Study On The Mechanisms Of NO-mediated Protein S-nitrosylation In The Lens-induced Myopia

Objective: This study intends to preliminarily explore the roles of protein S-nitrosylation of NO in the regulation of myopia by detecting the expression of n NOS and downstream S-nitrosylation,using the animal model of lens-induced myopia(LIM)in mice.Methods: The 3-week-old C57BL/6J mice were divided into three groups: group I: lens-induced 0-week group,group II: self-control group,group Ⅲ: lens-induced 4-week group.The diopter and axial length of each group were measured by streak retinoscopes method and OCT before and after modeling.The protein expressions and locations of n NOS and PSNOs were measured by Western Blot and immunofluorescence staining.Sitespecific proteomics for protein S-nitrolysation was used to detect the Snitrosylation proteins and sites in the retina of myopic and non-myopic mice.The GO,KEGG,and Motif enrichment analyses were performed.The differential sites were analyzed by GO,KEGG and Motif.Irreversible biotinylation procedure combined with protein purification and Western Blot were used to detect the protein expression of ENO1,a key player in the hypoxia-related signal pathway.Result:(1)Four weeks after the modeling,group Ⅲ showed shift to myopia(-0.800±1.849 D)and group Ⅱ showed shift to high hyperopia(+9.600±3.528 D,P<0.001).The axial length of the group Ⅲ(3.500±0.072 mm)was significantly longer than the group Ⅱ(3.403±0.073 mm,P<0.001).(2)The n NOS and PSNOs were highly expressed in ganglion cell layer,inner nuclear layer and outer nuclear layer.Compared to the group I and group II,the expression of n NOS and PSNOs at all retinal layers were lower in the group Ⅲ.(3)A total of 595 S-nitrosylated proteins,709 S-nitrosylated peptides,708 S-nitrosylated site were identified by sitespecific S-nitrolysation proteomics in the retina of myopic and control eyes.A total of 19 differentiation loci were screened,of which 13 sites were down-regulated and 6 sites were up-regulated in experimental eyes compared with the self-control group.(4)Specifically,the expression of SNO-ENO1 was significantly lower in retina of the group Ⅲ than that in the group I(P=0.006)and the group II(P=0.036).Conclusion: LIM induces the decrease of n NOS and PSNOs protein levels in the retina of myopic mice.NO-mediated non-classical protein Snitrosylation modification may play an important role in the regulation of lens induced myopia.ENO1 may be a key factor in the regulation of Snitrosylation modification of myopia.