Sphingosine-1-Phosphate Attenuates LPS-Induced Inflammatory Cardiac Injury in Association With RASGRP1-S100A9-NLRP3 Signaling.

Lipopolysaccharide (LPS) induces endotoxemia-associated inflammatory cardiac injury rather than classical viral or autoimmune myocarditis. Sphingosine-1-phosphate (S1P) regulates cardiovascular and immune responses, predominantly through S1P receptors, but whether it also modulates macrophage-associated inflammatory signaling during LPS-induced cardiac injury remains incompletely understood. This study investigated the role of the RASGRP1-S100A9-NLRP3 signaling axis in the cardioprotective effects of S1P. Transcriptomic and single-cell RNA-sequencing analyses were used to identify inflammation-associated candidate genes and their cellular distribution. Male Sprague-Dawley rats received a single intraperitoneal injection of LPS (8 mg/kg) followed by S1P treatment for 6 weeks. Histopathology, echocardiography, serum cardiac-injury markers, oxidative-stress indices, inflammatory cytokines, Western blotting, and cellular metabolic-flux assays were evaluated. RAW264.7 macrophages were used for mechanistic cellular studies. RASGRP1 or NLRP3 overexpression and S100A9-R101Q mutation were used to assess functional contributions. Molecular docking and molecular-dynamics simulations were complemented by SPR, MST, CETSA, DARTS, and Co-IP assays to evaluate S1P-RASGRP1 target engagement and pathway-associated protein interactions. Bioinformatic analyses identified RASGRP1 as a macrophage-enriched, inflammation-associated hub gene. In LPS-treated rats, S1P reduced myocardial inflammatory injury and collagen deposition, improved ejection fraction and fractional shortening, and decreased cardiac-injury, oxidative-stress, and inflammatory markers. In RAW264.7 macrophages, S1P attenuated inflammatory and oxidative-stress responses, restored oxidative phosphorylation and glycolytic capacity, and reduced the abundance of RASGRP1, S100A9, NLRP3, and ASC. SPR and MST supported a concentration-dependent biophysical interaction between S1P and RASGRP1; CETSA and DARTS provided complementary target-stability evidence. The molecular-dynamics trajectory showed substantial rearrangement of S1P from the starting pose before reaching a plateau and therefore supports conformational sampling rather than preservation of the original docked pose. Co-IP supported protein associations within the proposed inflammatory network. RASGRP1 or NLRP3 overexpression attenuated several S1P-associated protective effects. The S100A9-R101Q mutation also weakened several S1P-associated protective responses. SPR indicated a lower apparent dissociation constant for R101Q than for WT S100A9 after model-specific fitting, which is consistent with stronger or more persistent NLRP3 binding rather than loss of the S100A9-NLRP3 interaction. S1P mitigates LPS-induced inflammatory cardiac injury in association with altered macrophage RASGRP1-S100A9-NLRP3 signaling. Biophysical and target-stability assays support direct engagement of RASGRP1 by S1P. However, these findings do not establish RASGRP1 as the sole mediator of S1P activity, exclude contributions from canonical S1P receptor signaling, or demonstrate a strictly linear RASGRP1-S100A9-NLRP3 signaling cascade. Because the LPS model primarily represents endotoxemia-associated inflammatory cardiac injury, extrapolation of these findings to classical viral or autoimmune myocarditis should be made with caution.