Yao Min, Liu Yuting, Meng Dongdong, Zhou Xian, Chang Dennis, Li Lili, Wang Ning, Huang Qi
Department of Pharmacy, Anhui University of Chinese Medicine, Hefei, China.
National Institute of Complementary Medicine, Western Sydney University, Westmead, NSW, Australia.
Int Immunopharmacol. 2025 Feb 6;147:114040. doi: 10.1016/j.intimp.2025.114040. Epub 2025 Jan 10.
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.
背景：羟基红花黄色素A（HSYA）是从红花中分离出的一种活性成分，已证明对脑缺血/再灌注（I/R）损伤具有强大的保护作用。小胶质细胞极化在I/R中起关键作用。然而，HSYA调节小胶质细胞极化的机制尚不清楚。 目的：探讨HSYA在I/R损伤小鼠模型中对脂多糖（LPS）刺激的小胶质细胞表型极化的作用机制。 方法：分别用LPS损伤的BV2细胞和改良的大脑中动脉闭塞/再灌注（MCAO/R）模型在体外和体内模拟I/R。通过光学显微镜评估BV2细胞形态，使用CCK-8法评估细胞活力。使用Longa法、脑指数、氯化三苯基四氮唑和苏木精-伊红染色评估HSYA对MCAO/R小鼠的影响。按照制造商的方案，使用相应的ELISA试剂盒测量LDH、NO、IL-6、TNF-α和IL-10水平。通过蛋白质印迹法检测M1和M2型小胶质细胞标志物，包括CD86、CD16/32、iNOS、YM1/2、TGF-β和Arg。使用免疫荧光检测M1和M2细胞表面标志物（CD86和CD206）。应用分子对接、DARTS和CETSA研究HSYA与SIRT1之间的相互作用。使用免疫共沉淀检测HSYA在调节HMGB1与SIRT1结合中的作用。还通过蛋白质印迹法分析与HMGB1/NF-κB途径相关的蛋白质。 结果：HSYA促进LPS诱导的BV2细胞和MCAO/R小鼠中小胶质细胞从M1型向M2型极化。HSYA显著降低M1极化标志物，包括IL-6、TNF-α、CD86、CD16/32，同时增加IL-10、Arg、YM1/2、TGF-β的表达。此外，与MCAO/R组相比，HSYA显著改善神经功能评分、脑指数和梗死体积，并使核仁排列正常化。分子对接评估显示HSYA与SIRT1具有强结合力，并显著改善SIRT1与HMGB1之间的相互作用。HSYA还降低了细胞质-HMGB1的表达并降低了P-P65/P65比值。 结论：HSYA通过调节小胶质细胞极化减轻LPS诱导和MCAO/R诱导的炎症反应。这种作用与SIRT1介导的HMGB1/NF-κB信号通路有关。
CNS Neurol Disord Drug Targets. 2018
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