Myoglobin promotes macrophage polarization to M1 type and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway in CS-AKI

Crush syndrome (CS) is a life-threatening illness in traffic accidents and earthquakes. Crush syndrome-induced acute kidney injury (CS-AKI) is considered to be mainly due to myoglobin (Mb) circulation and deposition after skeletal muscle ruptures and releases. Macrophages are the primary immune cells that fight foreign substances and play critical roles in regulating the body's natural immune response. However, what effect does myoglobin have on macrophages and the mechanisms involved in the CS-AKI remain unclear. This study aims to look into how myoglobin affects macrophages of the CS-AKI model. C57BL/6 mice were used to construct the CS-AKI model by digital crush platform. Biochemical analysis and renal histology confirmed the successful establishment of the CS-AKI mouse model. Ferrous myoglobin was used to treat Raw264.7 macrophages to mimic the CS-AKI cell model in vitro. The macrophage polarization toward M1 type and activation of RIG-I as myoglobin sensor were verified by real-time quantitative PCR (qPCR), Western blotting (WB), and immunofluorescence (IF). Macrophage pyroptosis was observed under light microscopy. The interaction between RIG-I and caspase1 was subsequently explored by co-immunoprecipitation (Co-IP) and IF. Small interfering RNA (siRIG-I) and pyroptosis inhibitor dimethyl fumarate (DMF) were used to verify the role of macrophage polarization and pyroptosis in CS-AKI. In the kidney tissue of CS-AKI mice, macrophage infiltration and M1 type were found. We also detected that in the cell model of CS-AKI in vitro, ferrous myoglobin treatment promoted macrophages polarization to M1. Meanwhile, we observed pyroptosis, and myoglobin activated the RIG-I/Caspase1/GSDMD signaling pathway. In addition, pyroptosis inhibitor DMF not only alleviated kidney injury of CS-AKI mice but also inhibited macrophage polarization to M1 phenotype and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway. Our research found that myoglobin promotes macrophage polarization to M1 type and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway in CS-AKI.