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.
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。