Combined Nivolumab and Ipilimumab Therapy Promotes Immune-Mediated Cardiomyocyte Apoptosis Through TLR4-Myd88-NF-b-Driven Activation of the NLRP3 Inflammasome.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment but are associated with serious immune-related cardiac toxicities. In this study, AC16 human cardiomyocytes were exposed to nivolumab alone or in combination with ipilimumab. The expression of apoptosis- and inflammation-associated proteins and key components of the TLR4-MyD88-NF-B signaling pathway was examined by Western blotting. Apoptotic responses were evaluated using flow cytometry and immunofluorescence assays. BALB/c mice were administered nivolumab or nivolumab plus ipilimumab intraperitoneally for 4 weeks followed by histopathological assessment of cardiac tissue and measurement of myocardial injury biomarkers. Network pharmacology, protein-protein interaction analysis, GO/KEGG enrichment, and molecular docking were applied to identify active constituents of and their potential targets in immune-related cardiac injury. Combined treatment with nivolumab and ipilimumab markedly increased cardiomyocyte apoptosis and elevated the expression of NLRP3 and ASC. Increased phosphorylation of IKK and NF-B p65 indicated activation of the TLR4-MyD88-NF-B signaling cascade. Knockdown of either TLR4 or NLRP3 significantly mitigated apoptosis and reduced inflammatory protein expression. In vivo, combined ICI therapy led to higher levels of myocardial injury markers and proinflammatory mediators. Network pharmacology analysis identified six major compounds from , with sinomenine showing strong predicted binding to TLR4 (binding energies ranging from -9.6 to -5.7 kcal/mol). These findings demonstrate that under the present experimental conditions, combined nivolumab and ipilimumab treatment was associated with greater activation of the TLR4-MyD88-NF-B-NLRP3 axis and greater cardiomyocyte injury than nivolumab alone. However, because the combination group received a higher total antibody dose, the current study does not distinguish whether this difference reflects increased total drug exposure, additivity, or the specific contribution of CTLA-4 blockade. Bioactive constituents of , particularly sinomenine, may represent potential cardioprotective modulators against ICI-induced cardiac injury.