Effect Of Human Umbilical Cord MSC-derived Exosome(hucMSC-Ex) On Aerobic Metabolism Of ARPE-19 In Vitro

ObjectTo study the effect of human umbilical cord MSC?derived exosome?hucMSC?Ex?on aerobic metabolism of human retinal pigment epithelial cell?ARPE-19?induced by cobalt chloride,and to explore the protective mechanism of hucMSC?Ex on oxidative damage of ARPE-19 cells.Methods1.The hucMSC?Ex were extracted by the kit method;hucMSC?Ex were identified by transmission electron microscope?TEM?,and the marker proteins of hucMSC?Ex were identified by western blot.2.The CCK-8 method was used to establish CoCl₂-induced ARPE-19 cell oxidative injury model.3.The experimental groups:1)normal control group?normally cultured ARPE-19 cells?;2)non-intervention group?After CoCl₂ damages the cells,the original medium is not changed and the culture is continued for 24 hours?;3)different concentrations of hucMSC?Ex intervention Group?After CoCl₂ damages the cells,the original medium is discarded,and new culture medium with exosomal concentrations of 0ug/ml,25ug/ml,50ug/ml,and 100ug/ml is added to continue the culture for 24 hours?;the JC-1 method was used for detection.The mitochondrial membrane potential of each group of ARPE-19cells was determined by spectrophotometric method.The activity of aerobic enzymes?respiratory chain complex I,III,IV,V?and ATP synthesis in ARPE-19 cells were detected.Results1.using SBI ExoQuick-TC kit method to extract human umbilical cord mesenchymal stem cell exosomes;hucMSC?Ex were observed under transmission electron microscope as circular and elliptic membranous vesicles with diameters between 40-100 nm;Western blot results showed that hucMSC?Ex expressed specific marker proteins CD63 and CD9;2.CCk-8 assay for cell viability The results showed that ARPE-19 cells treated with100ug/ml?93.38±4.28%?,400?mol/L?50.71±2.33%?and 800?mol/L?25.06±3.75%?CoCl₂ concentrations.The survival rate was statistically different from that of the control group?CoCl₂ 0?mol/L?,P<0.05.When the concentration of CoCl₂ was 500?mol/L,the ARPE-19 cell survival rate was 50%,which was the best oxidative damage concentration.3.1)Respiratory respiratory chain complex I enzyme activity?nmol/min/10^4 cell?:100ug/ml exosomal group?0.066±0.00?>untreated group?0.052±0.052?>50ug/ml exosomes group?0.026±0.052?>25ug/ml exosomes group?0.018±0.052?=normal cell group?0.018±0.052?>0ug/ml exosomal group?0.015±0.00?;2)Respiratory chain complex III enzyme activity?nmol/min/10^4 cell?:untreated group?0.025±0.00?,0ug/ml exosomes group?0.025±0.00?,25ug/ml exosomes group?0.015±0.035?,50ug/ml exosomes group?0.025±0.00?and 100ug/ml exosomes group?0.025±0.00?,no significant difference between these groups,and both of them are lower than normal cell group?0.062±0.018?;3)Respiratory chain complex IV enzyme activity?nmol/min/10^4 cell?:100ug/ml exosomes group?0.0089±0.00?=normal cell group?0.0089±0.00?>50ug/ml exosomes group?0.0067±0.031?>untreated group?0.0044±0.00?=0ug/ml exosomes group?0.0044±0.00?=25ug/ml exosome group?0.0044±0.00?;4)Respiratory chain complex V enzyme activity?nmol/min/10^4 cell?:100ug/ml exosomes group?0.078±0.00?>untreated group?0.0020±0.00?=25ug/ml exosomes group?0.0020±0.00?>50ug/ml exosomes group?0.017±0.0077?>0ug/ml exosomes group?0.0010±0.00?=normal cell group?0.0010±0.00?;5)ATP synthesis?umol/10^4 cell*10?:100ug/ml exosomes group?0.020±0.0016?>untreated group?0.018±0.0041?>50ug/ml exosomes group?0.013±0.0013?>25ug/ml exosomes group?0.0044±0.00032?>0ug/ml exosomes group?0.0036±0.00063?>normal cell group?0.0020±0.00095?;The above data were statistically significant?P<0.05?.Conclusion1.hucMSC?Ex have protective effects on oxidatively damaged ARPE-19 cells induced by cobalt chloride in vitro.2.The protective mechanism of hucMSC?Ex on oxidatively damaged ARPE-19 cells may be to save its aerobic metabolic function,restore cellular ATP synthesis,improve cell repair damage and ability to deal with hypoxic environment.3.hucMSC?Ex may have clinical application prospects for the treatment of RPE-related retinal diseases.