Glycochenodeoxycholic acid induces the release of IL-1beta from L02 cells via the NLRP3/caspase-1/GSDMD pathway to activate LX2 cells

Purpose: Cholestatic liver diseases, characterized by hepatic bile acid accumulation, often progress to fibrosis through incompletely understood mechanisms. While hydrophobic bile acids like glycochenodeoxycholic acid (GCDCA) are known to induce hepatocyte apoptosis or necrosis and their role in pyroptosis—a pro-inflammatory programmed cell death—remains unclear. This study investigates whether GCDCA drives hepatic stellate cell (HSC) activation via NLRP3 inflammasome-mediated pyroptosis in hepatocytes. Patients and Methods: Using L02 hepatocytes and LX2 HSCs, we evaluated the effects of GCDCA on NLRP3 inflammasome activation, gasdermin D (GSDMD)-dependent pyroptosis, and interleukin-1beta (IL-1β) secretion. Cells were treated with GCDCA (25– 400 μM) ± lipopolysaccharide (LPS) to simulate cholestatic injury. Caspase-1 inhibition (Ac-YVAD-cmk), GSDMD knockdown (shRNA), and IL-1 receptor antagonist (IL-1RA) were employed to dissect mechanistic pathways. Pyroptosis (LDH release, Annexin V/PI staining), inflammasome components (NLRP3, ASC, caspase-1), GSDMD cleavage, IL-1β secretion (ELISA), and HSC activation (α-SMA, Collagen-I, proliferation/migration assays) were analyzed. Results: GCDCA alone upregulated NLRP3, pro-caspase-1, and pro-IL-1β in L02 cells but required LPS co-stimulation to trigger caspase-1 activation and GSDMD cleavage, indicating a dual-signal mechanism (priming + activation). This led to pyroptotic cell death (dose/time-dependent LDH release, Annexin V⁺/PI⁺ cells) and IL-1β secretion. GSDMD knockdown abolished pyroptosis and IL-1β release, while caspase-1 inhibition suppressed GSDMD-N-terminal (GSDMD-NT) generation. Conditioned media from pyroptotic hepatocytes activated LX2 HSCs, upregulating α-SMA and Collagen-I, and enhancing proliferation/migration—effects reversed by IL-1RA. Conclusion: GCDCA promotes HSC activation via NLRP3 inflammasome-mediated pyroptosis in hepatocytes, driven by caspase-1-dependent GSDMD cleavage and IL-1β paracrine signaling. This mechanism bridges bile acid toxicity to fibrogenesis, highlighting therapeutic potential for targeting NLRP3-GSDMD-IL-1β signaling in cholestatic liver diseases. Plain Language Summary: Cholestatic liver diseases, marked by toxic bile acid buildup, often progress to liver scarring (fibrosis), but the exact mechanisms are unclear. This study explored how a bile acid (GCDCA) triggers inflammatory cell death (pyroptosis) and drives fibrosis. Using lab-grown liver and scar-forming cells, the team exposed liver cells to GCDCA and bacterial toxins (LPS) to mimic disease conditions. They blocked key molecules to identify critical pathways. GCDCA activates the NLRP3 inflammasome, priming liver cells for pyroptosis. Combined with LPS, GCDCA triggers pyroptosis, releasing IL-1β, a protein that activates scar-forming cells. Blocking IL-1β reversed scar cell activity. This reveals how bile acids like GCDCA cause liver scarring through pyroptosis and IL-1β signaling. Targeting this pathway—NLRP3, GSDMD (a cell death protein), or IL-1β—could lead to new treatments for cholestatic liver diseases. Keywords: glycochenodeoxycholic acid, NLRP3 inflammasome, pyroptosis, gasdermin D, interleukin-1beta, hepatic stellate cell