Hydroxysafflor yellow A attenuates the inflammatory response in cerebral ischemia-reperfusion injured mice by regulating microglia polarization per SIRT1-mediated HMGB1/NF-κB signaling pathway

Background: Hydroxysafflor yellow A (HSYA), an active component isolated from Carthamus tinctorius L., has demonstrated potent protective effects against cerebral ischaemia/reperfusion (I/R) injury. Microglial polarisation plays a crucial role in I/R. However, the mechanism by which HSYA regulates microglial polarisation remains unclear.

Objective: To explore the mechanism of action of HSYA on the phenotypic polarisation of microglia stimulated by lipopolysaccharide (LPS) in a mouse model of I/R injury.

Methods: BV2 cells injured by LPS and a modified middle cerebral artery occlusion/reperfusion (MCAO/R) model were used to mimic I/R in vitro and in vivo, respectively. BV2 cell morphology was assessed by optical microscopy, and cell viability was evaluated using the CCK-8 assay. The effect of HSYA on MCAO/R mice was assessed using the Longa assay, brain index, triphenyl tetrazolium chloride, and haematoxylin and eosin staining. LDH, NO, IL-6, TNF-α, and IL-10 levels were measured using corresponding ELISA kits following the manufacturers' protocols. M1 and M2 type microglia markers, including CD86, CD16/32, iNOS, YM1/2, TGF-β, and Arg, were detected by western blotting. M1 and M2 cell surface markers (CD86 and CD206) were detected using immunofluorescence. Molecular docking, DARTS, and CETSA were applied to investigate the interactions between HSYA and SIRT1. The role of HSYA in regulating the binding of HMGB1 to SIRT1 was tested using co-immunoprecipitation. Proteins related to the HMGB1/NF-κB pathway were also analysed by western blotting.

Results: HSYA promoted microglial polarisation from M1 to M2 type in LPS-induced BV2 cells and MCAO/R mice. HSYA significantly reduced M1 polarisation markers, including IL-6, TNF-α, CD86, CD16/32, while increasing the expression of IL-10, Arg, YM1/2, TGF-β. Furthermore, compared to the MCAO/R group, HSYA significantly improved neurological scores, brain index, and infarct volume and normalised nucleolar arrangement. Molecular docking assessment showed that HSYA exhibited strong binding SIRT1 and significantly improved the interactions between SIRT1 and HMGB1. HSYA also decreased the expression of cytoplasm-HMGB1 and reduced the P-P65/P65 ratio.

Conclusions: HSYA attenuates LPS-induced and MCAO/R-induced inflammatory responses by modulating microglia polarisation. This effect is associated with the SIRT1-mediated HMGB1/NF-κB signalling pathway.