Neuroprotective potentials of Obestatin against ethanol-induced neurotoxicity in the SH-SY5Y and C8-D1A cell lines

Abstract

Ethanol negatively impacts the brain by causing structural and functional alterations leading to numerous disorders such as memory impairment, learning difficulties, cognitive decline, motor–skills delays, and psychosocial problems. As such, excessive ethanol intake is a vital risk factor for the development of many brain disorders. Moreso, excessive ethanol use is a significant health issue in South Africa with Foetal Alcohol Spectrum Disorder (FASD) being a common neurodevelopmental disorder associated with intra-uterine ethanol exposure. Ethanol exerts its damaging effects through the upregulation of oxidative stress, neuroinflammation, impairment of mitochondrial function, excitotoxicity and apoptosis. Given the effects of ethanol-induced neurotoxicity, investigations into possible ameliorative agents are needed. In this study, the neuroprotective capabilities of Obestatin against ethanol-induced neurotoxicity in the SH-SY5Y neuroblastoma cells and C8-D1A astrocytes were explored. Obestatin, a naturally occurring gastric hormone, has been shown to modulate and control cell differentiation and cellular metabolism, promote cell proliferation and survival, and suppress inflammation and apoptosis in different cells. SH-SY5Y and C8-D1A cells were treated accordingly with Obestatin to determine its impact on cell viability and neuroprotection against ethanol effects using MTT and trypan blue assays. Furthermore, the mechanism of neuroprotection of Obestatin following ethanol toxicity was determined by measuring the levels of adenosine triphosphate (ATP) activity with a cell-based assay kit. Moreover, the levels of reactive oxygen species (ROS) in the cells were determined using the fluorescent DCFH- DA staining probe following Obestatin and ethanol treatments. Autophagy was assessed using acridine orange staining by fluorescence microscopy and evaluating autophagy markers (LC3BII and p62) using Western blotting. Apoptosis was also evaluated using the nuclear stain DAPI by fluorescence microscopy, Western blotting with antibodies to Cleaved PARP and the expression of caspase-3 gene using quantitative PCR. The findings of this study demonstrated iii that Obestatin protected the SH-SY5Y and C8-D1A cells by reducing the depletion of ATP and mitigated ethanol-induced oxidative stress. Moreover, Obestatin attenuated apoptosis induced by ethanol through reducing chromatin condensation, the level of Cleaved PARP and the expression of caspase-3 gene. Further, Obestatin inhibited apoptosis in the cells and regulated autophagy through a reduction in acidic vesicular organelle (AVO) formation evidenced by acridine orange as well as the protein expression levels of p62 and LC3BII. This study thus provided evidence of the neuroprotective potentials of Obestatin against ethanol- induced neurotoxicity, in vitro, using the SH-SY5Y and C8-D1A cell lines suggesting Obestatin could be a potential neuroprotective agent that needs to be explored further. However, additional studies are needed to substantiate its neuroprotective effects against ethanol especially in in vivo models.