibitor)and MK-2206(Akt inhibitor)).Cells of the Control group were treated with DMEM;HD group were treated with 2,5-HD at a dosage of 25 m M for 24 hours.HD+MSC-CM group were treated with MSC-CM for 24 hours after being exposed to HD for 24 hours.HD+MSC-CM+inhibitor groups were pretreated with inhibitor for 1 hour before adding the supernatant.TUNEL assay was used to detect the cell apoptosis in VSC4.1 cells.ELISA was used to detect the concentration of NGF in MSC-CM.The alterations of Trk A,p-Trk A,Akt,p-Akt,Bad,p-Bad and cytochrome c proteins in spinal cord were detected by Western-Blotting.The activity of Caspase-3 was detected by activity kit.Distribution of cytochrome c in VSC4.1 cells was detected by Immunofluorescence double staining.The experimental results were analyzed by using SPSS19.0 statistical package.Results: The number of TUNEL positive cells in HD group was significantly higher than that in Control group(P < 0.05).The number of TUNEL positive cells in HD + MSC-CM group was significantly lower than that in HD group(P < 0.05),With the increase of MSC-CM concentration,TUNEL positive cells were getting lower and lower(P < 0.05).The apoptotic index of HD + MSC-CM group was 42%,30% and 17% respectively,which was significantly lower than that of HD group(P < 0.05).The level of OD in HD group was significantly lower than that in HD group(P < 0.05),and the OD of HD + MSC-CM group was significantly higher than that of HD group(P < 0.05),Increased dependency.There was a significant negative correlation between the quantification of cell viability and the apoptotic index of TUNEL,and the correlation coefficient was 0.8554.The activity of NGF in MSC-CM and the activation of its receptor were detected.The NGF content in the supernatant of the third generation MSCs was 66.08 pg / ml.The expression of p-Trk A protein in HD group was significantly lower than that in HD group(P < 0.05).The expression of p-Trk A protein in HD + MSC-CM group was significantly higher than that in HD group(P <0.05).Compared with the control group,the expression of Trk A protein in each group was not statistically significant(P > 0.05).The number of TUNEL positive cells in HD group was significantly higher than that in control group(P < 0.05).The number of TUNEL positive cells in HD + MSC-CM group was significantly lower than that in HD group(P < 0.05),But the protective effect of MSC-CM was significantly decreased after NGF neutralizing antibody(ab16161)was added(P < 0.05).Caspase-3 activity was also true.The expression of p-Akt and p-Bad protein in HD group was significantly lower than that in Control group(P < 0.05).The expression of p-Akt and p-Akt in HD + MSC-CM group was significantly higher than that in control group(P < 0.05),but the expression level of p-Akt and p-Bad increased by MSC-CM was reversed after the addition of Trk A receptor inhibitor(k252a)(P < 0.05).Cyt C was distributed in the mitochondria in the control group.The Cyt C of the HD group was diffuse in the whole cell,and the color was much higher than that of the control group.The distribution of Cyt C in MSC-CM group was similar to that of Control group.The expression of Cyt C protein in the mitochondria of HD group was significantly lower than that in the control group(P < 0.05),but the expression of Cyt C protein in the cytoplasm was opposite.The HD + MSC-CM group reversed this situation.The expression of Cyt C and the expression level of mitochondria and cytoplasm in HD + NGF group were similar to those in HD + MSC-CM group.However,after adding K252 a,The role of MSC-CM and NGF was reversed(P < 0.05).The number of TUNEL positive cells in HD group was significantly higher than that in HD group(P < 0.05).The number of TUNEL positive cells in HD + MSC-CM group was significantly lower than that in HD group(P < 0.05),but the protective effect of MSC-CM and NGF was significantly decreased after the addition of Akt inhibitor(MK-2206)(P < 0.05).Electron microscopy and Caspase-3 activity results were also true.Conclusion: MSC-CM can repress HD-induced mitochondria-dependent apoptosis of VSC4.1 cells via activating NGF/Akt/Bad pathway and NGF may be responsible for the protection of MSC-CM against HD-induced apoptosis.