Study On The Expression And Mechanism Of Fra-2 In Mouse Retinal Light Damage Model

Background With the rapid development of science and technology,the use of artificial lighting equipment continues to expand.Therefore,the light pollution has become one of the risk factors for human health.Excessive exposure to light in the environment can induce retinal damage.And in some common retinal degenerative ophthalmic diseases,such as age-related macular degeneration(AMD)and retinitis pigmentosa(RP)have shown the similar pathological lesions with light induced retinal damage.So the retina Light damage is a good model for studying many ophthalmic diseases.Intracellular intense stress can cause DNA damage,which induced a variety of intracellular reactions,including DNA repair,cell cycle arrest and apoptosis.However,severe DNA damage can lead to DNA repair failure,which ultimately resulting apoptosis.It was reported that(ADP-ribose)polymerase 1(PARP-1)can be activated rapidly by DNA damage,and promote the polymerization of ADP with PARP-1 and other nucleoproteins such as histone and topoisomerases.The 46 kDa Fra-2 is a member of the activator protein 1(AP-1)family,which is comprised of proteins related to the products of two proto-oncogenes,c-fos and c-jun.In mouse tissues,fra-2 plays a role in proto-oncogene,and elevated the expression of Fra-2 in mice disturbs the normal development of mouse eyes.Apoptosis inducing factor(AIF),as mitochondrial oxidoreductase,has the effect of catalyzing the electron transfer between cytochrome C and nicotinamide adenine and nucleotides(NAD).Excessive activation of PARP-1 induces cells synthesis of large amounts of PAR,which promoting the release and transfer of AIF from mitochondria into the nucleus.Finally,induces the activation of the apoptotic signaling pathway.In the present study,the possible mechanism of the Fra-2 gene in retinal light injury was investigated by establishing a model of mouse light injury and in vitro cell experiments.Part one Light exposure induced injury of mouse retinaObjective The effect of 2600 Lux visible light exposure on mouse retina was investigated by establishing a model of retinal light damage in mouse.Methods Incubating the mice with light intensity of 2600 Lux for 36 h to established mouse retina damage model.The histological changes of mouse retina were observed by HE-staining.The protein expression levels of PARP-1,Fra-2 and apoptosis-related proteins(Cyt-c,Bax,Blc-2 and Bcl-xL)in the retina of mice were detected by western blot.Results The results of the present study show that the retinal injury was observed after exposed to 2600 Lux light for 12 h.The inner and outer nuclear layers of retina were thinning;the cleavage and cell lysis of the tissue;the extracelluar phase of photoreceptors was disordered and apoptosis was observed in the ganglion cell layer.The expression of apoptosis related proteins Cyt-c and Bax in mouse retina increased significantly after exposure to light for 12 h,and the protein expression of Bcl-2 and Bcl-xL decreased significantly.Moreover,the protein levels of PARP-1 and Fra-2 in the retina of mouse were increased significantly after exposure to light.Conclusion These studies demonstrate that the serious damage and retinal photoreceptor cell apoptosis were observed in mouse retina exposed to 2600 Lux light for 36 h.PARP-1/Fra-2 signal pathway was involved in light induced retinal damage.Part two The effect of overexpression or interference expression Fra-2 on RGC-5 cellsObjective To investigate the effect of overexpressing or interfering with the expression of Fra-2 on RGC-5 cells.Methods The Fra-2 overexpression vector or interference vector was transfected into RGC-5 cells.Immunofluorescence assay was used to detect the expression of the plasmid.The expression of the relevant protein was detected by western blot.The cell proliferation was detected by MTT assay.Apoptosis was detected by flow cytometry.Results Immunofluorescence assay and western blot analysis confirmed that the Fra-2 overexpression plasmid or the interference plasmid was successfully transferred into RGC-5 cells.Overexpression of the Fra-2 significantly inhibited the proliferation of RGC-5 cells and induction of the cell apoptosis.The expression of PARP-1 protein in RGC-5 cells was promoted by overexpression of Fra-2 gene,and up-regulation of Fra-2 induced the transfer of AIF from cytoplasm to nucleus.Conclusion Over-expression of fra-2 gene in normal RGC-5 cells can inhibit cell proliferation and induce apoptosis and PARP-1/AIF signaling pathway is involved in fra-2 overexpression-induced RGC-5 cell injury.Part three Effect of interfering with Fra-2 expression on light induced RGC-5 cell injuryObjective To investigate the effect of interference Fra-2 expression on 2600 Lux visible light induced RGC-5 cell damage.Methods RGC-5 cells were transfected with Fra-2 interfering plasmid siFra-2 and incubated with 2600 Luxvisible light for 72 h.Normal RGC-5 cell cultured in normal environment used as control group(CON),normal RGC-5 cell with visible light exposure used as negative control(light exposure group,LE),RGC-5 cell treated with PARP-1 inhibitor NU1025 and exposure to light used as positive control group(LE+NU1025).RGC-5 cell apoptosis was detected by flow cytometry.MTT assay was used to detect the cell proliferation activity.The expression of PARP-1 and AIF was detected by western blot.Results Compared with normal control group,light exposure significant induced RGC-5 cell apoptosis and inhibited cell proliferation.However,compared with light exposure group,RGC-5 cell transfected with siFra-2 were significantly decreased cell apoptosis and increased cell proliferation.Compared with the normal control group,the protein level of PARP-1 was significantly increased after exposure to visible light,and AIF was transferred from the cytoplasm to the nucleus.Compared with the visible light exposure group,the expression of PARP-1 protein levels were significantly decreased,and AIF and cytoplasm to the nucleus of the transfer is also significantly inhibited.Conclusion Interference of Fra-2 in RGC-5 cells can reduce the release of PARP-1 in RGC-5 cells and reduce the transfer of AIF into the nucleus of RGC-5 cytoplasm,thus reducing exposure to RGC-5 cells induced by visible light exposure.