Objective To elucidate the mechanism by which Danhong Injection (丹红注射液, DHI) ameliorates mitochondrial dynamics imbalance and inhibits NOD-like receptor family pyrin domain containing 3 (NLRP3)-mediated pyroptosis in H9c2 cells subjected to oxygen-glucose deprivation based on AMP-activated protein kinase (AMPK)/dynamin-related protein 1 (Drp1) pathway. Methods Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was employed to identify the core active components of DHI. Molecular docking was performed using AutoDock Vina software to assess the affinity between DHI core components and target proteins, including AMPK, Drp1, optic atrophy 1 (OPA1) and cystein-asparate protease-1 (Caspase-1). An H9c2 cell oxygen-glucose deprivation model was established, control group, model group, DHI group, DHI + Compound C (AMPK inhibitor) group, AICAR (AMPK agonist) group, MCC950 (NLRP3 inhibitor) group and Mdivi-1 (Drp1 inhibitor) group were set up. The optimal intervention concentration of DHI was screened using CCK-8 assay. Mitochondrial membrane potential (MMP) was measured using JC-1 probe, and mitochondrial reactive oxygen species (mtROS) level was determined using MitoSOX^TM. Mitochondrial ultrastructure was observed via transmission electron microscopy. Immunofluorescence was used to detect the protein expressions of p-AMPK and p-Drp1. Western blotting was performed to detect the expressions of AMPK/Drp1 pathway-related proteins, mitochondrial dynamics-related proteins [mitochondrial fission 1 protein (FIS1), mitofusin 2 (MFN2) and OPA1] and pyroptosis-related proteins [NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1 and N-terminal fragment of gasdermin D (GSDMD-NT)]. ELISA was used to determine the levels of interleukin-1β (IL-1β), IL-18 and adenosine triphosphate (ATP). Results UHPLC-MS identified eight active components of DHI, including salvianolic acid, lithospermic acid B, danshensu, caffeic acid, sodium danshensu, etc. The molecular docking results showed that each target and active ingredient had strong affinity. The in vitro experimental results showed that compared with model group, mitochondrial morphology in DHI group was significantly improved, with continuous outer membrane and relatively intact cristae structure, increased quantity, and matrix density restored to near normal. MMP and ATP levels were increased (P < 0.01), mtROS release was decreased (P < 0.01), p-AMPK/AMPK value was increased (P < 0.01), p-Drp1/Drp1 value was decreased (P < 0.01), MFN2 and OPA1 protein expressions were upregulated (P < 0.01), FIS1, NLRP3, ASC, Caspase-1 and GSDMD-NT protein expressions were downregulated (P < 0.01), and IL-1β and IL-18 levels were significantly decreased (P < 0.01), consistent with the effects of AICAR, MCC950 and Mdivi-1. Compound C could partially inhibit the above-mentioned effects of DHI. Conclusion DHI activates AMPK/Drp1 pathway, inhibits mitochondrial excessive division and promotes mitochondrial fusion, reduces NLRP3 inflammasome activation and pyroptosis, thereby alleviating hypoxia/hypoglycemia induced H9c2 cell damage.

Danhong Injection  /  AMPK/Drp1 pathway  /  mitochondrial dynamics  /  pyroptosis  /  mitochondrial fusion  /  mitochondrial fission  /  salvianolic acid  /  lithospermic acid B  /  danshensu  /  caffeic acid  /  sodium danshensu  /  Boc-D-glutamine  /  D-galactose 1-[2-(2-azidoethoxy)ethoxyethyl]-2,3,4,6-tetra-O-acetate  /  guanosine