Preliminary study on prevention and treatment of experimental autoimmune uveitis by blocking CD73 detachment from the surface of retinal pigment epithelial cells with matrix metalloprotein-9 inhibitor / 中华眼底病杂志

ABSTRACT

Objective:To preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis(EAU).Methods:RPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73 -/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/Ladenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4 + T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73 -/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data. Results:When the stimulation mode was AMP, the proliferation of CD4 + T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the nonintervention group ( F=13.28, P<0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4 + T cells between the two groups ( F=7.78, 6.58; P>0.05). There was no statistically significant difference in the proliferation capacity of CD4 + T cells between the CD73 -/- MMP-9 inhibitor intervention group and the non-intervention group ( F=5.24, 6.12, 7.04; P>0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group( F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group ( F=15.24, P<0.01). Conclusion:MMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.