| ObjectiveThe "local control theory of the retina" is received special attention in a variety of theories about myopia pathogenesis, in which it was thought that myopia signal factor (such as retinoic acid, RA) released from retina after perception of the change of external environment, activated retinal pigment epithelium and promoted its releasing of secondary signaling factors(such as TGF-β2) to regulate the scleral growth leading to myopia. This study was to investigate the roles of retinoic acid receptor beta antagonist,LE540, on secretion of basic fibroblast growth factor(bFGF) and transfer growth factor-β2(TGF-β2) in cultured retinal pigment epithelial cells, and to explore whether the secretion of transforming growth factor B2 was regulated by activator protein-1 in retinoic acid signaling pathway.MethodsPart one:Cultured human retinal pigment epithelial cells D407 was administrated with all trans retinoic acid at concentration of 5×10-6M,10×10-6M, 40×10-6M. Cell proliferation was detected by MTT while the most suitable concentration will be used to the following experiment. Cells were divided into 6 groups:10×10-6mol/L of ATRA was added in medium in 24 hours after the first medium exchange as the first group,5×10-6mol/L of LE540 was added in medium as the second group, The same dose of DMSO or ddH2O was used for control group.The bFGF and TGF-B2 secreted by RPE cells were detected using ELISA method at 2,4,6,8,16 hours after the addition of ATRA and LE540. Part two:Cultured human retinal pigment epithelial cells D407 were divided into 3 groups.The ap-1 and TGF-β2 secreted by RPE cells were detected at 4,8,16 hours after the addition of 10×10-6 M ATRA in group one; The ap-1 and TGF-β2 secreted by RPE cells were detected at 4,8,16 hours after the addition of 10×10-6M ATRA and 2.5×10-6M LE540 in group two; The TGF-B2 secreted by RPE cells were detected at 4,8,16 hours after the addition of 2.5×10-6M LE540 in group three. ELISA method was used to detect the secretion of TGF-B2 in cell culture medium.Western blotting was used to detect the secretion of protein c-fos in cell lyssates. The statistical analyses were performed by the SPSS 11.5 software. The differences between groups were analyzed with independent samples T-test or ANOVA. All measurement data was expressed as mean±standard deviation (x±s).ResultsPart one:The MTT result showed that the RPE cells proliferation was inhibited by 40×10-6MATRA compared with the control groups (P<0.05).while no significant differences were seen in 5×10-6M and 10×10-6M ATRA groups compared with the control.The secretion of TGF-B2 was significantly increased after addition of ATRA in 2,4,6,8,16 hours in comparison with control group, while the secretion of bFGF was significantly reduced (P<0.05). Difference was statistically significant.The secretion of TGF-β2 were significantly reduced after addition of LE540 in 2,4,6,8,16 hours in comparison with control group (P<0.05). Difference was statistically significant. While the secretion of bFGF were not significantly change (P =0.178), no statistically significant difference.Part two:The secretion of TGF-B2 were significantly reduced after administration of ATRA+LE540 group in comparison with ATRA group (P<0.05). The secretion of TGF-β2 were significantly reduced after addition of ATRA+curcumin group in comparison with ATRA group (P<0.05). Difference was statistically significant. While there was no significant difference for secretion of TGF-B2 between ATRA+curcumin group and ATRA+LE540 group. It was shown c-fos/B-actin was significantly increased after addition of LE540 and RA in 4,8,16 hours in comparison with ATRA group (P<0.05). Difference was statistically significant(t=-51.16, P<0.001;t=-40.226, P<0.001;t=-31.706, P<0.001).ConclusionRetinoic acid signaling pathway play an important role in the pathogenesis of myopia. We hypothesize that increased secrection of retinoic acid functioning on retinoic acid receptorβof retinal pigment epithelial cells, c-fos protein of AP-1 expression was downregulated by which transforming growth factor-β2 was increasingly expressed through a number of cascades in cells, promoting formation of myopia. It was observed that after addition of retinoic acid, basic fibroblast growth factor expression was declined; however, this effect was by retinoic acid receptorβ. The specific mechanism of action remains to be further studied.The effect on TGFβ2 secrection of retinoic acid can be not completely inhibited by retinoic acid B-receptor antagonist LE540,and AP-1 receptor antagonist curcumin. |
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