Abstract：
OBJECTIVE: PAX6 is a key regulator of eye development and epithelial homeostasis in the cornea. When deficient, chronic corneal inflammation, neovascularization and limbal stem cell deficiency can occur. Here we investigated the potential of duloxetine, a generic serotonin reuptake inhibitor that can upregulate PAX6 in vitro, for its in vivo activity in the context of corneal inflammation.
METHODS: Duloxetine tolerance was tested in a human limbal stem cell line and isogenic CRISPR-knockout PAX6+/- cells. C57BL/6-Wildtype mice were administered duloxetine eye drops at concentrations of 1 μM - 2 mM and tested for toxicity and corneal PAX6 expression. In LPS-induced corneal inflammation in mice, duloxetine\'s effect on PAX6 expression, corneal opacification and inflammatory responses were evaluated by in vivo corneal imaging, immunostaining, and whole-transcriptome microarray analysis.
RESULTS: No toxicity was observed in vitro for duloxetine concentrations up to 10μΜ. In vivo, duloxetine drops were well-tolerated up to 50 μM. Duloxetine drops at 10μΜ significantly upregulated PAX6 protein levels in the cornea by 30 % within 2 days. In the LPS model, duloxetine resulted in a sustained 33 % PAX6 protein upregulation in the cornea at 7 days, and in reduced opacity within 2 days, accompanied by a significant dampening of IL-17A signaling, neutrophil degranulation, microglial activation, macrophage markers, and MMP expression, despite non-significant changes in total inflammatory cell infiltration.
CONCLUSIONS: Short-term administration of a repurposed generic drug, duloxetine, upregulates PAX6 protein levels in the cornea of mice and exerts an anti-inflammatory activity by dampening innate immune responses.
METHODS: Duloxetine tolerance was tested in a human limbal stem cell line and isogenic CRISPR-knockout PAX6+/- cells. C57BL/6-Wildtype mice were administered duloxetine eye drops at concentrations of 1 μM - 2 mM and tested for toxicity and corneal PAX6 expression. In LPS-induced corneal inflammation in mice, duloxetine\'s effect on PAX6 expression, corneal opacification and inflammatory responses were evaluated by in vivo corneal imaging, immunostaining, and whole-transcriptome microarray analysis.
RESULTS: No toxicity was observed in vitro for duloxetine concentrations up to 10μΜ. In vivo, duloxetine drops were well-tolerated up to 50 μM. Duloxetine drops at 10μΜ significantly upregulated PAX6 protein levels in the cornea by 30 % within 2 days. In the LPS model, duloxetine resulted in a sustained 33 % PAX6 protein upregulation in the cornea at 7 days, and in reduced opacity within 2 days, accompanied by a significant dampening of IL-17A signaling, neutrophil degranulation, microglial activation, macrophage markers, and MMP expression, despite non-significant changes in total inflammatory cell infiltration.
CONCLUSIONS: Short-term administration of a repurposed generic drug, duloxetine, upregulates PAX6 protein levels in the cornea of mice and exerts an anti-inflammatory activity by dampening innate immune responses.
摘要：
■PAX6是角膜中眼睛发育和上皮稳态的关键调节因子。当不足时，慢性角膜炎症，可发生新生血管形成和角膜缘干细胞缺乏。在这里，我们调查了度洛西汀的潜力，一种通用的5-羟色胺再摄取抑制剂，可以在体外上调PAX6，在角膜炎症的情况下的体内活性。
方法：在人角膜缘干细胞系和等基因CRISPR敲除PAX6+/-细胞中测试了度洛西汀耐受性。向C57BL/6-野生型小鼠施用浓度为1μM-2mM的度洛西汀滴眼剂,并测试毒性和角膜PAX6表达。在LPS诱导的小鼠角膜炎症中，度洛西汀对PAX6表达的影响，通过体内角膜成像评估角膜混浊和炎症反应，免疫染色，和全转录组微阵列分析。
结果:对于浓度高达10μΜ的度洛西汀,在体外没有观察到毒性。在体内,度洛西汀滴剂在高达50μM时耐受性良好。度洛西汀以10μM下降,在2天内使角膜中的PAX6蛋白水平显著上调30%。在LPS模型中,度洛西汀在7天时导致角膜中持续33％的PAX6蛋白上调，并在2天内减少不透明度，伴随着IL-17A信号的显着抑制，中性粒细胞脱颗粒，小胶质细胞激活，巨噬细胞标记物,和MMP表达，尽管总炎性细胞浸润没有显着变化。
结论：短期给药再利用的仿制药，度洛西汀,上调小鼠角膜中PAX6蛋白水平，并通过抑制先天免疫反应发挥抗炎活性。
方法：在人角膜缘干细胞系和等基因CRISPR敲除PAX6+/-细胞中测试了度洛西汀耐受性。向C57BL/6-野生型小鼠施用浓度为1μM-2mM的度洛西汀滴眼剂,并测试毒性和角膜PAX6表达。在LPS诱导的小鼠角膜炎症中，度洛西汀对PAX6表达的影响，通过体内角膜成像评估角膜混浊和炎症反应，免疫染色，和全转录组微阵列分析。
结果:对于浓度高达10μΜ的度洛西汀,在体外没有观察到毒性。在体内,度洛西汀滴剂在高达50μM时耐受性良好。度洛西汀以10μM下降,在2天内使角膜中的PAX6蛋白水平显著上调30%。在LPS模型中,度洛西汀在7天时导致角膜中持续33％的PAX6蛋白上调，并在2天内减少不透明度，伴随着IL-17A信号的显着抑制，中性粒细胞脱颗粒，小胶质细胞激活，巨噬细胞标记物,和MMP表达，尽管总炎性细胞浸润没有显着变化。
结论：短期给药再利用的仿制药，度洛西汀,上调小鼠角膜中PAX6蛋白水平，并通过抑制先天免疫反应发挥抗炎活性。
