Calycosin inhibited MIF-mediated inflammatory chemotaxis of macrophages to ameliorate ischemia reperfusion-induced acute kidney injury

Acute kidney disease caused by ischemia-reperfusion (IRI-AKI) is characterized by ectopic inflammation and tubular injury, in which macrophage infiltration and inflammatory activation play a critical pathogenic role. Calycosin is an active flavone from the root of Astragalus membranaceus and shows anti-inflammatory effects in various diseases. In this study, we investigated the renoprotective role of calycosin against IRI-AKI and the underlying mechanism. Our results showed that calycosin treatment reduced the levels of serum creatinine and urea nitrogen along with attenuated tubular necrosis and cast formation in IRI-AKI mice. Calycosin significantly suppressed the activation of NF-?B signaling and the expression of inflammatory mediators IL-1ß and TNF-a in IRI-AKI kidneys. In vitro, calycosin inhibited LPS-induced inflammatory activation in RAW 264.7 cells. Interestingly, RNA-seq revealed that calycosin remarkably down-regulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was down-regulated 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 combination with target prediction, molecular docking, and surface plasmon resonance, we showed that calycosin can directly bind to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-?B signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells without influencing its expression. Collectively, calycosin protects from IRI-AKI by suppressing MIF-mediated macrophage inflammatory chemotaxis. Calycosin could be a promising candidate medicine for clinical treatment of IRI-AKI. Overall design: To study the anti-inflammatory effect of calycosin in macrophage, the RAW 264.7 cells were pretreated with 100 ug/mL of calycosin for 24 hours followed by stimulation with 300 ng/mL LPS for 6 hours. RNA was collected for RNA-seq. Transcriptome difference was analyzed in vehicle-treated cells, cells treated with LPS, and cells treated calycosin and LPS.