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中华眼科医学杂志(电子版)

中华眼科医学杂志(电子版) ›› 2018, Vol. 08 ›› Issue (05) : 216 -222. doi: 10.3877/cma.j.issn.2095-2007.2018.05.004

所属专题： 文献；

论著

吡非尼酮抑制大鼠角膜急性碱烧伤后新生血管的实验研究
邹志康1, 吴共发2,†(), 邱丽浈3, 刘钰君2, 曾宇婷2, 黄绮亭2   
  1. 1. 511300　广州市增城区人民医院急诊科
    2. 511300　广州市增城区人民医院病理科
    3. 511300　广州市增城区人民医院体检中心
  • 收稿日期:2018-10-08 出版日期:2018-10-28
  • 通信作者: 吴共发
  • 基金资助:
    广东省医学科研基金(A2016422); 广州市增城区人民医院优秀医学人才培育基金(2017－YX－001)

The study of the inhibiting effect of pirfenidone on rat corneal neovascularization by acute alkali burn

Zhikang Zou1, Gongfa Wu2,†(), Lizhen Qiu3, Yujun Liu2, Yuting Zeng2, Qiting Huang2   

  1. 1. Emergency department, Zengcheng District People's Hospital of Guangzhou City, Guangzhou 511300, China
    2. Department of Pathology, Zengcheng District People's Hospital of Guangzhou City, Guangzhou 511300, China
    3. Physical examination center, Zengcheng District People's Hospital of Guangzhou City, Guangzhou 511300, China
  • Received:2018-10-08 Published:2018-10-28
  • Corresponding author: Gongfa Wu
  • About author:
    Corresponding author: Wu Gongfa, Email:
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引用本文：

邹志康, 吴共发, 邱丽浈, 刘钰君, 曾宇婷, 黄绮亭. 吡非尼酮抑制大鼠角膜急性碱烧伤后新生血管的实验研究[J]. 中华眼科医学杂志(电子版), 2018, 08(05): 216-222.

Zhikang Zou, Gongfa Wu, Lizhen Qiu, Yujun Liu, Yuting Zeng, Qiting Huang. The study of the inhibiting effect of pirfenidone on rat corneal neovascularization by acute alkali burn[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2018, 08(05): 216-222.

目的

研究吡非尼酮(PFD)对大鼠角膜急性碱烧伤后角膜新生血管(CNV)的抑制作用。

方法

碱烧伤法制备CNV模型，模型制作成功后，将健康的斯泼累格·多雷(SD)大鼠20只随机平均分为PFD组和磷酸缓冲盐溶液(PBS)组，PFD组给予3 mg/ml PFD滴眼液，PBS组给予磷酸缓冲盐溶液，4次/d、连续14 d。裂隙灯显微镜观察角膜炎症反应评分、角膜混浊程度和角膜新生血管生长情况。第14 d处死大鼠取角膜，行苏木精－伊红(HE)染色法染色观察角膜病理形态，免疫组化CD34染色检测角膜微血管密度(MVD)。角膜炎症反应和新生血管情况在不同时间点，PFD和PBS两组间的比较采用两因素重复测量方差分析。

结果

第14 d PFD组的角膜混浊评分是(1.00±0.00)分，PBS组的角膜混浊评分是(3.40±0.52)分，PFD组的角膜混浊评分低于PBS组，差异有统计学意义(t＝－14.697，P<0.05)。第7 d和第14 d PFD组的角膜炎症评分分别是(3.50±0.53)分和(2.2±0.42)分，PBS组的角膜炎症评分分别是(4.9±0.57)分和(3.3±0.48)分，PFD组的角膜炎症评分均低于PBS组，差异有统计学意义(t＝－5.715，－5.425；P<0.05)。PFD组，随时间延长，角膜透明度增加，炎症下降。该组第7 d与第3 d比较，差异有统计学意义(t＝7.56，P<0.05)。第14 d分别与第3 d及第7 d比较，差异均有统计学意义(t＝13.17，6.08；P<0.05)。PBS组，第7 d与第3 d比较，差异有统计学意义(t＝2.64，P<0.05)。第14 d分别与第3 d及第7 d比较，差异均有统计学意义(t＝8.74，6.79；P<0.05)。第3 d、第7 d及第14 d PFD组的CNV面积分别是(6.06±0.93)mm2、(17.94±1.89)mm2及(23.89±1.84)mm2，PBS组的CNV面积分别是(9.08±1.42)mm2、(27.11±2.02)mm2及(31.01±2.04)mm2，PFD组的CNV面积均低于PBS组，差异均有统计学意义(t＝－5.609，－10.452，－8.202；P<0.05)。PFD组大鼠角膜CNV模型的CNV面积，第7 d比第3 d增大，差异有统计学意义(t＝17.83，P<0.05)；第14 d比第3 d及第7 d，差异均有统计学意义(t＝27.38，7.13；P<0.05)。PBS组，第7 d与第3 d的CNV面积比较，差异有统计学意义(t＝23.09，P<0.05)；第14 d比第3 d及第7 d，差异均有统计学意义(t＝27.90，4.29；P<0.05)。HE病理染色显示PBS组的角膜基质有大量炎症细胞浸润及纤维组织增生，而PFD组炎症细胞数量极少，基质排列较规则。免疫组化CD34染色结果显示PFD组的MVD为(3.17±1.17)条/mm2，PBS组的MVD为(10.83±2.48)条/mm2，PFD组的MVD低于PBS组，差异有统计学意义(t＝－6.842，P<0.05)。

结论

PFD能减轻大鼠角膜急性碱烧伤模型中的角膜炎症反应，并抑制角膜新生血管形成，有望为临床防治角膜新生血管类疾病提供一种新方法。

关键词: 吡非尼酮, 角膜新生血管, 碱烧伤
Objective

To study the inhibitory actin of pirfenidone (PFD) on corneal neovascularization (CNV) in rat caused by acute alkali burn.

Methods

Alkali burn was used to prepare a CNV model. Then twenty adult healthy Sprague Dawley (SD) rats were equally divided into PFD and phosphate buffered saline (PBS) two groups. The PFD group was treated with pirfenidone eye drops 3 mg/ml and PBS group was treated with phosphate buffered saline, the treatments were administered daily 4 times and continued for 14 days. The level of corneal inflammation reaction, corneal opacity, and the growth of CNV were analyzed by the slit-lamp microscopy. At the 14th day of post-therapy all rats were killed and remove their corneas. The structure of corneal tissue by hematoxyli-eoitaiig (HE) staining. The MVD was observed by immunohistochemical CD34 staining. Corneal inflammation reaction and the growth of CNV at different time periods, the comparison between PFD and PBS two groups adopt two factor repeated measurement of variance analysis.

Results

At the 14th day, the corneal opacity of PFD group was (1.00±0.00) scores, the corneal opacity of PBS group was (3.40±0.52) scores, PFD group compared with PBS group, the corneal opacity was effectively inhibited (t=-14.697, P<0.05). At the 7th day and the 14th day, the level of corneal inflammation reaction of PFD group were (3.50±0.53) scores and (2.2±0.42) scores, the level of corneal inflammation reaction of PBS group were (4.9±0.57) scores and (3.3±0.48) scores, PFD group compared with PBS group, the corneal inflammation reaction were effectively inhibited (t=-5.715, -5.425; P<0.05). For PFD group, With the extension of time, increased corneal transparency and inflammation decline. The 7th day compared with the third day, the difference was statistically significant (t=7.56, P<0.05). The 14th day compared with the third day and the 7th day, the differences were all statistically significant (t=13.17, 6.08; P<0.05). For PBS group, The 7th day compared with the third day, the difference was statistically significant (t=2.64, P<0.05). The 14th day compared with the third day and the 7th day, the differences were all statistically significant (t=8.74, 6.79; P<0.05). At the third day, the 7th day and the 14th day, the CNV areas of PFD group were (6.06±0.93) square millimeters, (17.94±1.89) square millimeters and (23.89±1.84) square millimeters, the CNV areas of PBS group were (9.08±1.42) square millimeters, (27.11±2.02) square millimeters and (31.01±2.04) square millimeters, PFD group compared with PBS group, the differences were all statistically significant (t=-5.609, -10.452, -8.202; P<0.05). HE staining showed that the corneal tissue in PBS group have many inflammatory cell infiltration and the proliferation of fibrous tissue, however, the morphology and structure of corneal tissues in the PFD group was no significant pathological changes. Immunohistochemical CD34 stainings showed that the MVD of PFD group was (3.17±1.17) bars per square millimeter, the MVD of PBS group was (10.83±2.48) bars per square millimeter, PFD group compared with PBS group, the difference was statistically significant (t=-6.842, P<0.05).

Conclusion

PFD can significantly reduce inflammation reaction and inhibit the growth of corneal neovascularization, which may provides a new methods for the treatment of corneal neovascularization.

Key words: Pirfenidone, Corneal neovascularization, Alkali burn
图1 两组大鼠角膜碱烧伤后不同时间点的角膜炎症评分
表1 两组大鼠碱烧伤后不同时间点角膜炎症反应情况的比较(分)
分组 眼数 时间点
3 d 7 d 14 d
PFD组 10 5.60±0.70 3.50±0.53 2.20±0.42
PBS组 10 5.67±0.71 4.90±0.57 3.3±0.48
t值 ? 0.48 －5.715 －5.425
P值 ? >0.05 <0.05 <0.05
图2 碱烧伤实验SD大鼠第14 d角膜新生血管生长外观的彩色图像　A图显示吡非尼酮组大鼠角膜仅见少量较长的角膜新生血管，细小血管萎缩；B图显示磷酸缓冲盐溶液组大鼠角膜的新生血管呈垂柳状生长，已达角膜中央
表2 两组大鼠碱烧伤后不同时间点角膜新生血管面积比较(mm2)
分组 眼数 时间点
3 d 7 d 14 d
PFD组 10 6.06±0.93 17.94±1.89 23.89±1.84
PBS组 10 9.08±1.42 27.11±2.02 31.01±2.04
t值 ? －5.609 －10.452 －8.202
P值 ? <0.05 <0.05 <0.05
图3 碱烧伤实验SD大鼠在Olympus－BX43生物显微镜下的病理切片图像　 A图显示吡非尼酮组大鼠角膜基质层浅表位置见少量CD34阳性的新生小血管且分布稀疏；B图显示磷酸缓冲盐溶液组大鼠角膜基质内见大量CD34阳性的新生血管形成且分布密集(×200，免疫组化染色法)
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