Title : Chrysin targets ceruloplasmin to modulate zinc homeostasis in cerebral ischemia reperfusion injury
Abstract:
As a plant flavonoid compound, chrysin comes from Scutellaria Baicalensis Georgi (Huangqin), which has a powerful immunoprotective effect to suppress immune inflammatory responses, and plays an outstanding role in the treatment of various brain diseases. This study aims to explore whether chrysin regulates zinc homeostasis by reducing oxidative stress and targeting Ceruloplasmin (CP), thereby alleviating Cerebral Ischemia-Reperfusion Injury (CIRI). In vivo and in vitro experiments were conducted on tMCAO model SD rats and OGD/R PC12 cell model. The therapeutic effects of chrysin on CIRI were evaluated by TTC staining, Nissl staining, neurological function scores, cell counting kit-8 and transmission electron microscope. To investigate the mechanism, TPEN was used to reduce the level of zinc ions in vivo, adeno-associated virus and siRNA were used to increase and decrease the expression of ceruloplasmin, respectively, and HIF-1α was overexpressed by dimethyloxallyl glycine. Molecular dynamics simulation, infrared spectroscopy, DARTS, and CETSA experiments were used to explore the binding characteristics of chrysin and CP in a zinc environment. RT-qPCR, immunohistochemistry, and Western blot were used to detect mRNA and protein expression levels.
Results: Chrysin significantly reduced the cerebral infarction rate and increased the survival rate of cells, while reducing zinc ion and oxidative levels in vivo. Mechanistically, both chrysin and the zinc chelator TPEN could reverse the promoting effect of zinc overload on CIRI, and overexpression of CP synergistically enhanced the therapeutic effect of chrysin on tMCAO rats, while CP knockdown exacerbated OGD/R injury. Zinc ions enhanced the affinity of chrysin and CP by stabilizing the coordination bonds of the chrysin-CP complex, which was confirmed by ex vivo experiments. In addition to directly binding zinc ions, chrysin directly binds CP, enhancing its activity and zinc-chelating capacity, thereby reducing zinc overload. Concurrently, chrysin suppresses HIF-1α expression, lowering CP levels and restoring physiological balance. Additionally, HIF-1α is the direct upstream regulatory molecule of CP. This study is the first to demonstrate the molecular mechanism by which chrysin regulates zinc homeostasis and oxidative stress through targeting CP in CIRI.
