NLRP1 as a Novel Pyroptosis Biomarker in Irreversible Pulpitis: A Laboratory Investigation and Animal Model Study.

AIM: Irreversible pulpitis poses a significant clinical burden due to progressive inflammatory pulp damage. While inflammatory mechanisms are central to its pathogenesis, they remain incompletely characterised. This study aims to elucidate the role of Nod-like receptor thermal domain associated protein 1 (NLRP1) in irreversible pulpitis pathogenesis.

METHODOLOGY: Transcriptomic analysis of public dataset GSE77459 identified dysregulated pyroptosis-related genes (PRGs), with machine learning prioritising hub genes for experimental validation. Key targets were experimentally verified through an integrated approach: in vitro models using lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs) analysed by qRT-PCR, ELISA and Western blot and specific pyroptosis assays (PI uptake, Caspase-1 cleavage); in vivo rat pulpitis models; and clinically validation with human irreversible pulpitis tissues analysed via H&E staining, immunohistochemistry (IHC) and immunofluorescence (IF). Functional roles were further assessed via NLRP1 knockdown and overexpression in hDPCs.

RESULTS

Bioinformatics identified 11 differentially expressed PRGs, with machine learning highlighting six hub genes including unreported NLRP1. Specific pyroptosis assays confirmed that LPS induces membrane pore formation and Caspase-1 activation in hDPCs. Significant post-transcriptional regulation of NLRP1 was demonstrated by pronounced protein upregulation in LPS-stimulated hDPCs despite unaltered mRNA. Functional studies established NLRP1 as a positive regulator, where its knockdown attenuated the LPS-induced inflammatory-pyroptotic response, while its overexpression alone was sufficient to upregulate key mediators. Consistently, elevated NLRP1 expression was observed in rat models and human tissues, where IHC/IF localised prominent expression to inflammatory infiltrates.

CONCLUSION

NLRP1 is highlighted as a novel pyroptosis biomarker for irreversible pulpitis, with its dysregulation offering diagnostic value for inflammation. These findings suggest its potential involvement in the inflammatory mechanisms of pulpitis, providing a new molecular target for future therapeutic exploration.