冬凌草甲素通过与HMGB1共价结合抑制HMGB1/TLR4/MyD88/NF-κB信号通路减轻脑缺血再灌注损伤OA

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,oxygen-glucose deprivation/reperfusion(OGD/R)models were 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.