The Therapeutic Effect Of Copolymer Micelle-administered Melatonin On Experimental Dry Eye Through Regulating PINK1 Mediated Mitophagy

Purpose To investigate the role and molecular mechanism of melatonin(Mel)-loaded polymer polyvinyl caprolactam-polyvinyl acetate-polyethyleneglycol graft copolymer(PVCLPVA-PEG)micelles(melatonin-micelle,Mel-Mic)in dry eye disease(DED).Methods Mel-Mic was identified and evaluated by transmission electron microscope(TEM),particle size analysis,drug absorption and encapsulation rate.In vitro,TUNEL staining and flow cytometry(FCM)were used to detect the effect of Mel-Mic on cell apoptosis.The effects of Mel-Mic on oxidative stress and mitochondrial autophagy were detected by immunofluorescence staining and Western Blot(WB).PINK1 knockdown was analyzed by small interfering RNA(si RNA).In vivo,fluorescein sodium staining,tear secretion test and periodate schiff(PAS)staining were used to observe whether Mel-Mic could improve the clinical symptoms of DED.Immunofluorescence staining,TUNEL staining and transmission electron microscopy were used to detect the cell apoptosis and autophagy in the cornea.In addition,Luzindole and 4-P-PDOT,small molecule antagonists of melatonin receptors,were used to investigate whether melatonin type 1 and/or type 2 receptors(MT1/MT2)mediate Mel-Mic mediated mitochondrial autophagy in DED.Results Mel-Mic improved the solubility and biological activity of Mel in aqueous solution,and had good stability and bioavailability.Pretreatment with Mel-Mic can reduce the apoptosis of human corneal epithelial cells(HCECs)in hypertonic environment.In addition,the application of Mel-Mic inhibited ROS production in HCECs,improved mitochondrial function,and reduced the expression of oxidative stress related markers(COX-2 and 4-HNE).In addition,mitochondrial autophagy of HCECs increased after Mel-Mic treatment under hypertonic conditions.However,after PINK1 was knocked down in HCECs,mitochondrial autophagy was significantly weakened.In vivo,compared with the control group,clinical parameters of mice treated with local Mel-Mic solution were significantly improved,with increased tear secretion and reduced goblet cell loss in a dose-dependent manner.TEM showed that the number of autophagosomes in corneal epithelial cells of dry eye mice in Mel-Mic treatment group increased.Luzindole,a non-selective MT1/MT2 antagonist,significantly blocked the protective effect of Mel-Mic,while 4-P-PDOT,a selective MT2 antagonist,had no significant effect,suggesting that MT1 receptor mediated the protective effect of Mel-Mic on experimental dry eye.Conclusion Our findings demonstrated that Mel-Mic ameliorate ocular surface damage in DED through regulating PINK1 mediated mitophagy,and Mel-Mic has a significant protective effect against experimental dry eye mediated by MT1 receptor.