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.

Keywords:

• Dry eye disease
• melatonin
• PINK1
• hyperosmolarity
• mitophagy

Acknowledgments

The authors thank the Editage.com for its linguistic assistance during the preparation of this manuscript. We would like to thank Jiayi Liu for generous technical advice.

Author contributions

Jing Xu and Peng Chen contributed to sample testing, data analysis, and study design; Jing Xu, Susu Wei, and Qiqi Li contributed to sample testing and data analysis; Guangfen Zhao, Chuanlong Guo, Qilong Cao, and Xianggen Wu contributed to sample preparation; Guohu Di contributed to study design, data analysis, and manuscript preparation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.

Additional information

Funding

This work was supported by Error! Hyperlink reference not valid. [No. 81670829 and No. 81970782] and China Postdoctoral Science Foundation [No. 2019M652328].