Inhibitory effect of melatonin on pyroptosis of lens epithelium cells and its mechanism / 中华实验眼科杂志

ABSTRACT

Objective:To investigate the inhibitory effect of melatonin on pyroptosis of lens epithelium cells (HLECs) induced by hydrogen peroxide (H 2O 2) and its mechanism. Methods:The cultured HLECs were divided into normal control group, model control group, melatonin group, vitamin E group, and vitamin E solvent group.Cells in melatonin group, vitamin E group and vitamin E solvent group were pre-cultured with 1×10 -6 mol/L melatonin, 100 μmol/L vitamin E or equal volume of vitamin E solvent, then cultured with 100 μmol/L H 2O 2, respectively, and the cells in the normal control group and model control group were cultured with normal condition or 100 μmol/L H 2O 2, respectively.The HLECs transfected with nuclear factor erythroid 2-related factor 2 short hairpin RNA (shNrf2) or shNrf2 negtive control lentivirus and following with 100 μmol/L H 2O 2 treatment were served as shNrf2 group and shNrf2 negative control group, respectively; and the transfected cells treated with 1×10 -6 mol/L melatonin and subsequent 100 μmol/L H 2O 2 treatment were served as melatonin+ shNrf2 group and melatonin+ shNrf2 negative control group.The activity of lactic dehydrogenase (LDH) and the expression levels of interleukin (IL)-1β and IL-18 in the culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA) kit.The concentration of reactive oxygen species (ROS) was assessed by flow cytometry.The expression level of Nrf2, NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1 p20 and GSDMD-N proteins were evaluated by Western blot. Results:Compared with model control group, the activity of LDH and the concentrations of IL-1β and IL-18 were significantly decreased in melatonin group and vitamin E group, showing statistically significant differences (all at P<0.05). The ROS fluorescence intensities were 13 040.67±1 550.66 and 12 593.67±1 677.06 in melatonin group and vitamin E group, respectively, which were significantly lower than 18 310.33±1 248.01 in model control group (both at P<0.05). The relative expression levels of Nrf2 protein were 4.24±0.44 and 3.73±0.38 in melatonin group and vitamin E group, respectively, which were significantly higher than 2.28±0.34 in model control group, and the relative expression levels of NLRP3, ASC, caspase-1 p20 and GSDMD-N in melatonin group and vitamin E group were significantly decreased than model control group (all at P<0.05). The relative expression level of Nrf2 protein in shNrf2 group and melatonin+ shNrf2 group was significantly reduced, and the expression levels of LDH, IL-1β, IL-18, ROS content, NLRP3, ASC, caspase-1 p20 and GSDMD-N were significantly increased in comparison with shNrf2 negative control group and melatonin+ shNrf2 negative control group, respectively (all at P<0.05). Conclusions:Melatonin can inhibit the release of NLRP3 inflammasome by activating Nrf2, and has an inhibitory effect on the pyroptosis of HLECs.