The Mechanism Of BAG2 Expression Inhibition By High Glucose Concentration In Retinal Pigment Epithelium Aging

Objective: Age-related macular degeneration(AMD)is a highly disabling disease and one of the leading causes of blindness in the elderly population worldwide.Due to the lack of research on the relevant mechanisms,its treatment is still unclear.With an accelerated population aging process,it is expected that about 400 million elderly individuals will be affected by this disease by 2040.AMD is a progressive disease characterized by enlarged drusen(> 65μm).The accumulation of drusen in the macular area will lead to irreversible damage to the neural retina and abnormal retinal pigment epithelium(RPE),leading to central vision loss and finally blindness.The RPE plays an important role in maintaining retinal homeostasis by clearing the end products of photoreceptors and transporting glucose and other nutrients from the choroid circulation to photoreceptors.Therefore,the research on RPE,especially the protective mechanism of RPE under stress conditions,is very important for the development of new treatment methods.BAG2 is a member of Bcl-2-associated athanogene(BAG)anti-apoptosis gene family.BAG2 facilitated the PPIs by inhibiting the E3 ubiquitin ligase activities of C-terminus of Hsc70-interacting proteins(CHIP).BAG2 inhibits CHIP mediated ubiquitination of HSP72 in senescent cells.BAG2 inhibits PINK1 degradation in neurodegenerative diseases by blocking ubiquitination pathways,stabilizing PINK1 to trigger Parkin-mediated mitochondrial division and neurological stress resistance to1-methyl-4-phenylpyridine induced oxidative stress.BAG2 is associated with poor prognosis in breast cancer.BAG2 interacts with P53 mutants to form polymers and recruits HSP90 to maintain the stability and spread of polymers.This pathway inhibits mitochondrial apoptosis and leads to chemotherapy resistance in breast cancer.Recent reports mainly focus on the role of BAG2 in neurodegeneration and tumor drug resistance with no previous reports on retinal degenerative disorders.Therefore,the role of BAG2 in different disorders needs to be further studied.Previous studies of our research team found that BAG2 expression in retinal mechanisms remain to be further investigated.This paper looked into the cause and mechanism of down-regulation of BAG2 expression in Adult Retinal Pigment Epithelial cell line-19(ARPE-19)induced by high glucose.At the same time,the possible mechanisms of BAG2’s influences on mitochondrial morphology except PINK1 pathway was explored.Methods: 1.Western blot was used to detect the protein expression level of BA2 in ARPE19 cells cultured in high glucose and normal glucose.2.Real-time PCR was used to detect the mrna transcription level of BAG2 in ARPE19 cells cultured in high glucose and normal glucose.3.ARPE19 cell line with BAG2 knockdown was constructed by infecting cells with lentivirus.4.The luciferase reporter gene vectors containing different regions of BAG2 promoter were constructed,and the activity of specific regions of BAG2 promoter was detected by dual luciferase reporter gene assay.5.Western blot was used to detect the expression of FOXP2 and TWIST1 under high glucose and normal glucose treatment conditions.6.Ch IP-q PCR was used to detect the specific binding of FOXP2 and TWIST1 to different promoter regions of BAG2.7.The effect of BAG2 knockdown on mitochondrial morphology was observed by mitochondrial morphology electron microscope.8.the expression levels of mitochondrial fission and fusion markers in BAG2 knockdown ARPE-19 cell lines cultured in high glucose and normal glucose were detected by Western blot assay.Results: 1.BAG2 expression in ARPE-19 cells is decreased under high sugar treatment.2.Knocking down BAG2 expression will intensify β-galactosidase staining and improve the expression of age-related proteins P21 and P53.And more cells were blocked in G1 phase.High sugar can induce the aging model of ARPE-19,and BAG2 knockdown will intensify cell aging,so BAG2 has resistance to cell aging.3.Aging reduces the m RNA expression level of BAG2 and inhibits the activity of promoter-519/-363 of BAG2.The expression of BAG2 in aging ARPE-19 cells is inhibited at the transcriptional level.There are FOXP2 and TWIST1 binding sites in this promoter region.4.Decreased FOXP2 concentration in high glucose treatment mediated the inhibition of BAG2 expression in retinal epithelial cells during cellular aging,but TWIST1 had no effect on decreased BAG2 expression in high glucose treatment.5.BAG2 knockdown increased the expression of mitochondrial division genes DRP1 and MFF,but had no effect on the expression of mitochondrial fusion genes MFN2 and OPA1.BAG2 knockdown inhibits mitochondrial fusion in retinal epithelial cells.Conclusion: High glucose inhibits the expression of BAG2 in retinal epithelial cells,knockdown of BAG2 promotes senescence of retinal epithelial cells,high glucose inhibits the expression of BAG2 at the transcriptional level,FOXP2 plays a role in the inhibition of BAG2 expression in retinal epithelial cells,and BAG2 knockdown inhibits mitochondrial fusion in retinal epithelial cells.BAG2 has a protective effect on ARPE-19 cells under high glucose stress.