Fink Michael K, Giuliano Elizabeth A, Tandon Ashish, Mohan Rajiv R
Harry S. Truman Memorial Veterans Hospital, Columbia, MO, USA; College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Vet Ophthalmol. 2015 May;18(3):242-50. doi: 10.1111/vop.12194. Epub 2014 Jul 15.
To evaluate the safety and efficacy of Pirfenidone (PFD) in the treatment of equine corneal fibrosis using an in vitro model.
Healthy donor equine corneas were collected and used to generate primary equine corneal fibroblasts (ECFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Equine corneal myofibroblasts (ECMs), used as a model of equine corneal fibrosis, were produced by growing ECF cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue viability assays and changes in ECF morphology were utilized to determine the optimal PFD dose for this in vitro model. Trypan blue viability, phase-contrast microscopy, and TUNEL assays were used to evaluate the cytotoxicity of PFD. Scratch and MTT assays were used to evaluate the effect of PFD on cellular migration and proliferation. Real-time PCR, immunoblot analysis, and immunocytochemistry were employed to determine the efficacy of PFD to inhibit ECM formation in vitro.
Topical PFD application at 200 μg/mL successfully decreased αSMA expression when compared to the TGFβ1 only treatment group (P < 0.01). PFD application ≤ 200 μg/mL did not affect ECF phenotype or cellular viability and did not result in significant cytotoxicity.
Pirfenidone safely and effectively inhibits TGFβ1-induced equine corneal fibrosis in vitro. In vivo studies are warranted.
使用体外模型评估吡非尼酮（PFD）治疗马角膜纤维化的安全性和有效性。
收集健康供体马角膜，通过在添加10%胎牛血清的最低必需培养基中培养来生成原代马角膜成纤维细胞（ECF）。将ECF培养物在含有转化生长因子β1（1 ng/mL）的无血清培养基中培养，以此产生用作马角膜纤维化模型的马角膜肌成纤维细胞（ECM）。利用台盼蓝活力测定法和ECF形态变化来确定该体外模型的最佳PFD剂量。使用台盼蓝活力测定、相差显微镜检查和TUNEL测定法评估PFD的细胞毒性。使用划痕试验和MTT试验评估PFD对细胞迁移和增殖的影响。采用实时PCR、免疫印迹分析和免疫细胞化学方法来确定PFD在体外抑制ECM形成的效果。
与仅用转化生长因子β1处理的组相比，以200 μg/mL局部应用PFD成功降低了αSMA表达（P < 0.01）。应用≤200 μg/mL的PFD不影响ECF表型或细胞活力，且未导致明显的细胞毒性。
吡非尼酮在体外能安全有效地抑制转化生长因子β1诱导的马角膜纤维化。有必要进行体内研究。
