Astragaloside IV Synergizes with Mitochondrial or Inflammasome-Targeted Agents to Attenuate Cardiomyocyte Pyroptosis in Sepsis

<b>C</b><b>ontext:</b> Septic cardiomyopathy is a severe condition with limited effective therapeutics. Astragaloside IV (AS-IV) has been reported to exert antioxidant and anti-inflammatory activities.<b>Objective</b><b>:</b> This study investigated the protective mechanism of AS-IV against sepsis-induced pyroptosis.<b>M</b><b>ethods:</b> H9C2 cells were divided into seven groups: Control, LPS, and LPS co-treated with AS-IV, Mito-TEMPO (a mitochondrial reactive oxygen species [ROS] scavenger), MCC950 (an NOD-like receptor thermal protein domain associated protein 3 [NLRP3] inhibitor), or their combinations. Pyroptosis rate, mitochondrial ROS levels, and mitochondrial permeability transition pore (MPTP) opening were assessed by flow cytometry. Cardiac injury markers (Troponin I [TnI] and lactate dehydrogenase [LDH]) were measured by Enzyme-Linked ImmunoSorbent Assay. Protein expression (Bcl-2 Homologous Antagonist/Killer [BAK], NLRP3, Gasdermin-D, caspase-1/pro-caspase-1) was analyzed by western blot, and mitochondrial ultrastructure was observed via electron microscopy.<b>Results: </b>LPS significantly elevated TnI/LDH release, pyroptosis, mtROS, MPTP opening, and the expression of BAK, NLRP3, Gasdermin-D, and caspase-1/pro-caspase-1, while impairing mitochondrial ultrastructure. These effects were attenuated by AS-IV, Mito-TEMPO, or MCC950 alone. Notably, co-administration of AS-IV with either inhibitor conferred superior protection. Specifically: 1) AS-IV + Mito-TEMPO synergistically suppressed mitochondrial ROS and NLRP3; 2) AS-IV + MCC950 synergistically suppressed Gasdermin-D; 3) Both combinations synergistically reduced TnI/LDH, pyroptosis, MPTP opening, mitochondrial damage, and expression of BAK and caspase-1.<b>C</b><b>onclusion:</b> AS-IV alleviates LPS-induced pyroptosis by ameliorating mitochondrial dysfunction and inhibiting mtROS-NLRP3 activation. The superior efficacy of its combination with Mito-TEMPO or MCC950 reveals a synergistic mechanism, proposing a promising therapeutic strategy for septic cardiomyopathy.