A Correlation Research Between Retinal Pigment Epithelium Exosomes Secretion Induced By Blue Light And NLRP3 Inflammasome Activation

Objectives To observe the relativity between exosomes secreted by the blue light induced human retinal pigment epithelium(RPE) cells and NLRP3 inflammatory. In our study, we acted the exosomes secreted by the blue light induced RPE cells on the normal RPE cells, observe it’s effect and detect the NLRP3 expression level in affected RPE cells for exploring the influence of exosmes secreted by photo oxidative damaged RPE cells on the pathogenesis of AMD. These will provide objective basis for exosomes using on treatment, early diagnosis and prognosis of AMD.Methods 1. Establish the model of blue light induced RPE cells and evaluate its effectiveness: Cultured ARPE-19 cells were divided for 2 groups, of which one group creating Blue light induced RPE model, the RPE cells were exposed to blue light irradiation for 6 hours, another group was normal control which cultured in routine culture environment. Using MTT assay to detect the proliferation activity of two groups of cells.2. Isolation and identification of exosomes and detect the NLRP3 level of exoxomes: Both the experimental group and the control group respectively using progressively ultracentrifugation in low-temperature to isolate exosomes, then using transmission electron microscope to identify the form of exosomes. Western blot were used to detect the specific marker protein—CD63 that on the surface of exosomes. Compared the NLRP3 correlative proteins—IL-1β、IL-18 and caspase-1 levels between the experimental group and the control group.3. Effects of exosomes secreted by the blue light induced RPE cells on normal RPE cells: The exosomes isolate both by experimental group and control group was added in normal RPE cells. Using MTT assay to detect the proliferation activity of two groups of cells after acted. Western blot was used to compare the level of IL-1β,IL-18 and caspase-1 levels of two groups of RPE cells. immunofluoressent assay testing IL-1β, IL-18 and caspase-1 of two groups of cells. RT- PCR detecting the expression level of NLRP3 m RNA of two groups of cells.Results 1. Successfully constructed the blue light induced photo oxidative damage model of RPE cells: After injury,RPE cells became plump and loss multi angle shape, the cell body became long. Compared with the control group, the proliferating activity of the RPE cells in the experimental group significantly weakened(P < 0.05).2. Acquire exosomes by ultracentrifugation the cell culture fluid and transmission electron microscope identify that the characteristics of exosome we acquired are conform to the previously descriptions. Western blot showed that CD63 can be detected on the surface of exosomes we acquired, the level of NLRP3 correlative proteins— IL-1β, IL-18 and caspase-1 of exosomes in experimental group were significantly higher than those of the control group(P < 0.05).3. We added the exosomes secreted by the blue light induced RPE cells to normal RPE cells, the proliferation viability of RPE cells decreased significantly(P < 0.05).Western blot showed that the level of NLRP3 correlative proteins—IL-1β, IL-18 and caspase-1 of RPEcells in experimental group were significantly higher than those of the control group(P < 0.05), Immunofluorescence showed that the level of NLRP3 correlative proteins—IL-1β, IL-18 and caspase-1 of RPEcells in experimental group were significantly higher than those of the control group(P < 0.05).RT-PCR showed that the relative value of NLRP3 m RNA of PRE cells in experimental group and control group were(1.0±0.069)and(0.2±0.01),the level of NLRP3 m RNA in experimental group were significantly higher than those of the control group(P < 0.05).Conclusions1. Blue light induced RPE cells were exposed to light oxidative damage and the expression of NLRP3 correlative proinflammatory cytokines—IL-1β, IL-18 and caspase-1 was upregulated in the exosomes secreted by the blue light induced RPE cells.2. The NLRP3 inflammatory related cytokines IL-1β, IL-18 and caspase-1 expression up-regulation in the RPE cells after added with the exosomes which have high expression of NLRP3 inflammatory related proteins, the NLRP3 m RNA in these RPE cells were also up-regulated.