Cardiotoxin from Naja atra Activates the NLRP3/Caspase-1/GSDMD Pyroptosis Pathway to Induce Skin Tissue Injury

Cardiotoxin (CTX) from Naja atra venom is a principal virulence factor responsible for progressive local tissue necrosis and systemic inflammation following snakebite. Despite its clinical importance, the molecular mechanisms underlying CTX-induced skin injury remain poorly defined.

We employed a two-arm strategy combining transcriptome-guided discovery with mechanistic functional validation. In the transcriptomic arm, C57BL/6 mice received intradermal CTX injection (120 μg/50 μL), and skin tissues were harvested at 6, 12, and 24 h post-injection for RNA-seq analysis. Differentially expressed genes (DEGs) were screened and subjected to KEGG/GO enrichment and ssGSEA-based cell death mode scoring. In the functional validation arm, a separate cohort of mice was assessed at 72 h post-injection, with gross necrosis area quantified, followed by H&E staining, immunohistochemistry (IHC), and Western blot. In vitro validation was performed in human HaCaT keratinocytes using CCK-8 cytotoxicity assay, optical microscopy, transmission electron microscopy (TEM), propidium iodide/DAPI (PI/DAPI) dual staining, ROS detection, ELISA for IL-1β, LDH release assay, and pharmacological inhibition with MCC950 (NLRP3 inhibitor), VX-765 (caspase-1 inhibitor), and N-acetylcysteine (NAC, ROS scavenger).

RNA-seq identified 2,490 DEGs (|log2FC| > 1, FDR < 0.01; 1,047 upregulated, 1,443 downregulated). KEGG enrichment revealed that the NOD-like receptor signaling pathway was the most significantly enriched pathway (enrichment fold = 6.8, p_adj < 0.001, with 42 differentially expressed genes annotated to this pathway). Among eight assessed cell death modalities, ssGSEA demonstrated that pyroptosis had the highest activation score (p < 0.05). Six canonical NLRP3/caspase-1/GSDMD pathway genes-Nlrp3, Pycard, Gsdmd, Il18, Nfkb, and Tlr4-were continuously upregulated from 6 to 24 h. Western blot confirmed both full-length GSDMD and its cleaved N-terminal fragment (GSDMD-N), along with NLRP3 inflammasome activation, in CTX-treated skin tissues and HaCaT cells. In vitro, CTX induced characteristic pyroptotic morphology and pyroptotic bodies (1-5 μm by TEM). Western blot confirmed NLRP3/GSDMD-N upregulation in HaCaT cells. CTX also induced dose-dependent intracellular ROS accumulation (DCFH-DA fluorescence). Importantly, all three inhibitors-NAC (ROS scavenger), MCC950 (NLRP3 inhibitor), and VX-765 (caspase-1 inhibitor)-significantly attenuated CTX-induced LDH release, IL-1β secretion, and GSDMD cleavage (all p < 0.0001), confirming the mechanistic hierarchy: ROS → NLRP3 → caspase-1 → GSDMD.

CTX induces intracellular ROS accumulation that activates the NLRP3/caspase-1/GSDMD pyroptotic cascade as an important mechanism contributing to skin tissue necrosis through membrane pore formation and inflammatory amplification. Pharmacological inhibition (NAC, MCC950, VX-765) confirmed a hierarchical ROS → NLRP3 → caspase-1 → GSDMD cascade. The ROS-NLRP3-caspase-1-GSDMD axis constitutes a tractable therapeutic target for Naja atra envenomation.