The Study On The Protective Mechanism Of VEGF-B On Glaucomatous Retinal Cells By Quantitative Proteomics

Objective:Glaucoma is one of the blinding eye diseases.The incidence rate is increasing year by year.The patients' optic ganglion cells are irreversibly damaged by multiple factors.At present,the means of treating glaucoma are mostly to reduce intraocular pressure,but there are still unknown factors affecting the occurrence of glaucoma and development.In recent years,studies have found that VEGFB(Vascuar endothelial growth factor B,VEGFB)has protective effects on retinal ganglion cells,but the specific mechanism is still unclear.The purpose of this study was to quantify proteomics to study the protective mechanism of VEGF-B on glaucomatous retinal cells.Methods:We used the optic nerve crush method to construct the C57BL/6 mouse glaucoma model.The retinas ofsham-operated mice,and the 2-day model group and the 7-day model group were subjected to HE staining to verify the success of the mouse glaucoma model.Secondly,we used real-time quantitative polymerase chain reaction(qRT-PCR),Western blot and immunohistochemistry to confirm the up-regulation of VEGFB in retinal cells of glaucoma mice.Mass spectrometry was then used to extract 5 retinal tissues from each group.The nano-LC and QExactive Plus system were used to analyze the differentially expressed proteins by label-free quantitative proteomics.In-depth analysis of bioinformatics was performed on mass spectrometry data,and bioinformatics software such as KEGG,GO,IPA,Cytoscape and PANTHER were used to predict the signaling pathway involved in VEGFB.The biological expression method was used to verify differentially expressed proteins at the tissue level by qRT-PCR,Western Blot and ELISA.At the cellular level,RNAi was used to reduce the expression of VEGFB in ganglion cells(RGC5),using xCELLigence real-time cell analysis.The RTCA system detects cell proliferation.The expression of related proteins was detected by qRT-PCR,Western Blot and ELISA.Results:C57BL/6 mouse glaucoma model was successfully established by optic nerve crushing method,and the expression of VEGFB was up-regulated in the retina of glaucoma model.A total of 1585 proteins were detected,and 48 proteins were differentially expressed in the retinal tissue of the sham-operated group and the retinal tissue of the glaucoma model,and were statistically significant Among them,10 proteins showed high expression in the retinal tissue of glaucoma model,and 20 proteins showed low expression.Bioinformatics analysis showed that VEGFB affected the expression of Glutamate decarboxylase 1(GAD1)and Argininosuccinate synthase 1(ASS1),and it was verified at the tissue level that VEGFB was positively correlated with the expression of GAD1 and ASS1.Further cell experiments showed that after knockdown of VEGFB expression,GAD1 and ASS1 expression was down-regulated and the proliferation function of the cells was inhibited.At the same time,down-regulation of VEGFB expression reduced the secretion of r-aminobutyric acid(GABA)in cells.Conclusion:We successfully established a glaucoma mouse model to detect differentially expressed proteins in the retinal tissue of sham-operated and glaucoma model mice by quantitative proteomics.Bioinformatics analysis revealed that VEGFB is involved in the regulation of GAD1 and ASS 1 expression.At the tissue and cell level,it was confirmed that VEGFB has a significant positive regulation effect on GAD1.It is speculated that VEGFB regulates the expression of GAD 1 mainly through the receptor VEGFR1,affecting the metabolism of glutamate,and reducing the toxicity of glutamate to the retina.The role.