1. Characteristics of exosomes
Electron microscopy demonstrated that HUCMSCs-Exo were round or oval membranous vesicles with diameters between 40~100nm (Fig. 1A-D). Western blot analysis confirmed that the HUCMSCs-Exo expressed CD63 and CD9 (Fig .1E).
2. Optimal damage concentration of CoCl2 in ARPE-19 cell model
Cells were treated with six different concentrations (0μg/mL, 50μg/mL, 100μg/mL, 200μg/mL, 400μg/mL and 800μg/mL) of CoCl2 for 24 hours and then incubated with 10 μL CCK8 for 30min. The survival rate of ARPE-19 cells were (80.94±6.87)%, (87.37±2.81)%, (93.38±4.28)%, (80.26±4.67)% , (50.71±2.33)% and (25.06±3.75)%.
As shown in Fig .2, survival rate of ARPE-19 cells treated with 100 μg/mL,400 μg/mL and 800 μg/mL CoCl2 concentrations were statistically significant compared with the control group (P<0.05). 400μmol/L and 800μmol/L CoCl2 concentrations significantly decreased the survival rate of ARPE-19 cells (P<0.01). When the CoCl2 concentration is 400μmol/L, the survival rate of ARPE-19 cells is 50%, Which was chosen to be the optimal concentration for oxidative damage.
3. Effect of exosomes on the morphology of APRE-19 cells injured by CoCl2
Normal ARPE-19 cells were fusiform or polygonal monolayer adherent cells with a clear outline. The cytoplasm might contain pigment, namely lipofuscin, which was brown and mostly located in the inner side of the cell (Fig .3A). When 400μg/mL CoCl2 was added, the number of ARPE-19 cells decreased, the space widened, the arrangement was disordered, the cells shrank and the nuclei aggregated (Fig .3B). After treated with exosome (0μg/mL, 25μg/mL, 50μg/mL and 100μg/mL) respectively, ARPE-19 cells morphology was similar to the normal group, with clear cell boundaries and increased cell numbers (Fig. 3C-F). The cell morphology of the 50μg/mL and 100μg/mL exosome intervention groups were similar to that of the normal group, with clear cell boundaries and increased cell number.
4. Effect of exosome on the MMP of ARPE-19 cells injured by CoCl2
MMP detection of the three groups treated with different concentrations of exosomes (0 μg/mL, 25μg/mL, 100μg/mL) and non-intervention group showed that the proportion of apoptosis were (25.5 ±0.56)%, (21.4 ±0.28)%, (12.00 ±0.71)% and (11.32 ±0.21)%. Compared with the normal group(6.57±0.24)%, there were statistically significant difference (P<0.01). The result of the 50μg/mL exosome intervention group was (6.45 ±0.35) %, which was not significantly different from that of the normal group (P>0.05). There was significant difference between the 0μg/mL and 25μg/mL exosome intervention groups with non-intervention group (P<0.01). Additionally, there was a significant difference between 50μg/mL and 100μg/mL exosome intervention groups with the 0μg/mL exosome intervention group (P<0.01) (Fig.4).
5. Effect of exosome on the activity of oxidative respiratory chains complex in ARPE-19 cells injured by CoCl2
Respiratory chain complex I, namely NADH-Co Q reductase or NADH dehydrogenase, is the main part of O2.32- generated in the respiratory electron transport chain. Its activity reflects the state of respiratory electron transport chain and reactive oxygen species (ROS) production.
Based on statistical analysis (Fig.5A), the enzyme activities of respiratory chain complex Ⅰ in non-intervention group and 100μg/mL exosome intervention group were respectively (0.052±0.052) nmol/min/10^4 cell and (0.066±0.00) nmol/min/10^4 cell, which were significant differences between them and the normal group (P<0.05). The activities of respiratory chain complex Ⅰ in (0, 25, 50)μg/mL exosome intervention group were respectively(0.015±0.00, 0.018±0.052, 0.026±0.052)nmol/min/10^4 cell, which had no statistical difference with the normal groups (P>0.05). However, there were significant differences between (0, 25, 50 and 100) μg/mL exosome intervention group and non-intervention group (P<0.05). 50μg/mL and 100μg/mL exosome intervention group had significant differences with 0μg/mL exosome intervention group (P < 0.05).
Respiratory chain complex Ⅲ, namely CoQ-cytochrome C reductase, is a common component of the main circuit and branch of mitochondrial respiratory electron transport chain. The activities of respiratory chain complex Ⅲ in (0, 25, 50 and 100) μg/mL exosome intervention groups were (0.025 ±0.00, 0.015 ±0.035, 0.025 ±0.00, 0.025 ±0.00) nmol/min/ 10^4 cell respectively (Fig.5B), which had significant difference from normal group and non-intervention group (P < 0.05). 25μg/mL exosome intervention groups also had significant difference with 0μg/mL exosome intervention groups (P < 0.05).
Respiratory chain complex IV (cytochrome C oxidase), is a common component of the main and branch pathways of the mitochondrial respiratory electron transport chain. Activities of respiratory chain complex Ⅳ in non-intervention group, 0μg/mL exosome intervention group and 25μg/mL exosome intervention group were respectively (0.0044 ±0.00, 0.0044 ±0.00, 0.0044±0.00) nmol/min/10^4 cell (Fig.5C), which was significant difference with the normal group (P<0.05). There was no significant difference between normal group and 50μg/mL or 100μg/mL exosome intervention group (P>0.05), activity of respiratory chain complex Ⅳ of which were (0.0067 ±0.031) nmol/min/10^4 cell and (0.0089 ±0.00) nmol/min/10^4 cell respectively. However, there was significant differences between 100μg/mL exosome intervention group with 0μg/mL exosome intervention group and the non-intervention group (P < 0.05)
Respiratory chain complex V, also known as F1F0-ATP synthase, is the key enzyme for mitochondrial oxidative phosphorylation to synthesize ATP. Activities of respiratory chain complex V in non-intervention group, 0μg/mL and 25μg/mL exosome intervention group were (0.0020 ±0.00, 0.0010 ±0.00 and 0.0020 ±0.00) nmol/min/10^4 cell(Fig.5D), which were no significant difference with the normal group (P>0.05). The activities of respiratory chain complex V in 50μg/mL and 100μg/mL exosome intervention group were (0.017±0.0077) and (0.078±0.00) nmol/min/10^4 cell, and had significant differences with the other groups (P<0.05).
6. Effect of exosome on ATP synthesis of ARPE-19 cells injured by CoCl2
ATP is a complex high-energy compound, which widely exists in animals, plant microorganisms, and cultured cells. ATP is involved in many life processes and referred to as the "monetary molecular unit" for intracellular energy transfer. Measuring ATP content and calculating energy charge can reflect the state of energy metabolism. As shown in Fig. 6, normal group had significant differences (P<0.01) with the non-intervention group, 50μg/mL and 100μg/mL exosome intervention group, whose ATP synthesis were (0.018±0.0041, 0.013±0.0013, 0.020±0.0016)μmol/10^4 cells*10. However, there was no significant difference between normal group and 0μg/mL or 25μg/mL exosome intervention group (P>0.05), whose ATP synthesis were (0.0036±0.00063) and (0.0044 ±0.00032) μmol/10^4 cells*10. There was significant difference between 0μg/mL, 25μg/mL, 50μg/mL exosome intervention group with the non-intervention group (P<0.05). 0μg/mL exosome intervention group had significant difference with 25μg/mL and 50μg/mL exosome intervention group (P<0.05).