Aspirin reprograms platelet signaling and the intrahepatic microbiome to suppress RyR2-driven inflammation and fibrosis in preclinical chronic liver disease

Platelet deactivation by aspirin possibly helps in regression of liver fibrosis, though the mechanisms are unclear. We administered aspirin in a murine model of liver fibrosis and studied molecular signatures associated with fibrosis regression; both in vivo (murine model/ patients) and in vitro . Increase in intrahepatic PF4, p-selectin, PDGFR-β levels (platelet activation) correlated with increase in liver fibrosis (p < 0.05, r 2 >0.3). Aspirin reduced the number and activation of intrahepatic platelets, inflammation and fibrosis (p < 0.05). Platelet deactivation using aspirin in murine model increased autophagy, glutathione, energy metabolism and decreased arachidonic acid and butanoate metabolism (p < 0.05). Aspirin modulated liver microenvironment and showed decrease in intrahepatic immune cell activation (blood transcription module) which correlated with histidine and tryptophan metabolism (r2 > 0.5, p < 0.05). The intrahepatic microbiome post-aspirin showed increased abundance of Firmicutes (Ruminococcaceae, Lachnospiraceae, and Clostridiaceae) and improved functionality (p < 0.05). Aspirin caused decreased expression of Ryanodine-receptor-2 (RyR2), Arginase-1 and Kynurenine-3-monooxygenase, which correlate with reduction in α-SMA and degree of hepatic fibrosis (r2 > 0.75; p < 0.05). In addition, pan specific blocking of RyR2 by carvedilol/flecainide markedly inhibited HSC activation and proliferation in-vitro by reducing Ca 2+ overload, ER/mitochondrial stress (p < 0.05). Further, RyR2 blockade in HSCs reduced its activation by activated platelet secretome or TGFβ1 (p < 0.05). Platelet deactivation using aspirin regresses hepatic fibrosis by decreasing intrahepatic platelet accumulation/activation, inflammation and modulation of intrahepatic microbiome. Induction of RyR2 is critical for fibrosis development and pharmacological inhibition of RyR2 could ameliorate liver fibrosis. • Aspirin reduces intrahepatic platelet activation and fibrosis in CCl₄ mice. • Multi-omics links aspirin response to redox balance and energy pathways. • Aspirin associates with favorable intrahepatic microbial peptide signatures. • RyR2 correlates with fibrosis stage in mice and human liver tissues. • RyR2 blockade lowers Ca²⁺ stress and suppresses stellate cell activation, thereby reducing fibrosis.