The protective role of melatonin in early hypoxic ischemic brain damage by enhancing mitochondrial autophagy

Abstract

Objective To study the effects of melatonin (MT) on mitochondrial autophagy in neonatal rats with hypoxic-ischemic brain damage (HIBD). Method Animal model of HIBD was established. Forty-five 7-day-old Sprague-Dawley (SD) rats were randomly assigned to sham operation group and HIBD group. Brain tissue were taken at 0, 2, 4, 6, 8, 12, 24 and 48 h after model preparation, and the expressions of mitochondrial autophagy-related protein Bnip3 and autophagy-related protein LC3-Ⅱ were detected. Seventy-two 7-day-old SD rats were randomly assigned to sham operation group, HIBD group and post-HIBD treatment group (3-MA, Mdivi-1, Rapa, MT, 3-MA+ MT, Mdivi-1+ MT, Rapa+ MT). The sizes of cerebral infarction after different treatment were detected using triphenyltetrazolium chloride staining (TTC). Primary cortical cells of fetal SD rats (embryonic day: 17~19 d) were cultured. JC-1 staining was used to detect mitochondrial membrane potential and immunofluorescence method was used to observe mitochondrial autophagy. The Oxygen glucose deprivation/reperfusion/R(OGD) model was prepared. Autophagy inhibitor 3-MA, mitochondrial autophagy inhibitor Mdivi-1, autophagy activator Rapa, and MT were applied and Bnip3 and LC3-Ⅱ expressions and CCK8 (Cell Counting Kit CCK 8) for cell viability assay were examined. Result TTC staining results showed significant white infarcts in the tissue of HIBD group after hypoxia-ischemia, especially in the 3-MA and Mdivi-1 groups, and the infarcts were smaller in Rapa group and groups with MT treatment, the differences were statistical significant (P<0.05). Compared with the sham operation group, the expressions of Bnip3 and LC3-Ⅱ in the HIBD group were significantly increased (P<0.05). Compared with the normal group, the expressions of Bnip3 and LC3-Ⅱ in the OGD/R group were increased (P<0.05). The activities of 3-MA and Mdivi-1 cells decreased significantly, the mitochondrial membrane potential decreased, and mitochondrial autophagy were decreased (P<0.05). The cell activity, mitochondrial membrane potential, and mitochondrial autophagy of Rapa group were increased (P<0.05). The cell viability, Bnip3 and LC3-Ⅱ expressions were increased in groups with MT intervention (P<0.05). Conclusion MT may play an important protective role in the early stage of brain injury by enhancing mitochondrial autophagy of HIBD, which provide a theoretical basis for the study of specific related mechanisms. Key words: Hypoxia-ischemia, brain; Autophagy; Mitochondria; Melatonin; Rat