Fig. 8

Acute melatonin attenuated the acute ethanol-induced apoptosis and neurodegeneration in vivo and in vitro ethanol-exposed HT22 neuronal cells. a Western blots analysis of Bax, Bcl2, Cyt.c, activated caspase-3 and PARP-1 antibodies. The bands were quantified using Sigma Gel software, and the differences are represented by a histogram. β-Actin was used as a loading control. n = 10 pups/group, and the number of experiments = 3. b Western blots and the densitometric analysis of Bax, Bcl2, Cyt.c, activated caspase-3 and PARP-1 expression levels in the HT22 cells that were subjected to Nrf2 siRNA and treated with ethanol (100 mM) and melatonin (100 μM) for 12 h. β-Actin was used as a loading control. The number of experiments = 3. c The representative image shows immunofluorescence reactivity of activated caspase-3 in the cortices and CA1 regions of the hippocampi in the rat pups. n = 5 pups/group, and the number of experiments = 3. Magnification × 40. Scale bar = 50 μm. d Shown images indicate the TUNEL immunohistochemical staining in the cortices and CA1 regions of the hippocampi in the rat pups. n = 5 pups/group, and the number of experiments = 3. Magnification × 20. Scale bar = 20 μm. e Shown images indicate the FJB immunohistochemical staining in the cortices and CA1 regions of the hippocampi in the rat pups. n = 5 pups/group, and the number of experiments = 3. Magnification × 40. Scale bar = 50 μm. f Representative photomicrograph of Nissl staining in the cortices; and DG and CA1 regions of the hippocampi in the rat pups. n = 5 pups/group, and the number of experiments = 3. Magnification × 20. Scale bar = 20 μm. g Apo-Tox Glo™ assay in the neuronal HT22 cells using Nrf2 siRNA. (a–c) The cell viability, cytotoxicity and activation of caspase-3/7 respectively in the HT22 cells that were subjected to Nrf2 siRNA and treated with ethanol (100 mM) and melatonin (100 μM) for 12 h. The number of experiments = 3. The data are expressed as the mean ± SEM. The data are presented relative to control values. Significance = P < 0.05. σ Significantly different from the control saline-treated rat pups; Φ significantly different from the ethanol-treated rat pups. Similarly for in vitro studies, σ significantly different from the non-treated HT22 cell; Φ significantly different from the ethanol-exposed HT22 cells and ω significantly different from the ethanol + melatonin-exposed HT22 cells