Study On Hypoxic Pretreatment Of OM-MSCs Promotes HMC-3 Autophagy Clearance Of ?-synuclein

OBJECTIVE: Recent studies have found that mesenchymal stem cells(MSCs)have achieved some results in the treatment of Parkinson's disease,but their mechanism of action has not been fully clarified.We cultured human microglia cell line(HMC-3)and neuronal cell line(SH-SY5Y)in vitro by ?-synuclein,to simulate the location of neuronal cells in the black striatum of Parkinson's patients Immune environment;olfactory mucosal mesenchymal stem cells(OM-MSCs)pretreated with hypoxia were co-cultured with the above cells to observe the changes of HMC-3 phenotype,detect the content of ?-synuclein and the changes of corresponding immune factors Apoptosis of SH-SY5 Y,so as to explore the mechanism of hypoxic pretreatment of OMMSCs on the autophagy clearance of ?-synuclein and the protective effect on neurons.METHODS:(1)Acquisition and cultivation of human OM-MSCs;identification of OMMSCs using flow cytometry and immunofluorescence;(2)The ?-synuclein was used to pre-treat HMC-3 to polarize it;the hypoxic device was used to pre-treat OM-MSCs in the hypoxic group.(3)Co-cultivation of HMC-3,SH-SY5 Y and OM-MSCs in vitro using Transwell chamber;(4)After co-cultivation,observe the morphology of HMC-3 in each group;Western Blot and immunofluorescence to detect the content of ?-synuclein,IL-1?,CD206,lysosome and other related markers of HMC-3 in each group;transmission electron microscope The formation of HMC-3 autophagolysosome was observed in each group;the apoptosis of HMC-3 was detected by immunofluorescence and flow cytometry apoptosis.Observe the morphology of HMC-3 in each group after co-cultivation;Western Blot and immunofluorescence to detect the content of ?-synuclein,IL-1?,CD206,lysosome and other related markers of HMC-3 in each group;Transmission electron microscopy was used to observe the formation of HMC-3 autolysosome in each group;immunofluorescence and flow cytometry apoptosis were used to detect the apoptosis of HMC-3.RESULTS:(1)The ?-synuclein-treated HMC-3 is polarized from M2 type antiinflammatory type to M1 type pro-inflammatory type;(2)Normal oxygen OM-MSCs can promote the removal of intracellular ?-synuclein by HMC-3,and the effect of hypoxic pretreatment of OM-MSCs is more significant;(3)Normal oxygen OM-MSCs remove ?-synuclein by promoting the formation of HMC-3 autophagolysosome,and the effect of hypoxic pretreatment of OM-MSCs is more significant;(4)OM-MSCs inhibit the apoptosis of SH-SY5 Y by regulating the autophagy clearance mechanism of HMC-3.CONCLUSIONS:(1)OM-MSCs can reverse M1-type HMC-3 activated by ?-synuclein;(2)OM-MSCs can enhance the ability of HMC-3 to clear ?-synuclein,and its mechanism is related to the formation of autophagolysosomes;(3)OM-MSCs protect SH-SY5 Y and inhibit neuronal apoptosis caused by ?-synuclein;(4)Hypoxia pretreatment can enhance the ability of OM-MSCs to promote the autophagy clearance of ?-synuclein by HMC-3.