Discovery of a novel MARK4 antagonist from safflower to improve ischemic stroke by inhibiting microglial NLRP3 inflammasome activation.

BACKGROUND

Neuroinflammation has been proven to be a significant factor linked to stroke, with NLRP3 inflammasome activation being a common neuroinflammatory manifestation. Microtubule affinity regulated kinase 4 (MARK4) plays a key role in the activation process of NLRP3 inflammasome. However, the impact of MARK4 on neuroinflammation in stroke remains inadequately understood.

OBJECTIVE

To elucidate the effect and potential mechanism of MARK4 in microglial NLRP3 inflammasome activation, and to identify MARK4 antagonist from safflower, an herb frequently employed in stroke treatment.

METHODS

The effects of safflower extracts (SFFE) and MARK4 on NLRP3 inflammasome activation were evaluated using BV-2 cells stimulated by oxygen-glucose deprivation/reperfusion (OGD/R). A homogeneous time-resolved fluorescence (HTRF) assay was conducted to screen MARK4 antagonists from SFFE. To investigate the neuroprotective properties of the screened compound, an ischemic stroke model utilizing middle cerebral artery occlusion (MCAO) in C57BL/6J mice was implemented, and its intrinsic mechanisms were further explored.

RESULTS

SFFE inhibited the activation of NLRP3 inflammasome in OGD/R-induced BV-2 cells. Concurrently, the inhibition of MARK4 significantly blocked microglial NLRP3 inflammasome activation. Through HTRF screening, kaempferol was identified as a novel MARK4 antagonist from SFFE, exhibiting an IC value of 11.54 µM. Furthermore, kaempferol was found to directly interact with MARK4, resulting in the down-regulated interaction between MARK4 and NLRP3. Subsequently, kaempferol inhibited NLRP3 inflammasome activation in a MARK4-dependent manner in both BV-2 cells and MCAO mice. Furthermore, kaempferol obviously improved the neurological dysfunction in MCAO mice through multiple signaling pathways.

CONCLUSION

Kaempferol is a natural MARK4 inhibitor exhibiting therapeutic potential for diseases associated with microglial NLRP3 inflammasome activation, highlighting MARK4 as a promising target for the treatment of ischemic stroke.