Study On The Activation And Neuroprotection Of Müller Cells Depended On The Interleukin 17A In Oxygen-induced Retinopathy

Purpose:Interleukin 17A(IL-17A),as a proinflammatory cytokine,has been implicated in several autoimmune diseases.However,it is unclear whether IL-17A is involved in oxygen-induced retinopathy(OIR),in which neuroinflammation is a vigorous response to hypoxia.The purpose of this study is to assess the levels of IL-17A produced in the murine model of OIR and elucidate its potential roles in Müller cell activation as well as the neuroprotective function during the hypoxia induced apoptosis of photoreceptor in vivo and in vitro.Methods:1.Widetype C57BL/6J mice were prepared and randomly divided into normal control group and OIR model group.IL-17A and cytokines associated with Müller cell activation in retinas were evaluated by western blots and immunofluorescence(IF) staining.WT Müller cells were isolated and cultivated in hypoxia,RT-PCR,western blots,ELISA and IF staining were performed to detect the expression of GFAP,GS and IL-17A in vitro.2.The apoptosis of photorecptor cells induced by hypoxia were examined by western blot and TUNEL in vivo and in vitro.Co-culture systems of WT Müller cells and 661W cell lines were perpormed in hypoxia in vitro.After that,the apoptosis of photorecptor cells were examined.3.WT and IL-17A^-/-OIR model groups were established respectively.The expression levels of GFAP,GS and IL-17A were examined by western blot and IF staining in the above two gene groups.Similarly,they were examined by realtime RT-PCR,western blot and IF staining in the Müller cells isolated from the different genotypes with hypoxia stimulation in vitro.IL-17^-/-Müller cells were cultivated in hypoxia with different concentrations of rmIL-17A(0,10,50,100,200 ng/ml)and then GFAP were evaluated by western blot.Furthermore,the apoptosis of retinal cells was compared between the WT and IL-17^-/-OIR models by western blot and TUNEL.Besides,co-culture systems of Müller cells and 661W cell lines were separately perpormed with the above two genotype Müller cells in hypoxia,and the apoptosis of 661W cells was also examined in vitro.4.WT and IL-17A^-/-OIR model groups were established respectively.The expression levels of NT3 and proNT3 were examined by western blot.Similarly,they were examined by realtime RT-PCR and western blot in the different genotype Müller cells in hypoxia in vitro.IL-17^-/-Müller cells were cultivated in hypoxia with different concentrations of rmIL-17A(0,10,25,50,100,200 ng/ml).NT3 and proNT3 were evaluated by western blot.The expression levels of p75NTR and Trk C in 661W cells were also examined in the co-culture systems in vitro.Finally,we compare the MAPKs protein expressions between the WT and IL-17^-/-Müller cells in hypoxia,and the inhibitor of MEK was applied to detect the variation of Müller cells activation.Results:The expression of GFAP was increased by the activated Müller cells in hypoxia;in the presence of activated WT Müller cells,highly expressed IL-17A,the apoptosis of photoreceptor cells induced by hypoxia decreased.The expression of GFAP was decreased in the IL-17A^-/-Müller cells in hypoxia,but increased with the addition of rmIL-17A.The apoptosis of photoreceptor cells coexisting with the IL-17A^-/-Müller cells did not decrease in hypoxia.Decreased expression of NT3 by IL-17A^-/-Müller cells was promoted by low and medium concentration of rmIL-17A,but at the high concentration of rmIL-17A the expression of proNT3 was increased.Phospho-ERK1/2 expression was low in the IL-17A^-/-Müller cells.Inhibition of MEK pathway reduced the expression of GFAP,NT3 and proNT3 in WT Müller cells in hypoxia.Conclusions:Moderate expression of IL-17A through autocrine mode could suppress the apoptosis of photoreceptor cells in hypoxia by regulating the activation of Müller cells and the secretion of NT3 from them.The mechanism may include the phosphorylation of ERK pathway.