{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T18:35:18Z","timestamp":1786473318251,"version":"build-2736575974"},"update-to":[{"DOI":"10.1371\/journal.pntd.0014495","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:00:00Z","timestamp":1786406400000}}],"reference-count":40,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T00:00:00Z","timestamp":1783900800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["Grant nos.81560318 and 81860346"],"award-info":[{"award-number":["Grant nos.81560318 and 81860346"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Training Project of \u201c139\u201d Program for High-Level Medical Talents in Guangxi","award":["No. G201903034"],"award-info":[{"award-number":["No. G201903034"]}]},{"name":"Research and Demonstration Application of Snakebite Bigdata Platform Based on Virtual Standardized Snakebite Treatment Model","award":["No. 22QYCX0046"],"award-info":[{"award-number":["No. 22QYCX0046"]}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Cardiotoxin (CTX) from\n                      <jats:italic>Naja atra<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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 \u03bcg\/50 \u03bcL), 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&amp;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-1\u03b2, LDH release assay, and pharmacological inhibition with MCC950 (NLRP3 inhibitor), VX-765 (caspase-1 inhibitor), and N-acetylcysteine (NAC, ROS scavenger).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      RNA-seq identified 2,490 DEGs (|log\n                      <jats:sub>2<\/jats:sub>\n                      FC|\u2009&gt;\u20091, FDR\u2009&lt;\u20090.01; 1,047 upregulated, 1,443 downregulated). KEGG enrichment revealed that the NOD-like receptor signaling pathway was the most significantly enriched pathway (enrichment fold\u2009=\u20096.8, p_adj\u2009&lt;\u20090.001, 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\u2009&lt;\u20090.05). Six canonical NLRP3\/caspase-1\/GSDMD pathway genes\u2014Nlrp3, Pycard, Gsdmd, Il18, Nfkb, and Tlr4\u2014were 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\u20135 \u03bcm 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\u2014NAC (ROS scavenger), MCC950 (NLRP3 inhibitor), and VX-765 (caspase-1 inhibitor)\u2014significantly attenuated CTX-induced LDH release, IL-1\u03b2 secretion, and GSDMD cleavage (all p\u2009&lt;\u20090.0001), confirming the mechanistic hierarchy: ROS\u2009\u2192\u2009NLRP3\u2009\u2192\u2009caspase-1\u2009\u2192\u2009GSDMD.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      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\u2009\u2192\u2009NLRP3\u2009\u2192\u2009caspase-1\u2009\u2192\u2009GSDMD cascade. The ROS\u2013NLRP3\u2013caspase-1\u2013GSDMD axis constitutes a tractable therapeutic target for\n                      <jats:italic>Naja atra<\/jats:italic>\n                      envenomation.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pntd.0014495","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T17:37:19Z","timestamp":1783964239000},"page":"e0014495","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cardiotoxin from Naja atra Activates the NLRP3\/Caspase-1\/GSDMD Pyroptosis Pathway to Induce Skin Tissue Injury"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-6256-7588","authenticated-orcid":true,"given":"Nianying","family":"Qin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiling","family":"Zhai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongying","family":"Cao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunyang","family":"Dong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxiang","family":"Liao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guanyao","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhou","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhengzhuang","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"He","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jincheng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3941-4860","authenticated-orcid":true,"given":"Wei","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,7,13]]},"reference":[{"key":"pntd.0014495.ref001","article-title":"How and why snakebite became a global health priority: a policy analysis","author":"S Bhaumik","year":"2023","journal-title":"BMJ Glob Health"},{"issue":"10385","key":"pntd.0014495.ref002","doi-asserted-by":"crossref","first-page":"1382","DOI":"10.1016\/S0140-6736(23)00002-8","article-title":"Clinical aspects of snakebite envenoming and its treatment in low-resource settings","volume":"401","author":"DA Warrell","year":"2023","journal-title":"Lancet"},{"key":"pntd.0014495.ref003","author":"R Ralph","year":"2022","journal-title":"BMJ"},{"key":"pntd.0014495.ref004","doi-asserted-by":"crossref","first-page":"100103","DOI":"10.1016\/j.lansea.2022.100103","article-title":"Vulnerability to snakebite envenoming and access to healthcare in the Terai region of Nepal: a geospatial analysis","author":"C Ochoa","year":"2023","journal-title":"Lancet Reg Health Southeast Asia"},{"issue":"2","key":"pntd.0014495.ref005","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pntd.0007059","article-title":"Strategy for a globally coordinated response to a priority neglected tropical disease: Snakebite envenoming","volume":"13","author":"DJ Williams","year":"2019","journal-title":"PLoS Negl Trop Dis"},{"issue":"5","key":"pntd.0014495.ref006","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1042\/BCJ20160577","article-title":"Venomics: integrative venom proteomics and beyond","volume":"474","author":"JJ Calvete","year":"2017","journal-title":"Biochem J"},{"issue":"6","key":"pntd.0014495.ref007","doi-asserted-by":"crossref","DOI":"10.1111\/bcpt.70144","article-title":"Snake venom three-finger neurotoxins and neurotoxin-like proteins: insights into their structural and functional aspects along with their pharmacological potential","volume":"137","author":"M Choudhury","year":"2025","journal-title":"Basic Clin Pharmacol Toxicol"},{"key":"pntd.0014495.ref008","article-title":"Cobra three-finger toxins interact with RNA and DNA: nucleic acids as their putative biological targets","author":"AV Osipov","year":"2025","journal-title":"Int J Mol Sci"},{"key":"pntd.0014495.ref009","doi-asserted-by":"crossref","unstructured":"Hiu JJ, Yap MKK. 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