Erythropoietin Protects RPE Cells In Diabetic Rat By Regulating Autophagy

Background and purposeDiabetic retinopathy(DR)is a serious complication of diabetes in the eyes.Macular edema is the main cause of blindness and there is no effective intervention.The results show that the leakage of blood vessels caused by the destruction of blood retinal barrier is one of the main reasons for diabetic macular edema(DME).There are many researches focus on the abnormal function of retinal vascular endothelial cells and the mechanism of the destruction of internal barrier,but there are few studies focus on the diverse effects of diabetes on RPE cell function.However,the destruction of external barrier and abnormal RPE cell function were observed in clinical and experimental DR,but the exact mechanism is still not clear.RPE cells are one of the most active phagocytic cells in vivo.Abnormal autophagy or lysosomal degradation will lead to the deposition of metabolites in cells,and then lead to abnormal or death of RPE cells.More and more studies have shown that autophagy plays an important role in DR pathogenesis.It has been proved that injection of EPO can protect retinal cells and protect blood retinal barrier(BRB)and improve visual function.At present,the mechanism is still not clear.This project is intended to use the diabetic rat model,the primary RPE cell model to explore and clarify the changes of RPE autophagy function in DR timecourse and the protective mechanism of EPO by regulating autophagy.MethodsThe eyeballs of Dark Agouti(DA)rats and Sprague Dawley(SD)rats were taken at 10 days after birth(postnatal 10 days,PN10).RPE cells(15000 cells/cm2)were inoculated in cell culture dish and petri dish culture respectively.The culture medium was DMEM/F12 containing 10%FBS.The next day,the cells in the two types of dishes were divided into two parts,half of them were continued to use DMEM/F12 medium with 10%FBS,and the other half were used DMEM/F12 medium with N2 and B27.Therefore,the primary RPE cells were divided into four groups:1)cell culture dish,DMEM/F12 medium group(cell culture dish FBS,CC dish FBS)containing 10%FBS,2)cell culture dish,DMEM/F12 medium group containing N2 and B27 supplement,CC dish N2B27;3)Petri dish,DMEM/F12 medium group(Petri dish FBS)containing 10%FBS;4)Petri dish,DMEM/F12 medium group(Petri dish-N2B27)containing N2 and B27 supplements.The cell morphology was observed and photographed at 4 weeks.Meanwhile,the expression of RPE related genes(RPE65,CRALBP and Bestrophin)and EMT(N-cadherin,Fibronctin and α-SMA)were detected by quantitative real time polymerase chain reaction(Q-PCR)and the expression of mTOR,Akt and ERK were detected by Western blot.SD rats were divided into three groups:control group,diabetic group and EPO group.EPO(16mU/eye)was injected into the vitreous within two hours after the model was established in SD rats.The eyeball samples were collected at 4 weeks after modeling and EPO intervention.The expression of autophagy related proteins(Beclin-1,ATG and LC3)and the expression patterns of autophagy flow marker protein P62 were detected by Western blot and immunofluorescence staining.RPE cell morphology and the distribution of photoreceptor outer segments in RPE cells were observed by RBCC staining.The expression of RPE-specfic and autophagy related proteins,including mTOR,AKT and ERK were detected by Western Blot method.ResultsAfter isolation and cultured in vitro,RPE cells in high density showed regular polygon structure,while RPE cells in low density group lost regular polygon structure of epithelial cells completely.In cell culture dish,RPE cells proliferated obviously,compared with that of the petri dish.Because of its material properties,the petri dish is not conducive to cell adhesion and diffusion,so RPE cell proliferation was significantly inhibited.The cell morphology and gene expression of RPE cells also different under different culture conditions.The primary RPE cells can maintain good morphology under the condition of Petri dish-N2B27 culture.The expression level of RPE specific marker genes(RPE65,CRALBP and bestrophin)in Petri dish-N2B27 was significantly lower than that of the Petri dish-FBS,cell culture dish-FBS and cell culture dish-N2B27,while the expression of EMT related genes(N-cadherin,fibronettin and α-SMA)showed a contrary trend.The density of cell inoculation also affected the expression of mTOR signaling pathway related proteins.The phosphorylation level of mTOR signaling pathway protein in low density cultured-primary RPE cells was significantly higher than that in high density group.The change of culture conditions also affected the mTOR signal pathway in RPE cells.Under the condition of Petri dish-N2B27,the phosphorylation level of mTOR signaling pathway protein was significantly lower than that of cell culture dish-FBS,cell culture dish-N2B27 and Petri dish-FBS.Compared with the control group,the expressions of ATG,Beclin-1 and P62 in RPE cells of diabetic rats were significantly increased.The results of RBCC staining showed that the OS degradation ability of RPE cells was significantly reduced,and the expression of autophagy signal pathway related proteins increased.EPO intervention treatment inhibited the expression of autophagy related protein in RPE cells of diabetic rats.Glyoxal treatment significantly inhibited the activity of primary RPE cells in rats,and the number of RPE apoptosis increased significantly.Meanwhile,the results of protein electrophoresis showed that glyoxal treatment blocked the normal autophagy flow of RPE cells and activated the autophagy related signaling pathway.EPO intervention treatment can protect RPE cell activity,improve autophagy flow and improve RPE related gene expression to some extent.ConclusionsThe digestion of dispase and the cutting of retina/RPE complex into small pieces and then incubating in the medium can make RPE cell layer separate from retina without artificial mechanical stripping.It is easy to operate,and it is a rapid and effective method for separating RPE cells from newborn rats.To some extent,increasing the density of primary RPE cells can maintain the phenotype of RPE cells and delay the EMT process.The results showed that the combination of the two can effectively inhibit EMT and maintain the phenotype of rat RPE cells.The degradation ability of RPE cells to the photoreceptor cells was also significantly decreased in diabetic rats,and the expression of autophagy signal pathway related proteins increased and autophagy flow decreased.EPO intervention treatment inhibited the expression of autophagy related protein in RPE cells of diabetic rats to some extent and improved autophagy flow.Glyoxal treatment inhibited the activity of primary RPE cells and increased apoptosis of RPE cells,and the expression of autophagy related proteins was also significantly increased.EPO treatment can protect RPE cell activity.