BET溴域抑制通过阻断VEGFR2介导的PAK1和eNOS激活抑制糖尿病大鼠视网膜病变机制

BET bromine suppression inhibits the mechanism of retinopathy in diabetic rats by blocking VEGFR2-mediated PAK1 and eNOS activation

目的探究BET溴域抑制通过阻断血管内皮生长因子受体2(VEGFR2)介导的p21激活激酶1(PAK1)和内皮型一氧化氮合成酶(eNOS)激活抑制糖尿病大鼠视网膜病变机制。方法选择36只雄性SD大鼠,随机选取10只大鼠作为对照组,余下大鼠采用单次注射链脲霉素60 mg/kg建立糖尿病模型。将建模成功的20只大鼠随机分为模型组和BET抑制组,每组10只。对照组大鼠不建模。BET抑制组大鼠建模后,腹腔注射80 mg·kg^(-1)·d^(-1)剂量的JQ1。使用ZEISS数码显微镜和免疫组织化学分析大鼠视网膜心血管生成。通过蛋白质印迹法分析视网膜屏障功能相关蛋白的表达。通过FITC-葡聚糖泄漏实验检测大鼠内外视网膜屏障完整性。通过RT-qPCR分析大鼠视网膜炎症因子的表达。通过酶联免疫吸附试验试剂盒检测大鼠房水VEGF浓度。通过蛋白质印迹法分析视网膜VEGFR2/PAK1/eNOS的蛋白表达。结果与对照组相比,模型组和BET抑制组视网膜新血管生成量、CD34表达、albumin蛋白表达、内外视网膜FITC-葡聚糖泄漏量、IL-1β、iNOS和ICAM-1 mRNA表达、VEGF浓度、视网膜P-PAK1、P-VEGFR2和eNOS蛋白表达均升高,ZO-1和occludin蛋白表达均降低,差异均有统计学意义(P<0.05);与模型组相比,BET抑制组视网膜新血管生成量、CD34表达、albumin表达、内外视网膜FITC-葡聚糖泄漏量、IL-1β、iNOS和ICAM-1mRNA表达、VEGF浓度、P-PAK1、P-VEGFR2和eNOS蛋白表达均降低,ZO-1和occludin蛋白表达均升高,差异均有统计学意义(P<0.05)。结论BET溴化域通过调节VEGFR2介导的PAK1和eNOS信号通路激活抑制血管生成,降低血管通透性,改善糖尿病大鼠视网膜病变。抑制BET溴化域的策略可能为限制血管生成相关疾病提供一种新的治疗方法。

Objective To explore the mechanism of BET bromine domain inhibition on diabetic rat retinopathy by blocking vascular endothelial growth factor receptor 2(VEGFR2)-mediated activation of p21-activated kinases1(PAK1)and nitric oxide synthase(eNOS).Methods Thirty six male SD rats were selected,10 of them were randomly selected as the control group,and the rest of the rats were given a single injection of streptozotocin 60 mg/kg to establish the diabetes model.Twenty successfully modeled rats were randomly divided into a model group and a BET inhibition group,with 10 rats in each group.The control group rats were not modeled.After modeling the BET inhibition group rats,a dose of 80 mg·kg^(-1)·d^(-1) JQ1 was intraperitoneally injected.Rat retinal cardiovascular generation was analyzed using ZEISS digital microscopy and immunohistochemistry.Expression of proteins related to retinal barrier function was analyzed by westen blotting.The integrity of the retinal barrier inside and outside rats was determined by FITC-glucan leak assay.The expression of rat retinal inflammatory cytokines was analyzed by RT-qPCR.Rat aqueous VEGF concentration was determined by an ELISA kit.Protein expression of retinal VEGFR2/PAK1/eNOS was analyzed by protein blotting.Results Compared with the control group,retinal new blood vessel production,CD34 expression,albumin protein expression,FITC-glucan leakage,IL-1β,iNOS and ICAM-1 mrna expression,VEGF concentration,retinal P-PAK1,P-VEGFR2 and eNOS protein expression were increased in model group and BET inhibition group,ZO-1 and occludin expression were decreased,the differences were statistically significant(P<0.05).And compared with model group,retinal new blood vessel production,CD34 expression,albumin expression,FITC-glucan leakage,IL-1β,iNOS and ICAM-1 mrna expression,VEGF concentration,P-PAK1,P-VEGFR2 and eNOS protein expression in BET inhibition group were decreased,ZO-1 and occludin expression were increased,the differences were statistically significant(P<0.05).Conclusion BET brominated domain inhibits angiogenesis,reduces vascular permeability and improves retinopathy in diabetic rats by regulating VEGFR2-mediated activation of PAK1 and eNOS signaling pathways.Strategies that inhibit the brominated domain of BET may provide a new therapeutic approach for limiting angiogenesis related diseases.