Deoxynivalenol-induced circadian CLOCK oscillation disruption promotes RAW264.7 macrophage immunosenescence by unleashing cGAS-STING signaling

Abstract

Although deoxynivalenol (DON)-induced immunotoxicity is well-established, the mechanisms driving DON-mediated immunosenescence remain poorly understood. In this study, we examined the protective role of the circadian rhythm protein CLOCK against DON-induced immunosenescence using a RAW264.7 murine macrophage model and GAS-STING pathway involvement. After treatment of DON (2 mu M) for 24 h, the physiological oscillation of CLOCK expression was disrupted. DON exposure triggered increased SA-beta-gal activity, altered membrane morphology, cell cycle inhibitor upregulation (p21 and p16), and elevated IL-6, IL-8, and CCL-2 secretion. CLOCK blockage for 4 h and 12 h post-exposure exacerbated these senescent phenotypes and promoted apoptotic cell death, suggesting protection against DON-induced immunotoxicity. DON activated the cGAS-STING pathway, increasing cGAS expression, phosphorylated STING, and downstream components including p-TBK1, p-p65/NF-kappa B, and p-IRF3. CLOCK inhibition enhanced cGAS-STING activation. Conversely, pharmacological inhibition of STING significantly alleviated DON-induced senescence and apoptosis, confirming the role of cGAS-STING in mediating immunosenescence. Our results reveal a novel regulatory mechanism wherein DON disrupts CLOCK oscillation via HIF-1 alpha, activating the cGAS-STING pathway and driving RAW 264.7 cell immunosenescence. These findings position circadian CLOCK as a key negative regulator in this process and suggest its potential as a therapeutic target for counteracting DON-induced immunosenescence.