Purpose: Abnormal glycolysis of immune cells contributed to the development of inflammatory response. Inhibition of this Warburg phenotype could be a promising strategy for preventing various inflammatory diseases. Iridin (IRD) is a natural isoflavone, and exerts anticancer, antioxidant, and anti-inflammatory effects. However, the underlying mechanism of IRD on acute inflammation remains unknown. In this study, the protective effects of IRD against lipopolysaccharide (LPS)-induced inflammation were investigated in murine macrophage RAW264.7 cells and in mice.
Methods: The inhibition of IRD on NO production in culture medium was detected by Griess assay while the levels of TNF-α, IL-1β, and MCP-1 were detected by ELISA assay. The effects of IRD on OCR and ECAR levels in LPS-treated macrophages were monitored by using Seahorse Analyzer. The apoptosis rate as well as the release of ROS and NO of RAW264.7 cells were analyzed by flow cytometric assay. The protective effects of IRD were investigated on LPS-induced inflammation in mice. The expressions of PKM2 and its downstream (p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2) in cells and in lung tissues were detected by Western blotting analysis.
Results: IRD treatment at the concentrations of 12.5– 50 μM significantly inhibited the productions of TNF-α, IL-1β, MCP-1, and ROS, and suppressed the levels of glucose uptake and lactic acid in LPS-treated RAW264.7 cells. Oral administration with IRD (20– 80 mg/kg) inhibited LPS-induced acute lung injury as well as inflammatory cytokine production in mice. Moreover, IRD targeted pyruvate kinase isozyme type M2 (PKM2) and suppressed its downstream p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2, which could be abolished by PKM2 agonist DASA-58 and antioxidant N-acetyl-L-cysteine, but partly be reversed by NF-κB activator CUT129 and JAK1 activator RO8191.
Conclusion: IRD alleviated LPS-induced inflammation through suppressing PKM2-mediated pathways, and could be a potential candidate for the prevention of inflammatory diseases.
Keywords: isoflavone, glycolysis, Warburg effect, PKM2, JAK/STATs, NF-κB