Shikonin attenuates cerebral ischemia/reperfusion injury via inhibiting NOD2/RIP2/NF-κB-mediated microglia polarization and neuroinflammation

Objectives: Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, which still lacks effective therapeutic agents. Shikonin possesses anti-inflammatory and neuroprotective properties. However, its underlying mechanism remains elusive. This study aimed to investigate whether Shikonin confers protection against cerebral ischemia/reperfusion (I/R) injury by modulating microglial polarization and elucidate the associated mechanisms. Methods: This study employed an oxygen -glucose deprivation and reoxygenation (OGD/R) BV2 microglial cellular model and a middle cerebral artery occlusion/reperfusion (MCAO/R) animal model to investigate the protection and underlying mechanism of Shikonin against ischemic stroke. Results: The results demonstrated that Shikonin treatment significantly reduced brain infarction volume and improved neurological function in MCAO/R rats. Simultaneously, Shikonin treatment significantly reduced microglial proinflammatory phenotype and levels of proinflammatory markers (inducible -NO synthase (iNOS), tumor necrosis factor -alpha (TNF- alpha), interleukin-1 beta (IL-1 beta ), and IL -6), increased microglial anti-inflammatory phenotype and levels of anti-inflammatory markers (Arginase-1 (Arg1), transforming growth factor -beta (TGF- beta ), and IL -10), reversed the expression of Nucleotide -binding oligomerization domain 2 (NOD2) and phosphorylation receptor interacting protein 2 (p-RIP2), and suppressed nuclear factor kappa -B (NF- kappa B) signaling activation in the ischemic penumbra regions. These effects of Shikonin were further corroborated in OGD/R-treated BV2 cells. Furthermore, overexpression of NOD2 markedly attenuated the neuroprotective effects of Shikonin treatment in MCAO/R rats. NOD2 overexpression also attenuated the regulatory effects of Shikonin on neuroinflammation, microglial polarization, and NF- kappa B signaling activation. Conclusion: This study illustrates that Shikonin mitigates inflammation mediated by microglial proinflammatory polarization by inhibiting the NOD2/RIP2/NF- kappa B signaling pathway, thereby exerting a protective role. The findings uncover a potential molecular mechanism for Shikonin in treating ischemic stroke.