Evobrutinib mitigates neuroinflammation after ischemic stroke by targeting M1 microglial polarization via the TLR4/Myd88/NF-κB pathway.

Background: Evobrutinib, a third-generation Bruton's tyrosine kinase (BTK) inhibitor, shows great promise for treating neuroinflammatory diseases due to its small molecular size, ease of absorption, and ability to cross the blood-brain barrier. Although previous studies have confirmed significant BTK expression in microglia, the potential of Evobrutinib to treat ischemic stroke by modulating microglial function and its underlying mechanisms remain to be elucidated.

Methods: Male C57BL/6 mice with cerebral ischemia was established to evaluate the effects of oral Evobrutinib treatment. Assessments included TTC staining, behavioral experiments, and pathological examinations were used to evaluate cerebral ischemic injury. Western Blot, flow cytometry, and qPCR were employed to monitor changes in BTK and pBTK expression in microglia and the impact of Evobrutinib on neuroinflammation following the stroke. In vitro, primary microglia were generated to determine the effects of Evobrutinib on the TLR4/ Myd88/NF-κB pathway and on the polarization of microglial subtypes.

Results: The expression of BTK and pBTK is upregulated in microglia under conditions of cerebral ischemia and oxygen-glucose deprivation (OGD). Evobrutinib treatment not only reduced infarct volume in mice but also ameliorated pathological damage and facilitated neurological function recovery. Flow cytometry revealed that Evobrutinib decreased inflammatory cell infiltration and promoted M2 microglia polarization post-stroke. In vitro studies demonstrated that Evobrutinib downregulated the proportion of pro-inflammatory microglia and curtailed the secretion of inflammatory factors under OGD conditions. Mechanistically, Evobrutinib attenuated the OGD-induced upregulation of TLR4/Myd88/NF-κB expression, an effect that was further enhanced by the addition of the TLR4 pathway inhibitor TAK242.

Conclusions: Evobrutinib inhibits the expression and activation of BTK in microglia, reducing M1 microglia-mediated neuroinflammation and alleviating ischemic injury following stroke. This effect is mechanistically linked to the inhibition of TLR4/Myd88/NF-κB-mediated M1 polarization of microglia.