二十碳五烯酸对大鼠角膜新生血管核转录因子κB及细胞因子表达的抑制作用

Inhibitory effects of eicosapentaenoic acid on expression of nuclear factor-KB and cytokine in rat corneal neovascularization

背景角膜新生血管（CNV）是炎症性角膜病变导致视力下降和角膜移植后发生排斥反应的重要原因，其发生机制和治疗一直是国内外角膜病研究的热点。目的探讨二十碳五烯酸（EPA）对碱烧伤大鼠CNV核转录因子KB（NF—KB）、白细胞介素1α（IL一1αt）、IL一6表达的影响及作用机制。方法用浸有1mol／LNaOH的3mm圆形滤纸贴附于72只SD大鼠的右眼角膜中央30s制作角膜碱烧伤模型，用随机数字表法将大鼠随机分为3个组，另6只匹配的正常大鼠作为正常对照组。碱烧伤0．02mgEPA治疗组和碱烧伤0．03mgEPA治疗组大鼠分别于碱烧伤后即刻结膜下注射0．04ml剂量为0．02mg或0．03mgEPA，碱烧伤模型组结膜下注射0．04ml生理盐水，正常对照组大鼠未行结膜下注射。大鼠干预后每天裂隙灯下观察CNV的生长面积和角膜水肿。分别于造模后24h，4、7、14d过量麻醉处死动物后制备角膜标本，采用苏木精一伊红染色法检测各组大鼠角膜的组织病理学变化，免疫组织化学方法检测各组大鼠角膜组织中CD34的表达以计数血管内皮细胞，用逆转录聚合酶链反应（RT—PCR）及蛋白印迹法分别检测各组大鼠角膜组织中IL-1α-mRNA和IL．6mRNA的表达及NF—KBp65的蛋白表达。结果裂隙灯下见造模后2～4d，CNV缓慢生长，角膜水肿，上皮缺损；造模后7～10d，CNV面积增加，角膜水肿减轻；造模后14d，CNV面积最大，血管变细。苏木精一伊红染色显示，碱烧伤后7d碱烧伤组角膜上皮部分缺损，基质层水肿明显，可见较多炎性细胞及大量CNV；碱烧伤0．03mgEPA治疗组与碱烧伤0．02mgEPA治疗组均可见角膜上皮修复，基质层水肿减轻，少量炎性细胞，未见CNV。碱烧伤后7d和14d，碱烧伤0．02mgEPA治疗组CNV相对面积分别为（15．80±6．43）％、（11．06±2．14）％，碱烧伤0．03mgEPA治疗组为（16．10±7．41）％、（11．06±2．51）％，均明显小于碱烧伤组的（84．74±7．77）％、（89．63±7．50）％，差异均有统计学意义（P〈0．05）。碱烧伤后7d，碱烧伤组CD34在CNV的内皮细胞中呈强阳性表达，而在碱烧伤0．02mgEPA治疗组、0．03mgEPA治疗组角膜中未见表达。RT—PCR检测结果表明，碱烧伤后4d，碱烧伤0．02mgEPA治疗组、0．03mgEPA治疗组IL一1α、IL一6mRNA在角膜组织中的表达量明显低于碱烧伤组，蛋白印迹检测证实NF—KBp65蛋白在角膜组织中的表达低于碱烧伤组，差异均有统计学意义（P〈0．05）。结论EPA能够抑制NF—KB、IL一1α、IL一6的表达，从而抑制碱烧伤后CNV的生长。

Background Corneal neovascularization （CNV） is an important cause of visual impairment and graft rejection after allograft corneal transplantation in inflammatory corneal diseases. The mechanisms and therapy relating to CNV are intensely investigated at all times. Objective This study was to evaluate the effect of eicosapentaenoic acid （EPA） on CNV induced by alkali cauterization and its mechanism. Methods The animal models of corneal neovasculation were induced in the right eyes in 72 Sprayue-Dawley rats by putting a piece of 3 mm filter paper with 1 mol/L NaOH at the center of the cornea for 30 seconds. The rats were then divided randomly into the O. 02 mg EPA treatment group （24 rats） ,0.03 mg EPA treatment group （24 rats） , model group （24 rats） andnormal group （6 rats）. EPA of 0. 04 ml with doses of 0.02 mg or 0.03 mg or saline solution of 0.04 ml was injected subconjunctivally in model rats and immediately after cauterization. The presence of CNV and corneal edema were observed daily by slit lamp biomicroscope. 1,4,7 and 14 days after operation, corneal histopathological examination was performed by hematoxylin and eosin staining. The vascular endothelial ceils were stained with CD34 by immunohistochemistry,and the expression of IL-lα,IL-6 mRNA and the nuclear factor-KBp65 （NF-KBp65） proteins was measured by reverse transcription polymerase chain reaction （RT-PCR） and Western blot analysis. The use of animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by Hebei Province（version 1998）. Results Under the slit lamp, CNV grew slowly from days 2-4 with obvious corneal edema and defect of epithelium. Larger CNV area and less edema were seen from days 7-10. Maximal vessel growth was observed 14 days after injury with thinner vesseis in the model group. Histological examination showed that part of the corneal epithelium was damaged;serious corneal edema,more inflammatory cells and a lot of CNV in the stroma were presented in the model group. However, repairing of the corneal epithelium without CNV,light corneal edema and less inflammatory cells were found in both the 0.02 mg EPA and 0. 03 mg EPA treatment groups 7 days after alkali cauterization. The relative area of CNV in the 0.02 mg EPA treatment group was （ 15.80±6.43 ） % and （ 11.06± 2. 14）% ,and that in the 0.03 mg EPA treatment group was （16. 10＋7.41）% and （11.06＋2.51）% ,showing significant reduction in comparison with the model group [ （84. 74±7.77）% and （89.63±7.50） % ] 7 days and 14 days after operation （ P〈0. 05 ）. Stronger expression of CD34 in the vascular endothelial cells of the cornea stroma was observed in the model group and an absence of CD34 was observed in the EPA-treated groups on the 7th day. RT-PCR revealed that the expression of IL-lc~ mRNA and IL-6 mRNA was lower in the EPA treatment groups than the model group （P〈0.05） ,and Western blot analysis showed that the expression of NF-KB/p65 in the corneas in the EPA treatment groups was significantly lower than that in the model group on the 4th day after operation （ P〈0. 05 ）. Conclusion Topical application of EPA suppresses CNV induced by alkali burn possibly by inhibiting the expression of NF-KB,IL-lα and IL-6.