To investigate the role and mechanism of melatonin-loaded polymer polyvinyl caprolactam-polyvinyl acetate-polyethyleneglycol graft copolymer (PVCL-PVA-PEG) micelles (Mel-Mic) in dry eye disease (DED).
, the apoptosis and reactive oxygen species (ROS) generation in HCECs were analyzed by immunostaining and flow cytometry (FCM). The effect of Mel-Mic on autophagy and mitophagy was evaluated by immunostaining and western blots. PINK1 knockdown was analyzed by small interfering RNA (siRNA). , sodium fluorescein staining, tear secretion test, and periodic acid-schiff (PAS) 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.
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 knock down largely abolished its protective effects. , 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, TEM assay revealed increased autophagosome number in the corneal epithelium of Mel-Mic group. Moreover, luzindole, a non-selective MT1/MT2 antagonist, but not 4-P-PDOT, a selective MT2 antagonist, blocked the protective effect of Mel-Mic.
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.