Ethanol Directly Induced HMGB1 Release Through NOX2/NLRP1 Inflammasome In Neuronal Cells

Objective: Long-term alcohol consumption will result in significant alterations of brain structure and function. At last, cognition will be impaired and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, will develop. However, the mechanisms mediating the actions of ethanol on neuronal damage remain to be further elucidated. It has been suggested that brain HMGB1 is highly expressed in neurons and is released from neurons. Once secreted, HMGB1 is thought to drive inflammatory response, and take part in pathological processes in CNS. Therefore, our study attempts to test whether ethanol directly induced HMGB1 expression and release, and to explore the cellular and molecular mechanism mediating its action. Methods: The human neuroblastoma cell line SH-SY5 Y and primary rat cortical neurons were cultured. The levels of HMGB1 released into the growth medium after ethanol(50 mM) treatment at different time points were measured using sandwich enzyme immunoassays(ELISA). At the same time,the cell viability was detected by LDH method, and ROS production in cells was measured with fluorescent dye DCFH-DA. Meanwhile, we used western blot to detect the protein levels of HMGB1, NOX2 components gp91 and p47 phox, NLRP1, ASC and cleaved caspase 1 in ethanol-treated neuronal cells. What's more, with the same methods, we detected the expression and levels of these proteins and secreted HMGB1 in cells pretreated with ROS inhibitor, NADPH oxidase inhibitor and caspase-1 inhibitor before ethanol treatment. Results: 1. Ethanol induced HMGB1 release from neuronal cells. We found that when cells were treated with 50 mM ethanol for 12 h and 24 h, HMGB1 expression and release were significantly increased in cultured medium of SH-SY5 Y cells and cultured cortical neurons. In addition, the difference of the cell cytotoxicity after ethanol treatment at different time points measured by LDH tests was not statistically significant. 2. Ethanol-induced HMGB1 release was oxidative stress dependent. When neuronal cells were treated with ethanol, ROS production and the expression of components of NOX2 such as gp91 and p47 phox were significantly increased. Besides, cells were pretreated with NAC or Apocynin and then treated with ethanol. Summarized data suggested that both ROS inhibitor and NADPH oxidase inhibitor prevented ethanol-induced HMGB1 release. Our study confirmed that NOX2 mediated HMGB1 release in neuronal cells. 3. NLRP1 inflammasome was involved in ethanol-induced HMGB1 release. We found that ethanol treatment increased the expression of ASC and cleaved caspase 1. Inhibition of ROS or NOX2 prevented activation of NLRP1 inflammasome by ethanol. Moreover, we treated cells with z-YVAD-fmk, caspase-1 inhibitor, HMGB1 release was prevented, which suggested that ethanol-induced HMGB1 release was partly through inflammasomes. Conclusion: Ethanol directly induced neuronal cells to release HMGB1 through the activation of NOX2 and NLRP1 inflammasome, ultimately resulting in neuronal injury.