Modulation of glycolytic metabolic reprogramming and the TLR4/NF-κB/NLRP3 Pathway: comparative analysis of anti-inflammatory activity between two dosage forms of Pudilan xiaoyan.

ETHNOPHARMACOLOGICAL RELEVANCE: Pudilan xiaoyan oral liquid (PDL-L), a antipyretic and toxin-eliminating agent used in China for inflammatory conditions including sore throat and tonsillitis, is a standardized Chinese patent medicine. Pudilan formula granules (PDL-G), which share the same composition as PDL-L, are another dosage form of this medication. However, the difference in anti-inflammatory efficacy between PDL-L and PDL-G remains unevaluated, and their potential mechanisms require further exploration.

AIM OF THE STUDY: The present work systematically investigates comparative pharmacodynamic differences between PDL-L and PDL-G and their underlying mechanisms.

MATERIALS AND METHODS: Qualitative analysis of key bioactive constituents in PDL-G was conducted using high performance liquid chromatography (HPLC) to ensure pharmaceutical quality standards. The anti-inflammatory effects of PDL-L and PDL-G were compared using three established models: the mouse auricular swelling model, the abdominal capillary permeability model, and the rat paw edema model. Additionally, to establish an inflammatory phenotype, zebrafish embryos at 3-day-post-fertilization received microinjection of 2-nL LPS solution (3 mg/mL) into the yolk sac region for inflammatory challenge. Comparative evaluation of anti-inflammatory efficacy between PDL-L and PDL-G were compared by larval survival analysis and observation of neutrophils and macrophages migration. Quantitative real-time PCR (qRT-PCR) was employed to measure transcriptional levels of pro-inflammatory cytokines and TLR4/NF-κB/NLRP3 pathway components in LPS-challenged zebrafish. The inhibitory effects of PDL-L and PDL-G on LPS- and Poly (I:C)-induced acute lung injury (ALI) were evaluated in mice through enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. Pathological damage was evaluated via hematoxylin-eosin (H&E) staining. A concurrent evaluation was performed on glycolytic-related protein expression and TLR4/NF-κB/NLRP3 signaling activation in ALI mice.

RESULTS: Qualitative profiling of eight principal constituents in PDL-G and PDL-L was achieved through simultaneous chromatographic detection. Both PDL-L and PDL-G exhibited remarkable anti-inflammatory effects in several types of inflammation models. Both two formulations reduced ear swelling, abdominal capillary permeability, and paw edema with no significant difference in efficacy. In zebrafish inflammation model, PDL-L and PDL-G both significantly suppressed neutrophils infiltration and macrophages accumulation in LPS-challenged zebrafish larvae, reduced the release of LPS-stimulated inflammatory factors, and prolonged larval survival through the TLR4/NF-κB pathway inhibition coupled with NLRP3 inflammasome suppression. In murine models of LPS/Poly (I:C)-induced ALI, both PDL-L and PDL-G effectively mitigated pulmonary inflammation through suppression of pro-inflammatory mediator secretion in bronchoalveolar lavage fluid and lung tissues. Furthermore, these treatments downregulated the expression of glycolysis-associated proteins and suppressed the signaling activity of the TLR4/NF-κB/NLRP3 inflammatory cascade.

CONCLUSION: This investigation first provides experimental evidence that PDL-G demonstrates comparable or even superior efficacy to PDL-L in anti-inflammatory effects. Our results also show that PDL-G exerts anti-inflammatory activity through regulation of glycolysis and the TLR4/NF-κB/NLRP3 pathway. These findings substantiate PDL-G's clinical substitution potential for PDL-L, providing a molecular rationale for its therapeutic translation in inflammatory disorders.