Reprogramming lysosomal-purinergic crosstalk governs the synergistic attenuation of NLRP3 inflammasome activation by...

Steatohepatitis links cholesterol-crystal injury to ATP-gated purinergic signaling, culminating in NF-κB/NLRP3 inflammasome activation and metabolic dysfunction. We posited that dissolving cholesterol burden while concurrently blocking P2X7 would reprogram lysosomal-purinergic crosstalk and outperform single-pathway therapy. In a streptozotocin-high-fat-diet diabetic rat model, hydroxypropyl-β-cyclodextrin (HPβCD; cholesterol efflux/crystal dissolution) combined with the P2X7 antagonist coomassie brilliant blue G-250 (CBB) improved glucose tolerance, fasting glycemia, and HOMA-IR beyond either monotherapy. In addition, this dual intervention lowered transaminases and inflammatory cytokines; reduced hepatic fibrosis and lipid overload; and restored lysosomal and insulin-signaling readouts. Mechanistically, dual treatment most effectively suppressed the P2X7-Panx1 axis, restored lysosomal integrity (increased LAMP1, decreased cathepsin-B), and curtailed NF-κB/NLRP3-GSDMD signaling, indicating disruption of the feed-forward loop connecting lysosomal injury with purinergic activation. Synergy at fixed in-vivo doses was formally quantified by HSA and Bliss, with the largest Bliss gains at purinergic-inflammasome nodes (P27RX, Panx1, NLRP3, p-AKT/AKT) and coherent effects at lysosomal biogenesis markers (LAMP1, TFEB). Integrative network analysis intersecting cholesterol-regulated genes with the P2RX7 interactome revealed an overlap enriched for IL-1β processing, pyroptosis, and lysosomal organization, nominating a high-connectivity P2RX7/Panx1-LAMP1/CTSB-NLRP3 module selectively reprogrammed by the combination. This work provides in-vivo evidence that simultaneously targeting cholesterol-crystal-driven lysosomal injury and ATP-gated P2X7 signaling yields mechanistic and therapeutic synergy, while linking systems-level network topology to multi-arm pharmacodynamics to define a reprogrammable lysosomal-purinergic module. Collectively, the data establish a mechanistically anchored, clinically plausible combination strategy for diabetes-associated steatohepatitis that surpasses single-pathway interventions.