Chrysophanol exerts neuroprotection against cerebral ischemia- reperfusion injury by regulating IκBα-stabilized MLKL via ZNF460-TRAF6 axis

Background: With the aging of the global population, the incidence of ischemic stroke is gradual increasing, yet the available clinical prevention and effective treatment remain restricted. Chrysophanol (CHR) has potential anti-inflammatory properties on cerebral ischemic reperfusion injury (CIRI). However, the neuroprotective mechanisms of CHR remain obscure.

Purpose: In this present study, we investigate the neuroprotective effect and the precise pharmacological mechanism of CHR against CIRI, focusing on tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated Necroptosis.

Methods: C57BL/6 and conditional TRAF6-knockout mice were used to assess CHR's neuroprotection against middle cerebral artery occlusion (MCAO)-induced CIRI. Molecular Biology approaches explored its role in regulating Necroptosis. Oxygen-glucose deprivation/reoxygenation (OGD/R)-challenged BV2 microglia models elucidated mechanisms of CHR on IκBα-stabilized MLKL via ZNF460-TRAF6 axis modulation.

Results: Here, we reported that the therapeutic effects of CHR were evidenced, which were consistently associated with downregulation of TRAF6. Importantly, TRAF6 overexpression not only exacerbated neural damage, but also nullified the cytoprotective properties of CHR, reflecting TRAF6's notable function in ischemic pathogenesis. At the molecular level, CHR exerted potent inhibition of the necroptotic pathway by specifically disrupting MLKL oligomerization and subsequent plasma membrane translocation. This anti-necroptotic activity was mechanistically linked to CHR's capacity to modulate TRAF6 expression and function. Comprehensive mechanistic inquiry revealed that CHR can interfere with the TRAF6-IκBα and distinctly modulate K48- versus K63-linked ubiquitination patterns, which lead to stabilization of IκBα and diminished dissociation from p65, thus potently inhibiting the necroptotic pathway post-CIRI. A critical finding was identifying IκBα suppressed RIPK3 kinase activity via its ARD domain, reducing necroptosis-related MLKL-Ser³⁴⁵ phosphorylation, whereas TRAF6 abrogates CHR-enhanced IκBα-RIPK3 binding. Additionally, we firstly identified ZNF460 as a novel transcriptional activator of TRAF6. CHR potently suppressed ZNF460 expression and impaired its nuclear translocation, resulting in transcriptional repression of TRAF6.

Conclusions: These outcomes regarded CHR as a multi-target neuroprotective agent functioning through a coherent molecular mechanism: (1) suppression of the ZNF460-TRAF6 axis, (2) stabilization of IκBα protein, and (3) consequent inhibition of MLKL activation and oligomerization.

Cerebral ischemic reperfusion injury (CIRI); Chrysophanol (CHR); Mixed lineage kinase domain-like pseudokinase (MLKL); Necroptosis; Tumor necrosis factor receptor-associated factor 6 (TRAF6).