Fig. 5.

The inhibitory effect of melatonin on H₂O₂-triggered autophagic GC death is independent of ROS elimination. (A) Primary cultured GCs grown in medium containing 10 μM melatonin for 24 h were then rinsed in PBS, and exposed to 2 h of H₂O₂ (200 μM) incubation. The inhibitors of melatonin downstream antioxidants (AOI) were added 1 h prior to H₂O₂ treatment. The total antioxidation capability (T-AOC) was determined as described in Materials and Methods section. Data represent mean ± S.E; n = 3. *P < 0.05, **P < 0.01; NS, not significant, P > 0.05. (B) GCs were subjected to H₂O₂ exposure following melatonin and/or AOI treatment as mentioned above. The ROS levels were detected by dichlorofluorescein fluorescence (green), and nuclei were counterstained with DAPI (blue). Bar, 20 µm. (C) The optical density of intracellular ROS was quantified using ImageJ software. ** Represents P < 0.01 compared to control group; # Represents P < 0.05; N, not significant. (D) The immunoblotting detection of MAP1LC3B and SQSTM1 in GCs received the indicated treatments as described above. (E and F) Quantification of the MAP1LC3B-II accumulation and SQSTM1 degradation. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3 in each group. ** Represents P < 0.01 compared to control group. ## Represents P < 0.01 compared to H₂O₂-only-treated cells. N, not significant. (G) Primary cultured GCs treated with H₂O₂ at different concentrations as indicated for 2 h were collected for immunoblotting analysis of MAP1LC3B and SQSTM1. (H and I) The expression of MAP1LC3B-II and SQSTM1 were quantified by densitometric analysis. ** Represents P < 0.01 compared to control group; NS, not significant. (J) GCs pretreated with or without melatonin (10 μM) for 24 h were then rinsed in PBS, and incubated with H₂O₂ in various concentrations. 2 h later, the formation of ROS in GCs was observed under a laser confocal-scanning microscope. Bar, 20 µm. (K) Quantification of ROS levels by calculating the green fluorescence optical density in each GC. Experiments were repeated in triplicate, and three fields of each coverslip were selected at random for counting. Data represent mean ± S.E; n = 3. ** Represents P < 0.01 compared to control group; NS, not significant. (L) GCs cultured with 10 μM melatonin for 24 h were then rinsed in PBS, and exposed to 2 h of H₂O₂ (200 μM) incubation. For the inhibition of melatonin-induced activation of downstream antioxidative components, cells were treated with AOI 1 h before H₂O₂ exposure. Cell viability was examined as described above. **P < 0.01; NS, not significant, P > 0.05.