Neuroprotective Effects And Mechanism Of Skin-derived Precursor Schwann Cells On Parkinson’s Disease Model

Objective Parkinson’s disease(PD)is the second most common neurodegenerative disease related to aging,secondly only to Alzeheimer’s disease(AD).Parkinson’s clinical manifestations are mainly bradykinesia,resting tremor,muscular rigidity and abnormal posture gait.Since the 1990 s,extensive experimental research on stem cells have brought new direction to the treatment of Parkinson’s disease.This study was aimed to investigate the possible neuroprotective effects of Skin-derived Precursor Schwann cells(SKP-SCs)in 6-OHDA-induced Parkinson’s disease(PD)model.And to study whether SKP-SCs can play a role in promoting survival and inhibiting apoptosis through the PI3K/AKT/Bcl-2 signaling pathway.Furthermore,to analyze the conditioned medium of SKPSCs by mass spectrometry,and select specific components that may play a protective role,which provides a new theoretical basis for the treatment of PD.Methods We cultured and purified SKP-SCs and detected the expression of specific markers by immunostaining test.Then,we collected conditioned medium of SKP-SCs for subsequent experimental research.6-OHDA treatment of SH-SY5 Y cells was used to establish a PD cell model,which was divided into four groups:control group,6-OHDA injury group,6-OHDA + control medium group,6-OHDA + SKP-SCs conditioned medium group;CCK-8 test and Ed U test were used to detect the effect of the survival and proliferation of SH-SY5 Y cells;Western blotting test was used to detect the activated caspase-3 expression level,TUNEL test and livedead cell staining method were used to reflect the apoptosis levels of SH-SY5 Y cells;Western blotting experiments were used to detect the expression levels of PI3K/AK/Bcl-2 signaling pathway-related proteins in different groups,and JC-1 staining was used to detect changes in mitochondrial membrane potential;SKP-SCs conditioned medium was analyzed by mass spectrometry and KEGG pathway analysis to find molecules that may exert neuroprotective effects.Enzyme linked immunosorbent assay(ELISA)was used to detect insulin-like growth factor-2(IGF-2)content.Results1.SKP-SCs were identified by immunofluorescence as overexpressing their specific biological markers just like S100,GFAP,and P75,,which were consistent with the phenotypic characteristics of SKP-SCs.2.The cell viability reaches forty percent at 50μM 6-OHDA which was measured by CCK-8 assay,so this concentration was used for subsequent experiments.3.It was observed that the SH-SY5 Y cells in the control group which present normal morphology,round cell bodies,intact cell membranes and good neuron elongation under microscope.After 6-OHDA injury,neuronal processes of SH-SY5 Y cells disappeared,and vacuoles appeared in the cytoplasm.However,the morphology of SH-SY5 Y cells in the SKP-SCs group did not change significantly.Compared with the 6-OHDA injury group,SKP-SCs increased the survival rate of SH-SY5 Y cells and promoted its proliferation effect(*P<0.05).4.Compared with the control group,the expression of activated caspase-3 protein in the 6-OHDA group was significantly increased.However,SKP-SCs significantly reduced the expression of activation caspase-3 protein compared with the control medium group.Similarly,SKP-SCs can significantly reduce the ratio of positive and dead cells and TUNEL positive cells(**P<0.01).5.The mitochondrial membrane potential was higher in the control group,but it was significantly reduced in the 6-OHDA model group.However,SKP-SCs could reverse the changes of mitochondrial membrane potential in SH-SY5 Y cells(*P<0.05).6.Western blotting experiments showed that phosphorylated PI3K(p-PI3K),phosphorylated AKT(p-AKT),and Bcl-2 protein levels were expressed low in the 6-OHDA model group compared with the control group;but SKP-SCs up-regulated p-PI3 K,p-AKT,and Bcl-2 proteins expression(**P<0.01).7.Mass spectrometry analysis of SKP-SCs conditioned medium showed that compared with the control medium,a total of 43 differential proteins existed only in SKP-SCs,and the KEGG pathway analysis of these 43 differential proteins suggested that : the PI3K/AKT signaling pathway is likely to occupy a dominant position,which is involved in cell survival and proliferation.The PI3K/AKT signaling pathway involved six proteins,including insulin-like growth factor 2(IGF-2),collagen,laminin-gamma 1,laminin-beta 1,and Reelin.Only IGF-2 is an extracellular protein,which suggests that it is most likely secreted by SKP-SCs.In addition,the results of our ELISA experiments also confirmed that the content of IGF-2 in the SKP-SCs group was significantly higher than that in the control medium group.Conclusions1.SKP-SCs can promote the survival and proliferation of 6-OHDA-injured SH-SY5 Y cells.2.SKP-SCs can inhibit 6-OHDA-induced apoptosis of SHSY5 Y cells.3.The protective effect of SKP-SCs on SH-SY5 Y cells is related to the PI3K/AKT signaling pathway.4.SKP-SCs are likely to exert protective effects on 6-OHDAinjured SH-SY5 Y cells by secreting IGF-2 and other factors.