Guoji Yanke Zazhi (May 2021)
Effect of human umbilical mesenchymal stem cells derived exosomes on autophagy and expression of VEGF of RPE cells under a high-glucose condition
Abstract
AIM: To investigate the effect of human umbilical mesenchymal stem cells(hUMSC)exosomes on the autophagy and expression of vascular endothelial growth factor(VEGF)of ARPE-19 cells under the condition of high glucose.METHODS: Tissue explants adherent culture was adopted to cultivate umbilical cord mesenehymal stem cells. The ultrastmcture of exosomes was observed under transmission electron microscope and the expression of surface specific marker proteins CD63 on exosomes were determined by Western blot. ARPE-19 cells were divided into the control group, high-glucose group and exosomes+high-glucose group. The cells in the exosomes+high-glucose group were pretreated with 75μg/mL exosomes. The ultrastructure of autophagosomes formed in all cells of the three groups was detected under transmission electron microscopy. The viability of ARPE-19 cells was measured by MTT assay. Western blot was applied to detect the expression of autophagy-associated proteins in different groups, including microtubule related protein 1 light chain 3B(LC3B), Beclin-1 and p62. The expression of VEGF in the cell supernatants was examined by ELISA assay. RESULTS: Transmission electron microscopy showed that the shape of hUMSC exosomes was spherical with membranous structure. The diameter of exosomes ranged from 30-100nm. Western blot indicated that the surface marker protein CD63 of exosomes showed positive expression. The number of autophagosomes in high glucose group was more than that in control group, while it was less in exosome+high-glucose group than in high glucose group. Compared with the high-glucose group, the cell proliferation rate of exosomes+high-glucose group was significantly increased(P<0.01). In the control group, high-glucose group and exosomes+high-glucose group, the LC3B-Ⅱ/LC3B-Ⅰ ratio in ARPE-19 cells was 0.214±0.019, 0.461±0.067 and 0.332±0.079, respectively; the relative expression of Beclin-1 protein in the cells was 0.186±0.029, 0.615±0.044 and 0.464±0.046, respectively; the relative expression of p62 protein in the cells was 0.771±0.051, 0.364±0.016 and 0.547±0.039, respectively. Compared with the control group and exosomes+high-glucose group, the ratio of LC3B-Ⅱ/LC3B-Ⅰ and the protein expression levels of Beclin-1 were significantly increased in the high-glucose group(all P<0.05). Compared with the control group and exosomes+high-glucose group, the protein expression level of p62 were significantly decreased in the high-glucose group(all P<0.01). Compared with the high-glucose group, the expression levels of VEGF were significantly decreased in the exosomes+high-glucose group(P<0.01). CONCLUSION: High glucose conditions activated autophagy of ARPE-19 cells and promoted the expression of VEGF. hUMSC exosomes could effectively inhibit the autophagy level of ARPE-19 cells and the expression of VEGF in high-glucose environments.
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