蛋白酪氨酸激酶抑制剂对血-视网膜屏障损害的影响及其作用机制

背景白介素1p（IL-1β）等细胞因子与血一视网膜屏障损害关系密切，细胞因子的信号传导主要由蛋白酪氨酸激酶受体传导通路介导。目的研究蛋白酪氨酸激酶（PTK）抑制剂-Genistein对IL-1β诱导血一视网膜屏障损害的影响，探讨PTK信号转导通路在血一视网膜屏障损害中的作用和机制。方法健康sD大鼠24只玻璃体腔内注射2mg／LIL-1β10ng建立血一视网膜屏障损害的动物模型，再随机分为IL-1β模型组和0．2、1、5P,gGenistein干预组。Genistein干预组大鼠于造模后立即用1μl各剂量Genistein行玻璃体腔内注射，IL-1β模型组应用1μ1DMSO以同样的方法注射。分别于注射后3h和47h每组各取2只鼠经颈静脉10S内快速注射Evansblue（45ing／kg）并收集动脉血，检测血一视网膜屏障通透性的变化；分别于玻璃体注射后4h和48h收集视网膜，用苏木精一伊红染色观察视网膜的组织学变化，RT-PCR法观察视网膜中IL．8和单核细胞趋化蛋白-1（MCP-1）mRNA表达的变化，用免疫组织化学法染色观察视网膜中MCP-1蛋白表达的变化。结果大鼠玻璃体腔注射Genistein后，视网膜与血浆Evansblue比值明显下降，各组间的总体比较差异有统计学意义（F4h=7．510，P=0．010；F48h=5．960，P=0．019）；随着玻璃体腔注射Genistein的剂量增加，Genistein各剂量组的视网膜与血浆Evansblue比值逐渐下降，与IL-1β比较差异均有统计学意义（P〈0．05）。视网膜苏木精一伊红染色结果表明，IL-1β在玻璃体腔注射后4h视网膜血管扩张并有炎性细胞浸润，注射48h后上述症状减轻，而Genistein干预组视网膜血管反应轻，无明显炎性细胞浸润。RT-PCR法结果显示，各剂量Genistein组玻璃体腔注射后视网膜中IL-8和MCP-1mRNA的表达量均明显减少，与IL-1β组比较差异均有统计学意义（P〈0．05）。免疫组织化学染色提示Genistein大鼠神经视网膜中MCP-1蛋白表达与IL-1β组比较明显减弱。结论PTK抑制剂Genistein通过抑制IL-1β诱导的视网膜趋化因子的分泌及减少白细胞在视网膜中的浸润，进而减轻IL-1β诱导的血-视网膜屏障损害。Genistein的作用强度呈剂量依赖性。

Effects and mechanism of protein tyrosine kinase inhibitor on blood-retinal barrier breakdown

Background Several cytokines,especially interleukin-1β (IL-β) involve in the breakdown of blood-retina barrier,and the signal of cytokine is transduced through protein tyrosine kinase (PTK) pathway.Objective This study was to investigate the effects of PTK inhibitor,Genistein,on IL-1β-induced blood-retinal barrier breakdown and possible mechanism.Methods The animal models of blood-retinal barrier breakdown were induced through intravitreal injection of IL-1β(10ng) in 24 clean healthy SD rats and assigned to IL-1β group and Genistein group.5μl IL-1β+1μl Genistein with 0.2,1,5μg were intravitreally injected in 12 model rats and 5μl IL-1β (2mg/L)+1μl DMSO was used at the same way in other 12 models.Evans Blue was injected in rats via jugular vein in 1 hour before sacrifice of animals and the arterial blood was collected for the detect of serum Evans Blue.The retinas of the rats were obtained in 4 and 48 hours after injection of vitreous cavity to assay the content of Evans Blue in retina.The changes of vessels and infiltration of inflammatory cells were observed with hematoxylin-eosin stain.RT-PCR was employed to determine the expression of IL-8 and MCP-1mRNA in neuroretina after intravitreal injection.Expression of MCP-1 protein was localized by immunohistochemistry.Results The ratio of retinal Evans Blue and plasma Evans Blue was significantly decreased after intravitreal injection of different doses of Genistein among Genistein groups and IL-8 group with a statistical difference (4 hours:F=7.510,P=0.010;48 hours:F=5.960,P=0.019).With the increase of time after injection of Evans Blue,the ratio of retinal Evans Blue and plasma Evans Blue was gradually reduced in comparison to IL-1β group (P＜0.05).After injection of IL-1β,the dilation of retinal vessel and adhesion of leukocyte to vessel wall were seen under the light microscope,but infiltration of less inflammatory cells was found in Genistein group.The expressions of IL-8 and MCP-1mRNA were obviously declined in retina of rats in Genistein groups compared with IL-8 group (P＜0.05).Immunochemistry indicated that the expression of MCP-1 protein in neuroretina tissue was weaker in Genistein group compared with IL-8 group.Conclusion PTK inhibitor,Genistein,can decrease IL-1β-induced permeability of vessel and maintain the integrity of blood-retinal barrier by downregulating the expression of chemokines and infiltration of leukostasis in retinal vessels.This study imply that PTK pathway plays an important role in IL-1β-induced blood-retinal barrier breakdown.