The Effect Of PKC Activation On The Antioxidant Capacity Of Rabbit RPE Cells And Its Mechanism

Objective:In this study,the expression of NF-related factor 2 in RPE cells was detected by activating protein kinase C(PKC)in rabbit retinal pigment epithelium(RPE)and the expression of glutathione(GSH)in the cells to investigate whether the PKC signal system has an effect on the antioxidant capacity of RPE cells,and to treat age-related macular degeneration Macular degeneration,(AMD)to provide theoretical basis.Methods:Under the aseptic conditions,surgically removed of rabbit eyes and stripped of retinal nerve layer.The cells were isolated by enzymatic digestion,and identified by conventional cell culture method,primary cultured rabbit retinal pigment epithelial cells(RPE)and immunofluorescence staining.Three generations of RPE cells were used as experimental cells,divided into 3 groups:Group A(Control group):15%fetal bovine serum DMEM culture medium;Group B(PMA group):15%fetal bovine serum DMEM culture medium+phorbol12-myristate13-acetate(PMA)100 nmol/L;Group C(PMA+BST group):RPE cells were pretreated with 10μmol/L of Brusatol(Brusatol,BST)for 1 hour,then extracted and added with PMA100 nmol/L of 15%Fetal bovine serum in DMEM.The incubation time of each group was 24 h.Western blot was used to detect the expression of Nrf2 protein in cytoplasm and nucleus.The expression of GSH in RPE cells was detected by GSH kit.The expression of Nrf2 and intracellular GSH were analyzed by immunofluorescence staining.Nrf2 protein was detected by intracellular translocation happening.Results:1.The RPE cells of the third generation turned to long spindle-shape,a small amount of melanin granules in the cytoplasm,and anti-human keratin(AE1/AE3)immunofluorescence staining showed strong positive;2.Western blot showed that:compared with the control group,the expression of Nrf2 protein in cell cytoplasmic of PMA group and PMA+BST group were significantly reduced,however,the Nrf2 protein expression in cell nucleus were significantly increased.Moreover,the expression of Nrf2protein in the cytoplasm of PMA group decreased significantly more than PMA+BST group,andNrf2proteinexpressioninnucleuswassignificantly increased(cytoplasm:F=26.62,P<0.05;nucleus:F=29.88,P<0.05);3.GSH assay kit show that:compared with the control group,the expression of GSH in intracellular of PMA group and PMA+BST group were significantly increased,and the expression of GSH of PMA group was significantly higher than PMA+BST group(F=37.64,P<0.05).linear regression equation showed that Nrf2 protein expression in nucleus and GSH expression were significantly positive correlated(R~2=0.908,P<0.05);4.Immunofluorescence staining showed that:compared with the control group,the expression of Nrf2 protein in the nucleus of PMA group and PMA+BST group increased,the nuclear translocation were occurred.Compared with PMA+BST group,the nuclear translocation of PMA group was more significant.Conclusions:1.PKC activation,can make RPE nucleus Nrf2 expression increased,the occurrence of nuclear translocation,Nrf2 inhibitor can weaken the role of PKC;2.PKC activation can indirectly promote the expression of GSH,improve the antioxidant capacity of RPE cells;3.The expression of Nrf2 in the nucleus was positively correlated with the expression of GSH in the cells.