摘要背景 感染性角膜炎是中国常见的致盲眼病,临床上常见炎症失控反应的发生,其发病机制及治疗方法尚不明确. 目的 研究兔角膜铜绿假单胞菌性感染性炎症失控反应的临床表现和发生机制,探讨多西环素对兔角膜炎症失控反应的治疗作用.方法 选取60只1月龄新西兰白兔,采用铜绿假单胞菌液兔右眼角膜基质内注射法感染动物角膜,采用加替沙星滴眼液频繁点眼3d后,选取42只角膜炎症不能缓解或加重的眼作为角膜炎症失控反应动物模型.采用抽签法将模型眼随机分为加替沙星点眼组(15眼)、联合点眼组(加替联合多西环素点眼组,15眼)和平衡盐溶液(BSS)对照组(12眼),分别于分组后第1天用相应溶液点眼,1次/2 h,每日8次,连续用药14 d.裂隙灯显微镜下观察各组兔眼点眼后1、3、7和14d的眼部表现并进行炎症评分,计算角膜浸润面积;在上述各时间点获取各组兔眼角膜组织,采用常规组织病理学检查观察兔角膜组织的炎症细胞浸润情况;采用免疫组织化学法检测各组角膜组织中基质金属蛋白酶-2(MMP-2)和MMP-9的表达;采用ELISA法测定各组角膜匀浆中肿瘤坏死因子d(TNF-α)和白细胞介素1β(IL-1β)质量浓度的变化.结果 加替沙星点眼组和联合点眼组兔眼点眼后7d眼部炎症评分明显低于点眼前,角膜浸润面积明显较点眼前缩小,差异均有统计学意义(均P＜0.05);点眼后7d,加替沙星点眼组和联合点眼组眼部炎症评分明显低于BSS对照组,角膜浸润面积明显小于BSS对照组,差异均有统计学意义(均P＜0.05).组织病理学研究显示,联合点眼组兔眼点眼后14d,角膜组织中炎症细胞浸润明显少于点眼后5d,可见角膜胶原纤维增生和重新排列.免疫组织化学染色显示,联合点眼组点眼后5d可见角膜组织中MMP-2、MMP-9呈强阳性表达,随着点眼时间延长其表达强度减弱,点眼后14 d角膜组织中MMP-2、MMP-9表达量明显低于点眼后5d和BSS对照组,差异均有统计学意义(均P＜0.05).ELISA测定显示,点眼后5d,联合点眼组兔角膜组织中TNF-α及IL-1β质量浓度明显低于BSS对照组,差异有统计学意义(P=0.00、0.03),点眼后14d角膜中TNF-α及IL-1 β质量浓度明显低于点眼后5d和7d,差异均有统计学意义(均P＜0.05). 结论 采用铜绿假单胞菌角膜基质内注射并用加替沙星滴眼液频繁点眼法可成功建立角膜炎症失控反应动物模型.多西环素点眼可抑制炎症失控反应模型眼角膜中促炎因子及MMPs的释放,从而促进角膜组织的修复,对感染性角膜炎症失控反应损伤发挥治疗作用.

abstractsBackground Infectious keratitis is common blinding eye disease in China.Inflammatory runaway reaction often occurs in infectious keratitis, and its mechanism and treating approach are worthy of research.Objective This study was to investigate the clinical manifestation,mechanism and treatment outcome of doxycycline in overwhelming inflammation induced by Pseudomonas aeruginosa keratitis.Methods Pseudomonas aeruginosa suspension was intrastromal injected in 60 right eyes of 60 New Zealand white rabbits,and gatifloxacin eye drops was frequent instilled for consecutive 3 days to establish corneal inflammatory runaway reaction models,and 42 eyes with worse keratitis were defined as overwhelming inflammation.Then the models were randomly divided into gatifloxacin treatment group (15 rabbits), combined treatment group (gatifloxacin with doxycycline treatment, 15 rabbits) and balance salt solution (BSS) control group (12 rabbits), and corresponding eye drops was topically administered in the rabbits 8 times per day for 14 days.The symptoms of the models were observed under the slit lamp microscope on day 1,3,7 and 14 after treatment,and the corneal infiltration area was calculated.The corneal morphology was examined by using hematoxylin-eosin staining.The expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the corneal tissue were examined by immunohistochemistry.The contents of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in corneal homogenate were detected by ELISA.The use and care of the animals complied with the ARVO Statement.Results The inflammatory scores were lower and infiltration area of cornea was smaller 7 days after treatment than those before treatment in the combined treatment group and gatifloxacin treatment group (both at P＜0.05).Hematoxylin-eosin staining result showed that the inflammation response of cornea subsided 14 days in comparison with 5 days after treatment in the combined treatment group, with the proliferation and rearrangement of fiberal tissue.The MMP-2 and MMP-9 were strongly expressed in corneal tissue with overwhelming inflammation in day 5 after treatment, however, the expression intensity weakened in 14 days after treatment in the combined treatment,showing significant differences in the absorbance between them (all at P＜0.05).ELISA assay showed that the contents of TNF-α and IL-1β in corneal tissue were significantly lower in the combined treatment group than those in the BSS group (P =0.00,0.03).In addition, contents of TNF-α and IL-1β in corneal tissue were significantly declined at day 14 in comparison with day 5 and day 7 after treatment in the combined treatment group (all at P＜0.05).Conclusions Overwhelming inflammatory keratitis models can be successfully established by corneal intrastromal injection of Pseudomonas aeruginosa suspension and immediately frequent 3-day instillation of gatifloxacin eye drops in rabbits.The topical application of doxycycline plays treating effect on overwhelming inflammatory keratitis by inhibiting the release of pro-inflammatory cytokines and MMPs.