Study On The Repairing Mechanism Of Human Bone Marrow Mesenchymal Stem Cells Derived Exosomes In Oxidative Stress Injury Of SH-SY5Y Cells

Objective:To explore the repairing mechanism of bone marrow mesenchymal stem cell-derived exosome（h BMSCs-Exo）in oxidative stress injury of SH-SY5Y cells.To provide new ideas and therapeutic measures for clinical treatment of central nervous system（Central Nervous System,CNS）injury and its related sequelae.Methods:SH-SY5Y cells were divided into 6 groups:control group with equal volume phosphate buffer（Control group）,different concentrations of 1-methyl-4-phenylpyridine ion（MPP⁺group）（0.25、0.5、1、2、4 mmol/L）,0.25mmol/L MPP⁺+5mmol/LN acetylcysteine group（MPP⁺+NAC group）,0.25mmol/L MPP⁺+human bone marrow mesenchymal stem cell conditioned medium group（MPP⁺+h BMSCs-CM group）,0.25mmol/L MPP⁺+h BMSCs-Exo group（MPP⁺+h BMSCs-Exo group）,0.25mmol/L MPP⁺+Culture supernatant of human bone marrow mesenchymal stem cells without exocrine（MPP⁺+h BMSCs-CMexo-group）.The ability of cell proliferation,intracellular ROS content,apoptosis and related protein expression were detected by MTT,flow cytometry and Western Blot respectively.The experimental results of each group were analyzed,and the mechanism of h BMSCs-Exo protecting neurons against oxidative stress injury was discussed.Results:1.With the increase of MPP⁺concentration and treatment time,the survival rate of SH-SY5Y cells decreased gradually.There were significant differences between the concentration gradients in 24 h group and 48 h and 72 h groups（p<0.05）.There was no significant difference between 48h group and 72h group under each concentration gradient（p>0.05）.The intracellular ROS content and apoptosis rate in MPP⁺group were significantly higher than those in control group（p<0.05）.2.Compared with MPP⁺group,the cell activity of MPP⁺+NAC group recovered significantly,and the intracellular ROS content and apoptosis rate decreased significantly compared with MPP⁺group（p<0.05）.3.Compared with MPP⁺group,the cell survival rate of MPP⁺+h BMSCs-CM group recovered significantly,and the intracellular ROS content and apoptosis rate decreased significantly（p<0.05）.4.Compared with MPP⁺group,there was no significant difference in cell survival rate,intracellular ROS content and apoptosis rate in MPP⁺+h BMSCs-CM^exo-group（p>0.05）.5.Compared with MPP⁺group,the cell survival rate of MPP⁺+h BMSCs-Exo group was significantly restored,the intracellular ROS content and apoptosis rate were significantly decreased,the mitochondrial membrane potential was significantly increased,and the cell LDH release was significantly decreased.Compared with the control group,the expression of apoptosis-related protein Bax in MPP⁺group increased,while the expression of proliferation-related protein p-Akt and anti-apoptosis-related protein Bcl-2 decreased.Compared with MPP⁺group,the expression of Bax decreased and the expression of p-Akt and Bcl-2 increased in MPP⁺+h BMSCs-Exo group.Conclusion:1.MPP⁺can increase the content of reactive oxygen species in SH-SY5Y cells,promote apoptosis and inhibit cell survival.2.h BMSCs-Exo can inhibit the increase of intracellular reactive oxygen species induced by MPP⁺,maintain mitochondrial membrane potential,reduce LDH release and apoptosis,and improve the survival rate of SH-SY5Y cells.3.h BMSCs-Exo can activate AKT pathway,up-regulate the expression of Bcl-2protein and decrease the expression of Bax protein in SH-SY5Y cells.