Anti-asthmatic effects of Chai-Po-Tang in experimental models: chemical profiling, exploratory network pharmacology, and assessment of NLRP3/Caspase-1/GSDMD-related pyroptosis signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Chai-Po-Tang (CPT) is a traditional Chinese medicine formula derived from Xiao-Chai-Hu-Tang and Ban-Xia-Hou-Po-Tang, two classical prescriptions historically used for respiratory symptom patterns involving cough, dyspnea, chest oppression, and phlegm obstruction. However, whether the anti-asthmatic effects of CPT are associated with modulation of inflammasome- and pyroptosis-related signaling remains unclear.

AIM OF THE STUDY: The present study aimed to evaluate the anti-asthmatic effects of CPT and investigate whether these effects were associated with modulation of pyroptosis-related NLRP3/Caspase-1/GSDMD signaling, while using network pharmacology as a hypothesis-generating approach to identify candidate regulatory targets.

MATERIALS AND METHODS: The chemical constituents of CPT were identified using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-MS). Network pharmacology and molecular docking analyses were employed to predict its potential therapeutic targets and signaling pathways. Subsequently, LPS/ATP-induced BEAS-2B cell inflammasome activation model and an ovalbumin (OVA)-induced asthmatic mouse model were established. The effects of CPT were evaluated using cell viability assays, ROS measurement, transmission electron microscopy, inflammatory mediator quantification, pulmonary function testing, histopathology, immunofluorescence, and Western blotting, while pyroptosis-related proteins were assessed as mechanistic correlates rather than as evidence of direct causal dependence.

RESULTS: A total of 1155 chemical features/components were tentatively annotated in CPT, of which 102 candidate compounds were prioritized for network pharmacology using predefined in silico and literature-based criteria. Among the 102 prioritized candidates, 37 were supported by experimentally acquired MS/MS fragmentation spectra, whereas 65 were tentatively annotated on the basis of accurate mass, isotope distribution, database matching, and literature evidence without complete experimental MS/MS confirmation. Network pharmacology nominated IL6, FN1, and KIT as candidate targets, whereas molecular docking provided in silico support for potential ligand-target interactions only. In vitro experiments demonstrated that CPT-containing serum significantly enhanced the viability of BEAS-2B cells, reduced reactive oxygen species (ROS) levels, ameliorated ultrastructural damage, and inhibited the co-localization of NLRP3 with cleaved Caspase-1, as well as the co-localization of cleaved Caspase-1 with GSDMD-N. In vivo studies showed that CPT markedly improved clinical manifestations and pulmonary function in asthmatic mice, alleviated lung inflammation, airway remodeling, and mucus secretion. Additionally, CPT decreased the levels of IL-6, IL-1β, and IL-18, and significantly downregulated the expression of NLRP3, cleaved Caspase-1, and GSDMD-N in lung tissues. Western blot analysis further showed lower NLRP3 and ASC expression, together with lower GSDMD-N/GSDMD and cleaved Caspase-1/pro-Caspase-1 ratios, in CPT-treated lung tissues. These findings indicate that CPT treatment was associated with reduced NLRP3/Caspase-1/GSDMD pathway-related signaling in the experimental models.

CONCLUSION: In OVA-induced allergic asthma and LPS/ATP-stimulated BEAS-2B cells, CPT improved inflammatory and functional outcomes in parallel with reduced NLRP3/Caspase-1/GSDMD pathway-related signaling. These data support further investigation of inflammasome-associated pyroptosis as one plausible contributor to CPT activity, while direct causal dependence and the contribution of other predicted targets require dedicated assessment.