MEK/ERK和PI3K/Akt通路对大鼠脉络膜新生血管中DDR2和MMP-13表达的调控作用

Modulation of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in choroidal neovascularization

背景 研究表明盘状结构域受体2（DDR2）和基质金属蛋白酶-13（MMP-13）在肿瘤新生血管发生中具有重要作用,是相关疾病治疗的关键靶点,但DDR2和MMP-13在脉络膜新生血管（CNV）发生中的作用尚不清楚.目的 探讨DDR2和MMP-13在大鼠CNV中的表达特点以及MEK/ERK和PI3 K/Akt通路对DDR2及MMP-13表达的调控作用.方法 采用532 nm倍频激光视网膜光凝法制备棕色挪威（BN）大鼠CNV模型,将78只BN模型鼠按照随机数字表法分为正常对照组（n=7）、模型对照组（n=39）、PD98059（MEK1抑制剂）组（n=16）和LY294002（PI3K抑制剂）组（n=16）,PD98059组和LY294002组大鼠分别于光凝后1d、7d玻璃体内注射5 mmol/L PD98059 3μl或5 mmol/L LY294002 3μl.分别于光凝前和光凝后1、3、7、14 d用过量麻醉法处死动物并制备样本和切片,采用Western blot法检测各组大鼠眼杯中ERK、p-ERK、Akt和p-Akt蛋白的表达变化,采用逆转录PCR（RT-PCR）法检测各组大鼠眼杯中DDR2 mRNA和MMP-13 mRNA的表达变化,采用组织病理学方法测定各组大鼠CNV厚度,采用免疫组织化学和免疫荧光染色法分别定位和测定大鼠CNV区域DDR2和MMP-13的表达.结果 光凝后3d,光凝局部细胞增生,光凝后7 d CNV形成,至光凝后14 d CNV趋于稳定.免疫组织化学染色结果显示,DDR2在正常大鼠视网膜血管内皮细胞层、视网膜神经节细胞（RGCs）层和内核层细胞中呈弱表达,但在CNV中呈强阳性表达;MMP-13在正常对照组大鼠视网膜内界膜层、光感受器层和巩膜层均呈强阳性表达,在模型对照组中呈强阳性表达.免疫荧光结果显示,CNV组织中DDR2和MMP-13共表达.RT-PCR结果显示,模型对照组光凝后1、3、7、14 d DDR2 mRNA相对表达水平分别为55.22±4.03、47.74±2.23、14.82±4.56和5.59±2.47,MMP-13 mRNA相对表达水平分别为25.54±3.83、43.51±4.36、10.90±4.00和5.23±3.23,与正常对照组相比,差异均有统计学意义（均P＜0.05）.免疫荧光结果显示,模型对照组光凝后3、7、14 d大鼠CNV区DDR2相对荧光密度单位（RFU）值分别为2.73±0.53、5.21±0.31和3.22±0.33,MMP-13 RFU值分别为1.66±0.17、3.57±0.44和2.67±0.21,组间总体比较差异均有统计学意义（F=81.01,P＜0.05;F=40.31,P＜0.01）;PD98059组和LY294002组光凝后14 d CNV区DDR2 RFU值分别为1.14±0.19和1.03±0.14,与模型对照组比较,差异均有统计学意义（均P＜0.05）,MMP-13RFU值分别为1.37±0.25和1.24±0.20,与模型对照组比较,差异均有统计学意义（均P＜0.05）.Western blot结果显示,模型对照组光凝后7d眼杯中p-ERK和p-Akt水平升高,与正常对照组大鼠相比差异均有统计学意义（均P＜0.05）;至光凝后14 d恢复至近正常水平,与正常对照组比较差异均无统计学意义（均P＞0.05）.PD98058组大鼠眼杯p-ERK水平明显下降,注射后7d、14 d PD98058组大鼠眼杯中p-ERK表达的抑制率分别为71.58％和65.21％,PD98058组7d与模型对照组7d比较、PD98058组14 d与模型对照组14 d比较,差异均有统计学意义（均P＜0.05）.LY294002组大鼠眼杯中p-Akt表达水平明显下降,LY294002注射后7d、14d大鼠眼杯中p-Akt表达的抑制率分别为65.62％和67.04％,LY294002组7d与模型对照组7d比较、LY294002组14d与模型对照组14 d比较,差异均有统计学意义（均P＜0.05）.各组眼杯中ERK和Akt的表达水平总体比较差异均无统计学意义（F=0.62,P＞0.05;F=0.81,P＞0.05）.模型对照组、PD98059组和LY294002组光凝后14 d大鼠CNV厚度分别为（119.19±18.03）、（51.00±11.29）和（59.18±9.00） μm,总体比较差异有统计学意义（F=29.376,P＜0.05）,与模型对照组比较,PD98059和LY294002注射后CNV厚度分别减少57.21％和50.34％,差异均有统计学意义（均P＜0.05）.结论 DDR2和MMP-13在大鼠CNV中表达上调,参与CNV的形成;MEK/ERK和PI3K/Akt通路通过参与调控DDR2和MMP-13在CNV局部的表达而抑制CNV的发生和进展.

Background It is estimated that discoidin domain receptor 2 （DDR2） and matrix metalloproteinase-13 （MMP-13） play an important role in the development of tumor angiogenesis.However,their effects on choroidal neovascularizaiton （CNV） have not been clarified yet.Objective This study was to observe the expression pattern of DDR2 and MMP-13 in CNV and to further investigate the regulation role of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in CNV.Methods CNV models were established in 78 Brown Norway （BN） rats by retinal photocoagulation with 532 nm laser.Then the animals were randomly divided into the normal control group （n =7）,the model control group （n =39）,PD98059 （MEK1 inhibitor） group （n =16） and LY294002 （PI3K inhibitor） group （n =16）,and 5 mmol/L PD98059 or 5 mmol/L LY294002 3 μl was intravitreally injected 1 day and 7 days after photocoagulation in the PD98059 group or LY294002 group.The expression of DDR2 and MMP-13 mRNA and proteins in the CNV area were detected by using reverse transcription PCR （RT-PCR）,and the expression levels of p-ERK/ERK and p-Akt/Akt protein were detected by Western blot assay.CNV thickness was measured by pathological examination 14 days after photocoagulation,and the changes of CNV thickness,the expression levels of DDR2 and MMP-13 in CNV were compared among the model control group,PD98059 group and LY294002 group.Results Three days after photocoagulation,the cells within the lasered lesions proliferated,then CNV formed 7 days after photocoagulation and became stable 14 days after photocoagulation.Immunohistochemistry staining indicated that DDR2 was weakly expressed in the cells of ganglion cell layer,inner nuclear layer and vascular endothelial cells;while MMP-13 was strongly expressed in the cells of the inner limiting membrane layer,photoreceptor layer and sclera layer.Both DDR2 and MMP-13 were strongly expressed in CNV area.Double immunofluorescence staining revealed that MMP-13 and DDR2 co-expression in CNV area.RT-PCR revealed that the relative DDR2 mRNA levels at 1 day,3 days,7 days and 14 days after photocoagulation were 55.22±4.03,47.74±2.23,14.82±4.56 and 5.59±2.47 respectively,while the relative MMP-13 mRNA levels were 25.54±3.83,43.51±4.36,10.90±4.00 and 5.23±3.23 respectively.Compared with the normal control group,the expression of DDR2 and MMP-13 were significantly increased （all at P〈0.05）.Immunofluorescence staining showed that the relative fluorescence unit （RFU） values at 1 day,3 days,7 days and 14 days after photocoagulation were 2.73±0.53,5.21±0.31 and 3.22±0.33 for DDR2 and 1.66±0.17,3.57±0.44,2.67±0.21 for MMP-13,respectively.The RFU values in the PD98059 group and LY294002 group 14 days after photocoagulation were 1.14±0.19,1.03±0.14 for DDR2 and 1.37±0.25,1.24±0.20 for MMP-13,respectively.Compared with the model control group,the differences were statistically significant （both at P〈0.05）.Western blot results showed that,compared to the normal control group,the expression levels of p-ERK and p-Akt pretein increased at day 7 after photocoagulation （both at P〈0.05）,and returned back to the baseline at day 14 after photocoagulation （both at P〉0.05）.Both PD98059 and LY294002 treatment were able to attenuate the thickness of CNV to 57.21% and 50.34% at day 14 after photocoagulation and further decrease the expression levels of DDR2 and MMP-13 in CNV （all at P〈0.05）.Conclusions The expressions of DDR2 and MMP-13 up-regulate in laser-induced CNV.MEK/ERK and PI3K/Akt pathways suppress the development of CNV by regulating the expression of DDR2 and MMP-13.