Abstract：
BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism.
METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays.
RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1β and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells.
CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.
METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays.
RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1β and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells.
CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.
摘要：
背景：炎性巨噬细胞浸润在缺血再灌注（IRI-AKI）引起的急性肾脏疾病中起关键作用。毛蒜素是一种具有多种生物活性的天然黄酮。本研究旨在探讨calycosin对IRI-AKI的治疗作用及其机制。
方法：在具有IRI-AKI和脂多糖（LPS）刺激的RAW264.7细胞的C57BL/6小鼠中分析了calycosin的肾脏保护和抗炎作用。RNA-seq用于机制研究。通过计算机模拟方法筛选了calycosin的分子靶标，并通过表面等离子体共振（SPR）进行了验证。使用Transwell和琼脂糖凝胶斑点测定法分析巨噬细胞趋化性。
结果：Calycosin治疗可显著降低IRI-AKI小鼠的血清肌酐和尿素氮，并减轻肾小管破坏。此外,在IRI-AKI肾脏和LPS刺激的RAW264.7细胞中，calycosin显着抑制NF-κB信号激活和炎症介质IL-1β和TNF-α的表达。有趣的是,RNA-seq显示calycosin在RAW264.7细胞中显著下调趋化相关途径。在差异表达的基因中，CCl2/MCP-1是介导巨噬细胞炎性趋化的关键趋化因子,在LPS刺激的RAW264.7细胞和IRI-AKI肾脏中均下调。始终如一，calycosin治疗减轻了IRI-AKI肾脏中的巨噬细胞浸润。重要的是,计算机目标预测，分子对接,和SPR实验表明，calycosin直接与巨噬细胞迁移抑制因子（MIF）结合。功能上,calycosin在RAW264.7细胞中消除了MIF刺激的NF-κB信号激活和Ccl2表达以及MIF介导的趋化性。
结论：总之，calycosin通过抑制MIF介导的巨噬细胞炎性趋化作用减弱IRI-AKI，提示它可能是治疗IRI-AKI的有前途的治疗剂。
方法：在具有IRI-AKI和脂多糖（LPS）刺激的RAW264.7细胞的C57BL/6小鼠中分析了calycosin的肾脏保护和抗炎作用。RNA-seq用于机制研究。通过计算机模拟方法筛选了calycosin的分子靶标，并通过表面等离子体共振（SPR）进行了验证。使用Transwell和琼脂糖凝胶斑点测定法分析巨噬细胞趋化性。
结果：Calycosin治疗可显著降低IRI-AKI小鼠的血清肌酐和尿素氮，并减轻肾小管破坏。此外,在IRI-AKI肾脏和LPS刺激的RAW264.7细胞中，calycosin显着抑制NF-κB信号激活和炎症介质IL-1β和TNF-α的表达。有趣的是,RNA-seq显示calycosin在RAW264.7细胞中显著下调趋化相关途径。在差异表达的基因中，CCl2/MCP-1是介导巨噬细胞炎性趋化的关键趋化因子,在LPS刺激的RAW264.7细胞和IRI-AKI肾脏中均下调。始终如一，calycosin治疗减轻了IRI-AKI肾脏中的巨噬细胞浸润。重要的是,计算机目标预测，分子对接,和SPR实验表明，calycosin直接与巨噬细胞迁移抑制因子（MIF）结合。功能上,calycosin在RAW264.7细胞中消除了MIF刺激的NF-κB信号激活和Ccl2表达以及MIF介导的趋化性。
结论：总之，calycosin通过抑制MIF介导的巨噬细胞炎性趋化作用减弱IRI-AKI，提示它可能是治疗IRI-AKI的有前途的治疗剂。
