Abstract
Purpose :
To investigate the protective effect of melatonin on the corneal epithelial cells in experimental dry eye(EDE) and its underlying mechanisms.
Methods :
C57BL/6 mice were injected of subcutaneous scopolamine (0.5mg/0.2 mL) 3 times a day, and exposed to desiccating environment (humidity < 30%) for 7 consecutive days. Primary human corneal epithelial(HCE) cells and a HCE cell line were exposure to hydrogen peroxide(H2O2). Melatonin were used to treat EDE both in vivo and in vitro. Cell viability was detected by Cell Counting Kit-8 (CCK8) assay and Lactate Dehydrogenase (LDH) Leakage. Mitochondrial function was detected with the mitochondrial membrane potential (MMP) assay. Dichlorofluorescein diacetate (DCF-DA) assay was used to measure the cellular reactive oxygen species (ROS) levels. Apoptosis was analyzed by Annexin V- PI flow cytometry and TUNEL assay. Western blot assays and immunofluorescence staining were carried out to measure changes in protein expression. mRNA expression was investigated by RT-PCR and quantitative real-time PCR. Clinical parameters were measured using slit-lamp bio-microscopy. The number of goblet cells and tear volume was recorded.
Results :
Melatonin significantly protected cell viability and decreased apoptosis in HCE cells under H2O2. In addition, intraperitoneal injection of melatonin in dry eye mouse model showed significantly improved clinical parameters and increased tear volume. Interestingly, we found melatonin was able to reduce intracellular ROS production and maintain mitochondrial function via activating hemeoxygenase-1 (HO1). The protective effects of melatonin were largely abolished by SnPP, a HO1 inhibitor. Moreover, autophagic flux was impaired in EDE, which could be rescued by melatonin.
Conclusions :
Melatonin can protect HCE cells against oxidative damage via trigging HO1 expression and maintain normal autophagy. This study may provide potential treatment choices for dry eye disease.
This is a 2020 ARVO Annual Meeting abstract.