Melatonin Protects HT22 Hippocampal Cells from H2O2-induced Injury by Increasing Beclin1 and Atg Protein Levels to Activate Autophagy
Qiang Gao, Xiaocheng Guo, Yang Cao, Xiaotong Jia, Shanshan Xu, Chunmei Lu, Hui Zhu
Abstract
Background: The aging of hippocampal neurons leads to a substantial decline in memory formation, storage and processing. The neuroprotective effect of melatonin has been confirmed, however, its protective mechanism remains unclear. Objective: In this study, mouse hippocampus-derived neuronal HT22 cells were used to investigate whether melatonin protects the hippocampus from hydrogen peroxide (H 2 O 2 )-induced injury by regulating autophagy. Methods: Rapamycin (an activator of autophagy) and 3-methyladenine (3MA, an inhibitor of autophagy) were used to induce or inhibit autophagy, respectively. HT22 cells were treated with 200 μM H 2 O 2 in the presence or absence of 50 μM melatonin. Cell counting kit 8 (CCK-8), β-galactosidase and Hoechst staining were used to measure the viability, aging and apoptosis of cells, respectively. Western blot analysis was used to detect the levels of autophagy-related proteins. Results: The activation of autophagy by rapamycin alleviated H 2 O 2 -induced oxidative injury, as evidenced by morphological changes and decreased viability, while the inhibition of autophagy by 3MA exacerbated H 2 O 2 - induced injury. The inhibitory effect of melatonin on H 2 O 2 -induced injury was similar to that of rapamycin. Melatonin also alleviated H 2 O 2 -induced aging and apoptosis. Melatonin activated autophagy in the presence or absence of H 2 O 2 , as evidenced by an increased Lc3b 14/16 kd ratio and a decreased P62 level. In addition, H2O2 decreased the levels of Beclin1 and Atg5/12/16, which were reversed by rapamycin or melatonin. The effects of melatonin on H 2 O 2 -induced injury, autophagy and protein expressions were effectively reversed by 3MA. Conclusion: In conclusion, these results demonstrate that melatonin protects HT22 hippocampal neurons from H 2 O 2 -induced injury by increasing the levels of the Beclin1 and Atg proteins to activate autophagy.