ABSTRACT
Objective To examine the expression of stromal cell-derived factor-1 (SDF-1) in a rat model of retinal ischemia-reperfusion injury (RIRI),and investigate the protective effect of 17β-Estradiol (E2) on RIRI and explore the mechanism.Methods The RIRI model was established in Sprague-Dawley rats by increasing the intraocular pressure.Relative expression levels of SDF-1 mRNA and protein in the retina at 6 hours,12 hours and 24 hours following reperfusion was determined by RT-PCR and Western blot,respectively.E2 was administered to investigate the effects of estrogen on SDF-1 expression,and the estrogen receptor antagonist ICI 182-780 was administered to investigate the effect of estrogen receptor on the expression of SDF-1.Results SDF-1 expression in RIRI 6 hours group,12 hours group and 24 hours group was increased compared with normal control group (all P ＜ 0.05),with maximum expression at RIRI 12 hours group.As expected,pretreatment of RIRI rats with E2 had a protection on RIRI retina;SDF-1 expression was increased in RIRI + E2 group compared with IR control group and RIRI + vehicle group (all P ＜ 0.05).RIRI + E2 + ICI 182-780 group could decrease SDF-1 expression compared with RIRI + E2 group(all P ＜ 0.05).Conclusion E2 offers protection against RIRI by inducing an up-regulation in SDF-1 expression through activation of the estrogen receptor.
ABSTRACT
Background Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy (PVR),and the proliferation,migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy (DR).However,whether EMT could be induced in RPE cells under the high glucose condition has not been reported.Objective This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro.Methods Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10％ fetal bovine serum,and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group,and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0,24,48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) in the cells.Results Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group,but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching,migrating cells were seen in the scratching area,and the scratching area disappeared at 72 hours after scratching in the high glucose group,but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group,showing total differences among 3 groups and various time points (Fgroup =328.600,P =0.000 ; Ftime =773.270,P=0.000).Compared with the normal control group,the expression level of ZO-1 mRNA was significantly lower,and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group (all at P＜0.05).Conclusions High glucose induce the migration and EMT of RPE cells in vitro,which may be associated with the pathogenesis of proliferative diabetic retinopathy.
ABSTRACT
Background Sphingosine-l-phospate (S1P) is a bioactive lipid and important messenger molecule in cells.It participates in the regulation of many biological processes,such as cell proliferation,migration,survival,differentiation,apoptosis,etc.Hypoxia is a trigger factor of choriod neovascularization (CNV) and pathological basis of many diseases,and retinal pigment epithelial (RPE) cells are involved in formation of CNV.However,the effects of S1P on proliferation and apoptosis of RPE cells are below understood.Objective This study was to investigate the influence of S1P on proliferation and apoptosis of human RPE cells under hypoxic conditions.Methods Human RPE cells line-D407 cells were cultured and passaged and generation 3-5 cells were used and divided into 6 groups.The cells were regularly cultured in the blank control group using DMEM containing 10％ fetal bovine serum.CoCl2(200.00 μmol/L) was added into the colture medium for 2 hours in the hypooxic group.S1P of different concentrations (0.01,0.10,1.00,10.00 μmol/L) were added in culture medium 2 hours after the affection of 200.00 μmol/L CoCl2.The proliferative values of the cells were detected using WST-1 method as the absorbance (A value) and the proliferative rate of different groups were calculated.The apoptosis of the cells was assayed by Hoechst staining.The results were compared among different groups.Results Cultured cells showed the round-like in shape with clear nuclei and pigment.The proliferative values (A value) was 0.91 ±0.08,0.37±0.09,0.46±0.08,0.52±0.09,0.61 ±0.06,0.70±0.10 in the blank control group,hypoxic group and 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups (F=21.104,P=0.000),and A values in various S1P groups were higher than those in the hypoxiac group (all at P＜0.05).The proliferative rate was gradually raised with the increase of dose of S1P.Hoechst staining exhibited a few apoptosis cells in the blank control group,but in the hypoxic group,a lots of apoptosis cells were seen with the light-blue nuclei and condensable chromatin.However,the number of apoptosis cells was significantly decreased in various concentrations of S 1 P groups.The apoptosis rates were (1.21 ±0.08) ％,(8.99 ±0.09) ％,(6.60 ±0.08) ％,(5.95 ±0.09) ％,(4.81 ± 0.06)％ and (3.96±0.10)％ in the blank control group,hypoxic group and the 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups (F =25.070,P =0.000).Compared with the hypoxia group,the cellular apoptosis rates of various S1P groups were lower (all at P＜0.05).Conclusions Under the hypoxia condition,S1P can promote the proliferation of human RPE cells and inhibit apoptosis.