ABSTRACT
Objective To explore whether brimonidine has a protective effect on retinal ganglion cells (RGCs) through improving mitochondrial function under the oxidative stress.Methods Mouse RGC-5 cells were cultured in DMEM medium containing low concentration of glucose (1 g/L),10％ fetal bovine serum and 100 U/ml penicillin-streptomycin solution.The cells were divided into normal control group,H2O2-treated group and brimonidine+ H2O2 group.H2O2 at the concentration of 800 μmol/L was added into the medium in the H2O2-treated group,and 1 μmol/L brimonidine was added into the medium for 2 hours prior to the addition of H2O2 in the brimonidine+H2O2 group.The cells were sequently cultured for 24 hours.The morphology of the cell nucleus was examined by Hoechst fluorscence staining.The expressions of apoptosis-related protein in the cells were detected by Western blot assay.Mitochondrial membrane potential was assessed by JC-1 staining.Results The cell nuclei showed round or oval in shape with consistent size in the normal control group.The pycnosis and karyorrhexis of the cell nuclei were seen in the H2O2-treated group,and less abnormal nuclei were found in the brimonidine+H2O2 group.The relative expression level of bcl-2 protein in the cells was 0.76±0.15,0.50±0.13 and 0.75±0.17 in the normal control group,H2O2-treated group and brimonidine + H2O2 group,respectively,and the expression of bcl-2 protein in the H2O2-treated group was significantly lower than that in the normal control group and brimonidine+H2O2 group (both at P＜0.05).The relative expression level of bax protein in the cells was 0.65±0.13,0.83±0.07 and 0.70±0.10 in the normal control group,H2O2-treated group and brimonidine+H2O2 group,respectively,and the expression of bax protein in the H2O2-treated group was significantly higher than that in the normal control group and brimonidine+H2O2 group (both at P＜0.05).A strong orange fluorescence was seen in the mitochondrial membrane of RGC-5 in the normal control group with a coexpression with the green fluorescence of cell membrane.In the H2O2-treated group,the orange fluorescence intensity in the cells was evidently weakened,and the number of JC-1 responsed cells was considerably increased and the orange fluorescence intensity was enhanced in the brimonidine + H2O2 group.Conclusions Brimonidine can prevent RGCs from oxidative-stress damage by improving the mitochondrial function and therefore play a potential neuroprotective effect on optic nerve.
ABSTRACT
Background Mooren 's ulcer is an immune-related corneal inflammatory disease,and its pathogenesis remains below understood.Previous studies showed that the imbalance of T helper cell type 1 (Th1) and Th2 cell play important roles in the development of some autoimmune diseases.Thereby the influence of Th1/Th2 cells on the pathogenesis of Mooren's ulcer is being concerned.Objective This study was to investigate the change of Th1 and Th2 subsets in periphery blood of patients with active Mooren's ulcer.Methods Eleven consecutive patients with active Mooren's ulcer and 8 age-and gener-matched healthy controls were included in Zhongshan Ophthalmic Center,Sun Yat-sen University from January 2012 to July 2013 under the approval of Ethic Committee of this hospital and informed consent of each subject.The peripheral blood samples of all the subjects were obtained separately and periphery blood mononuclear cells (PBMCs)were isolated.The percentages of Th1 and Th2 in the PBMCs were assayed by flow cytometer.The relative expressions of T-bet mRNA,GATA-3 mRNA and signal transducer and activator of transcription 5 (Stat5) mRNA in the PBMCs were examined and compared by real-time fluorescence quantitative PCR (RT-qPCR) Results The percentage of Th1 cells in CD4+ T cells and Th1/Th2 value was 0.21％ (0.11％,0.31％) and 8.01 (4.49,12.01) respectively in the Mooren's ulcer group,which were significantly lower than 0.35％ (0.22％,0.71％)and 23.90 (22.49,33.49)in the normal control group,respectively (Z =-2.01,P =0.04 ; Z =-3.06,P =0.00).However,no significant difference was found in the percentage of Th2 between the two groups (Z=-1.98,P＞0.05).The relative expressions of T-bet mRNA and GATA-3 mRNA in PBMCs were significantly lower in the Mooren's ulcer group than those in the normal control group (Z =-3.47,-3.06,both at P=0.00) ;While the relative expression of Stat5 mRNA in PBMCs was insignificant changed between the two groups (Z =-1.05,P =0.33).Conclusions Th1 and Th2 cells are unbalanced in the active Mooren's ulcer patients.In addition,the down-expression of relevant transcription factors in peripheral blood also is seen in these patients.It is inferred that Th1 and Th2 cells may participate in the progress of Mooren's ulcer.