Icaritin induces resolution of inflammation by targeting cathepsin B to prevents mice from ischemia-reperfusion injury

Cerebral ischemic stroke is a major cause of mortality worldwide and severely affects the daily life of survivors. This study aimed to evaluate the therapeutic effects of icaritin (ICT) against cerebral ischemic stroke injury using a middle cerebral artery occlusion/reperfusion (MCAO/R) mice model, and investigate the pharmacological mechanisms via pharmacologically targeting molecular motor. Our results demonstrated that ICT significantly reduced the cerebral infarction size and brain edema of MCAO/R mice, and improved the neurological score at the same time. H&E and Nissl staining results confirmed that ICT alleviated the cerebral ischemia-induced damage in model mice and reduced the neuronal apoptosis caused by MCAO/R. More importantly, ICT pro-moted the expression of ZO1, Occludin and Claudin 5 by reducing the ratio of TIMP1/MMP2 and TIMP1/MMP9 in brain tissue to protect the blood-brain barrier. Further studies indicated that ICT inhibited the inflammatory level of brain tissue by regulating TLR4, MAPK and NLRP3 inflammasome signaling pathways. In combination with photochemically induced coupling reaction system, surface plasmon resonance, molecular docking and cellular thermal shift assay technology and Inhibitor Screening Kits, it was found that cathepsin B (CTSB) was the key target of ICT to attenuate neuroinflammation. To verify this conclusion, we inhibited the activity of CTSB in BV2 cells with CA-074 Me, and it demonstrated that ICT no longer had the inhibitory effect on glucose and oxygen deprivation-reoxygenation (OGD/R)-induced inflammation. When the mice were pretreated with CA-074 Me, ICT lost its protective effect on MCAO/R injury. Collectively, we reported ICT as a small-molecule targeting CTSB for anti-neuroinflammation. Morever, these findings broaden our knowledge of CTSB as a druggable target protein for neuroinflammation inhibition, and open a new avenue to novel therapy strategy for cerebral ische-mia-reperfusion injury.