Copolymer Micelle-administered Melatonin Ameliorates Hyperosmolarity-induced Ocular Surface Damage through Regulating PINK1-mediated Mitophagy.

ABSTRACT

PURPOSE: To investigate the role and mechanism of melatonin-loaded polymer polyvinyl caprolactam-polyvinyl acetate-polyethyleneglycol graft copolymer micelles (Mel-Mic) in dry eye disease (DED). METHODS: In vitro, the apoptosis and reactive oxygen species (ROS) generation in human corneal epithelial cells (HCECs) were analyzed by immunostaining and flow cytometry. The effect of Mel-Mic on autophagy and mitophagy was evaluated by immunostaining and western blots. PINK1 knockdown was analyzed by small interfering RNA. In vivo, sodium fluorescein staining, tear secretion test, and periodic acid-Schiff staining were used to determine whether Mel-Mic can alleviate the severity of DED. Small molecule antagonists were pretreated to investigate whether melatonin type 1 and/or 2 receptors (MT1/MT2) mediate the effects of Mel-Mic. RESULTS: Mel-Mic improved the solubility and biological activities of Mel in aqueous solutions. Treatment with Mel-Mic decreased the apoptosis of HCECs exposed to hyperosmotic medium, accompanied by downregulation of cleaved Caspase-3 and upregulation of Bcl-2. In addition, Mel-Mic application suppressed ROS overproduction, rescued mitochondrial function, and decreased the level of oxidative stress associated biomarkers (COX-2 and 4-HNE) in HCECs. Interestingly, HCECs treated with Mel-Mic exhibited increased levels of mitophagy markers (PINK1, PARKIN, Beclin 1, and LC3B) and restored impaired mitophagic flux under hyperosmolarity. While PINK1 knockdown largely abolished its protective effects. In vivo, compared to vehicle group, topical Mel-Mic-solution-treated mice showed significantly improved clinical parameters, increased tear production, and decreased goblet cells loss in a dose-dependent manner. Also, transmission electron microscopic assay revealed increased autophagosome number in the corneal epithelium of Mel-Mic group. Moreover, luzindole, a nonselective MT1/MT2 antagonist, but not 4-P-PDOT, a selective MT2 antagonist, blocked the protective effect of Mel-Mic. CONCLUSIONS: Our findings demonstrated that Mel-Mic ameliorates hyperosmolarity-induced ocular surface damage via PINK1-mediated mitophagy and may represent an effective treatment for DED possibly through acting MT1 receptor.