Janus Kinase Inhibition Ameliorates Cerebral Ischemic Injury and Neuroinammation through Reducing NLRP3 Inammasome Activation via JAK2/STAT3 Pathway Inhibition

Background Evidence shows that inammatory responses play multiphasic roles in stroke pathogenesis. Ruxolitinib (Rux), a selective oral JAK 1/2 inhibitor, is ecacious in COVID-19 by reducing inammation via the JAK2/STAT3 pathway. Here, we investigated whether JAK2 inhibition has neuroprotective effects against ischemic stroke (IS) in MCAO mice in vivo and in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model, and explored the potential molecular mechanisms. Rux was applied to MCAO mice. Immunouorescence staining, RT-qPCR, and western blots were used to measure the expression of NLRP3 inammation components and proinammatory cytokines as well as JAK2/STAT3 pathway. Local STAT3 deciency in brain tissue was established to investigate the interplay between NLRP3 and STAT3 signaling.


Abstract Background
Evidence shows that in ammatory responses play multiphasic roles in stroke pathogenesis. Ruxolitinib (Rux), a selective oral JAK 1/2 inhibitor, is e cacious in COVID-19 by reducing in ammation via the JAK2/STAT3 pathway.

Methods
Here, we investigated whether JAK2 inhibition has neuroprotective effects against ischemic stroke (IS) in MCAO mice in vivo and in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model, and explored the potential molecular mechanisms. Rux was applied to MCAO mice. Immuno uorescence staining, RT-qPCR, and western blots were used to measure the expression of NLRP3 in ammation components and proin ammatory cytokines as well as JAK2/STAT3 pathway. Local STAT3 de ciency in brain tissue was established to investigate the interplay between NLRP3 and STAT3 signaling.

Results
Rux treatment obviously improved neurological scores, decreased the infarct size and ameliorated cerebral edema 3 days after stroke. In addition, immuno uorescence staining and western blots showed that Rux application inhibited the expression of NLRP3 in ammasome components, proteins related to the NLRP3 in ammasome and phosphorylated STAT3 (p-STAT3) in neurons. Furthermore, Rux administration inhibited the expression of proin ammatory cytokines, including TNF-α, IFN-γ, HMBG1, IL-1β, IL-2, and IL-6 in middle cerebral artery occlusion (MCAO) model mice, suggesting that Rux may alleviate IS injury by inhibiting proin ammatory reactions via JAK2/STAT3 signaling pathway regulation.
Local STAT3 de ciency decreased histone H3 and H4 acetylation on the NLRP3 promoter and the NLRP3 in ammasome component expression, indicating that the NLRP3 in ammasome may be directly regulated by STAT3 signaling. Finally, the effect of Rux on the NLRP3 in ammasome was further assessed in a HT22 cell OGD/R model in vitro. Rux application markedly suppressed lipopolysaccharide (LPS)-induced NLRP3 in ammasome secretion and JAK2/STAT3 pathway activation in vitro the in OGD/R HT22 cell model.

Conclusion
JAK2 inhibition by Rux in MCAO mice decreased STAT3 phosphorylation, thus inhibiting downstream proin ammatory cytokines and H3 and H4 acetylation on the NLRP3 promoter, resulting in downregulation of NLRP3 in ammasome component expression.

Full Text
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed.
However, the manuscript can be downloaded and accessed as a PDF. Tables   Due to technical limitations, table 1 is only available       The data are shown as the fold change compared with the sham. n = 5. aP < 0.001 versus sham; bP < 0.01 versus vehicle; cP < 0.05 versus Rux.