| Objective To establish a hypoxia model of ARPE-19cell, investigate the the changes of MEK/ERK signaling pathway, tight junction protein and VEGF in RPE cell under hypoxia condition.Methods CoCl2was used to induce hypoxia model. Six dishes of ARPE-19cells were randomly divided into hypoxia group and non-hypoxia group. Cells of Hypoxia group was cultured in DMEM with200μM CoCl2to mimic chemical hypoxia while cells of non-hypoxia group cultured in DMEM with same dose of DMOS. Western blot analysis was performed to detect expression of HIF-1α, p-ERK/t-ERK and occludin, ELISA was performed to detect VEGF secreted to culture media.Results There was a significant elevation of HIF-lαin hypoxia group, about1.57times compared with non-hypoxia controls (p<0.001), which indicate that the hypoxia model was successful. Under the hypoxia condition, p-ERK and VEGF increased significantly, while occludin decreased compared with non-hypoxia controls (p<0.001).Conclusion CoCl2induced hypoxia model of ARPE-19cell was successful. Under the hypoxia condition, the MEK/ERK signaling pathway was activated, secretion of VEGF was up-regulated and expression of occludin protein was down-regulated. Objective To investigate the role of inhibiting MEK/ERK signaling pathway for tight junction between ARPE-19cells under hypoxi condition.Methods ARPE-19cells were randomly divided into hypoxia group and non-hypoxia group, and both groups then divided into PD98059(20μM) experimental group and DMSO control group. Western blot analysis was performed to detect expression of HIF-la, p-ERK/t-ERK and occludin, ELISA was performed to detect VEGF secreted to culture media. At the same time, we performed does course of PD98059with20-40μM concentrations.Result MEK/ERK inhibitor PD98059reduced ERK phosphorylation severely in hypoxia group, from5.09times of control reduced to2.44time of control, declined about52.06%, but there was no significant effect in non-hypoxia group (p>0.05). Pretreatment with PD98059was not effective in reducing hypoxia-induced HIF-1α in both hypoxia group and non-hypoxia group. On the other hand it blocked hypoxia-stimulated VEGF secretion from6hour time point in hypoxia group while have no effect on non-hypoxia group. Detect the tight junction protein occludin after hypoxia injury at24hour time point, there was protective effect of PD98059to promote the occludin content from46.7%(p<0.001) to80.3%(p<0.005) compared of non-hypoxia group. At the mean time, this protection is does dependent too, corresponding to the does dependent reduce of ERK phosphorylation.Conclusion Pretreatment with PD98059was not effective in reducing HIF-la under CoCl2induced hypoxia condition. MEK/ERK inhibitor PD98059reduced ERK phosphorylation severely, and it is also a does dependent effect. On the other hand it blocked hypoxia-stimulated VEGF secretion. PD98059protected the tight junction protein occludin from hypoxia injury, at the mean time, this protection is does dependent too, corresponding to the does dependent reduce of ERK phosphorylation. Objective To investigate the protective role of inhibiting MEK/ERK signaling pathway for oBRB break down in OIR mouse.Methods A well-established mouse model of OIR was used to investigate the role of MEK/ERK pathway.18newborn mouses were divided into OIR group, OIR+PD98059group and normal control group. The mouse of OIR+PD98059group was intraperitoneal injected with PD98059at a concentration of6.5mg/kg dissolved in10μL DMSO from12to16postnatal days while the control group intraperitoneal injected with the same volume of DMSO. Western blot analysis was performed to detect expression of HIF-la, p-ERK/t-ERK, VEGF and occludin from P13to P17in RPE layer of mouse eye ball. Immuno-fluorescence microscopy was used to detect the occludin location in RPE layer. The degree of outer-blood retina barrier breakdown in vivo was quantified by counting the number of severely damaged breakpoints of FITC-Dextran in the RPE.Result The western blot indicated that the p-ERK,HIF-1αand VEGF were activated in OIR model. Take the P13for statistic analyze, the increase of these protein were individually1.99times,2.2times and1.78times compare with normal control group (p<0.001). For the occludin, we detected the expression at P17. Western blot indicated that in OIR mouse occludin was decrease to almost36.2%of normal mouse. Using immunohistochemistry to detect the integrality of tight junction, results revealed that both content and localization of occludin at the plasma membrane were reduced in the OIR mice. After intraperitoneal injected with PD98059to OIR mouse, there was not effective in reducing hypoxia-induced HIF-1α in both OIR group and OIR+PD98059group. There were significant decrease of p-ERK and VEGF in OIR+PD98059group compared with OIR group. The Immunocytochemistry and western blot indicated that the localization and content of occludin at the plasma membrane was protected by the PD98059:the content of occludin was up-regulate about35.5%compared with OIR group, and the immune- oreactivity was intense in the in OIR+PD98059mice compared with OIR mouse. Using FITC-Dextran as the marker, we observed that there are several breakpoint that lead the FITC-Dextran leaking from the RPE to out segment of retina. After PD98059intraperitoneal injection, the breakpoint number was3.64±2.04vs10.06±2.91, the relative breakpoint ratio in significantly reduced73.4%.Conclusion MEK-specific inhibitor PD98059can inhibit the activity of MEK/ERK signaling pathway, then decrease the secretion of VEGF in RPE,reduce the losing of occludin, protect the breakdown of oBRB in hypoxia condition.
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