Assessment Of Genetic-related Myopia Due To An Lrpap1 Mutation And Dark-related Myopia Due To An Increase In Rhodopsin

Myopia is global public health problem that can cause visual dysfunction and even blindness.It is influenced by both genetic and environmental factors.Previous genetic studies have found that low-density lipoprotein receptor-related protein-associated protein 1（LRPAP1）is associated with high myopia based on high myopia families,but basic research is still lacking.In addition,outdoor time and light intensity are emerging as an important environmental factor affecting the prevalence of myopia.However,their regulation and mechanism of action remain poorly studied.From the perspective of heredity and environment,this study aims to preliminarily explore the effects of lrpap1 gene and low-light environment on myopia regulation of zebrafish and its mechanism,so as to explore potential therapeutic targets for myopia prevention and control.On the one hand,by constructing a zebrafish model with homozygous mutation of lrpapl gene,we explored the effect of gene function loss on myopia and its mechanism.The results showed that lrpap1 homozygous frameshift mutation（c.264₂68delinsTCTC,p.Lys35Asnfs*3）caused progressive myopic phenotype in zebrafish.In addition,RNA sequencing and bioinformatics analysis（KEGG、GO、GSEA）suggested enrichment of apoptosis-related pathways.We demonstrated increased apoptosis of eyeball cells of Irpapl homozygous mutant zebrafish by acridine orange staining and TUNEL staining.Finally,Western Blot and transforming growth factor-beta factor-β,TGF-β)agonist/inhibitor embryo incubation experiment showed that TGF-β level of mutant increased（P<0.05）and TGF-β agonist treatment of wild-type embryos can aggravate the degree of apoptosis of the eyeball,while TGF-β inhibitor can reduce the apoptosis of mutant embryos.These results indicated that homozygous frameshift mutation of Irpap1 gene was involved in the regulation of myopia through TGF-β-induced apoptosis.On the other hand,this study explored the effect of low light environment（<51ux）on zebrafish myopia from the perspective of environment factors.The results of diopter related parameters showed that relative refractive error（RRE）of zebrafish in dark-reared group shifted to myopia at 7,14,21 and 28 days after fertilization（P<0.05）,and the axis/body length increased at 21 and 28 days after fertilization（P<0.05）.RNA sequencing results showed that the photoconduction pathways were enriched（KEGG analysis）and the affected sites in the dark-reared group were mainly enriched in the outer photoreceptor segment and involved in biological processes such as eye development,visual system development and sensory perception of light stimulation（GO analysis）.HE staining and immunofluorescence analysis showed prolonged outer segment of rod cells,increased expression of Rhodopsin and choroid thinning.Meanwhile,zebrafish embryos overexpressing Rhodopsin showed RRE drift to myopia（P<0.05）and enlarged sclera,while 4-hydroxynonenal（4-HNE）accumulation was observed in the outer retina.In addition,ROS（P<0.05）and MDA（P<0.05）levels were increased in the dark-reared group,with lipofuscin and 4-HNE accumulated in the retina,and TUNEL positive cells increased significantly（P<0.05）.In addition,4-HNE level increased with the increase of Rhodopsin incubation concentration and time in ARPE-19 cell.Finally,4-HNE stimulation of ARPE-19 cells showed that low concentration of 4-HNE（20μM）could improve cell proliferation and TGF-βtranscription level（P<0.05）,and high concentration（40μM）could inhibit cell proliferation,inducing cell apoptosis and ferroptosis.These results suggest that RHO affects the function of retinal pigment epithelial cells through lipid peroxidation product 4-HNE,and thus mediates the occurrence of myopia.