Oridonin mitigates cerebral ischemic–reperfusion (I/R) injury via covalent binding to HMGB1 and inhibiting the HMGB1/TLR4/MyD88/NF-κB signaling pathway

Objective: Ischemic stroke (IS) is a leading cause of mortality and disability worldwide, and effective pharmacological treatments are limited. Oridonin (Ori) has demonstrated neuroprotective potential in IS; however, its underlying mechanisms are still poorly understood. Methods: In vitro, an oxygen-glucose deprivation/reperfusion (OGD/R) model was established using mouse neuroblastoma Neuro-2a cells and primary cortical neurons. In vivo, a transient middle cerebral artery occlusion (tMCAO) model was induced in male C57BL/6J mice to simulate cerebral ischemic/reperfusion (I/R) injury. The key targets of Ori were identified using activity-based protein profiling (ABPP). The binding affinity between Ori and its target protein was validated using multiple approaches, including Cellular Thermal Shift Assay (CETSA), molecular docking, and biolayer interferometry (BLI). Results: Ori significantly suppressed the expression of inflammatory cytokines in tMCAO- and OGD/R-treated neuronal cells. Target identification revealed that high-mobility group box 1 (HMGB1) protein is the key mediator of the protective effects of Ori against cerebral I/R injury. Mechanistically, Ori covalently binds to cysteine (Cys) 106 of HMGB1, reducing its secretion and proinflammatory activity. Additionally, Ori downregulated cytoplasmic HMGB1 levels and the expression of TLR4 and MyD88, as well as the p-p65/p65 ratio in both OGD/R and tMCAO models. Notably, the HMGB1 inhibitor NecroX-7 conferred protection against OGD/R-induced neuronal injury and tMCAO-induced brain damage in mice, which could not be further modulated by Ori treatment. Conclusions: Our findings demonstrate that Ori confers neuroprotection against brain I/R injury by covalently binding to HMGB1 at Cys106 via its reactive carbon–carbon double bonds, thereby eliminating the proinflammatory activity of HMGB1. This molecular interaction reduces HMGB1 secretion and inhibits the downstream HMGB1/TLR4/MyD88/NF-κB signaling pathway, ultimately attenuating neuroinflammation and ischemic damage.