他克莫司治疗碱烧伤致大鼠非特异性角膜炎的疗效及安全性

The efficacy and safety of tacrolimus for treating nonspecific keratitis in rats caused by alkali burn

目的比较他克莫司与地塞米松治疗碱烧伤所致大鼠非特异性角膜炎的疗效和安全性。方法实验研究。24只SD大鼠，体质量180—220g，按随机数字表法选取20只行单眼碱烧伤造模，造模后排除2只。将18只造模成功大鼠随机分为A、B、C碱烧伤组，每组6只，A组使用PBS缓冲液作为阳性对照组（20μl／次，3次，d），B组使用地塞米松滴眼液作为平行对照组，C组使用他克莫司滴眼液作为治疗组（20μl／次，3次，d），D组4只为空白对照组。于碱烧伤后不同时间（1、3、5、7、10、14d）使用裂隙灯显微镜动态观察角膜基质混浊程度、角膜新生血管面积、角膜上皮缺损面积、前房积血和角膜溃疡发生例数并记录，并取角膜组织（7、14d各一只）进行HE染色观察角膜炎症细胞浸润和免疫组化染色检查角膜组织中血管内皮生长因子（VEGF）。应用单因素方差分析和非参数U检验对相应时间点多组或2组数据进行统计分析。结果第14天C组角膜基质较B组清晰（U=2．00，P〈0．05）。B组、C组角膜新生血管面积第7、10、14天均明显较A组小（U=5．00、2．00，P〈O．05；U=O．00、0．00，P〈0．05；U=O．00、0．00。P〈0．05），且B组、C组间差异无统计学意义。A组和C组上皮缺损面积第10、14天明显较B组小（U=0．00、0．00。P〈0．05；U=O．00、0．00，P〈O．05）。全程C组前房积血发生例数最少，无角膜溃疡，B组前房积血例数与A组相当且角膜溃疡最严重。第7、14天HE和免疫组织化学染色示A组角膜基质炎症细胞浸润和VEGF阳性细胞数目最多，B组、C组均较A组少（炎症细胞：U=2．00、0．00，P〈0．05；U=0．00、0．00，P〈0．05。VEGF阳性细胞：U=0．00、0．00，P〈0．05；U=0．00、0．00，P〈0．05）。第7、14天C组较B组角膜基质炎症细胞数目少（U=0．00、0．00，P〈0．05），VEGF阳性细胞第7天2组差异无统计学意义，第14天C组较B组少（U=3．00，P〈0．05）。结论他克莫司对碱烧伤所致大鼠非特异性角膜炎有效，且安全性优于地塞米松。

Objective To investigate the safety and efficacy of tacrolimus （FK506） and dexamethasone in the treatment of nonspecific keratitis in rats induced by alkali burn. Methods A total of 24 rats were randomly divided into four groups in this experimental study： group A （PBS, n=6）, group B （dexamethasone, n=6）, group C （tacrolimus, n=6） and a control group （no treatment, n =4）. Biomicroscopic features, including the opacity of the corneal stroma, the area of corneal neovascularization and epithelial defect, the probability of hyphema, and the number of ulcer cases, were observed and recorded on days 1, 3, 5, 7, 10, 14 after surgery. On days 7 and 14, HE staining was done to observe corneal tissue with infiltrated inflammatory cells. An immunoreaction to vascular endothehal growth factor （VEGF） was detected. Data from multiple groups or two groups at corresponding times were analyzed using a one-way ANOVA and nonparametric Mann-Whitney U test. Results On days 14, the corneal stroma in group C was clear compared to group B （U=2.00, P〈0.05）. The areas of neovascularization in groups B and C on days 7, 10, and 14 were obviously smaller than the area in group A （U=5.00, 2.00, P〈0.05; U=0.00, 0.00, P〈0.05; U=0.O0, 0.00, P〈0.05）. The difference between groups B and C was not statistically significant. On days 10 and 14, the areas in groups A and C with epithelial defects were obviously smaller than the area in group B （U=0.00, 0.00, P〈0.05; U=0.00, 0.00, P〈0.05）. The possibility of hyphema in group C was the smallest and no ulcer cases were detected. The hyphema in group B was as severe as group A and many ulcer cases were detected. HE and immunohistochemical staining on days 7 and 14 revealed that the highest number of inflammatory cells infiltrating the corneal stroma and VEGF positive cells was detected in group A, followed by groups B and C （inflammatory cells, U=2.00, 0.00, P〈0.05; U=0.00, 0.00, P〈0.05. VEGF positive cells, U=0.00, 0.00, P〈0.05; U=0.00, 0.00, P〈0.05）. There were fewer inflammatory cells in group C than in group B on days 7 and 14 （U=0.00, 0.00, P〈0.05） and the difference in VEGF positive cells on day 7 was not statistically significant. On day 14, there were fewer VEGF positive cells in group C than in group B （U=3.00, P〈0.05）. Conclusion Tacrolimus is effective for nonspecific keratitis induced by alkali burn and superior to dexamethasone for safety.