ETHNOPHARMACOLOGICAL RELEVANCE: Neutrophilic asthma (NA) is a severe treatment-resistant phenotype characterized by neutrophilic airway inflammation, for which effective therapeutic interventions are currently lacking. Shuangpi Formula (SP) is an optimized formula derived from Xiebaisan, a classical prescription first recorded in Xiao'er Yaozheng Zhijue (A.D. 1119, Song Dynasty). According to this canonical text, Xiebaisan is indicated for "lung heat with wheezing and cough" - symptoms that directly correspond to the clinical manifestations of NA. However, its efficacy on NA and the underlying mechanisms remain unexplored.
OBJECTIVE: This study aimed to investigate the therapeutic effects of SP on NA and to explore the underlying mechanisms.
MATERIALS AND METHODS: NA was induced in rats by lipopolysaccharide (LPS) and ovalbumin (OVA). SP was administered, and its effects were assessed by measuring asthmatic latency, leukocyte counts and inflammatory cytokine levels in bronchoalveolar lavage fluid (BALF), and myeloperoxidase (MPO) expression in lung tissues. Transcriptomic analysis identified differentially expressed genes, with nuclear receptor subfamily 1 group D member 1 (NR1D1) upregulation confirmed by quantitative reverse transcription PCR (qRT-PCR) and immunofluorescence. Molecular docking evaluated binding affinities between NR1D1 and the main systemically absorbed bioactive components of SP. In vitro, the role of NR1D1 in SP's anti-inflammatory effects was examined using a pharmacological inhibitor.
RESULTS: SP treatment significantly prolonged asthmatic latency, reduced leukocyte counts and inflammatory cytokine levels in BALF, and decreased MPO expression in lung tissues. These effects were superior to those of dexamethasone, without the adverse effects observed with dexamethasone. Transcriptomic analysis revealed marked upregulation of NR1D1, validated by qRT-PCR and immunofluorescence. Molecular docking indicated strong binding affinities between NR1D1 and several SP components, including Liquiritin, Glycyrrhizic acid, Oxyresveratrol, Mulberroside A, Glycyrrhetinic acid, and Kukoamine B. Importantly, pharmacological inhibition of NR1D1 abolished SP's regulatory effects on chemokine (Chemokine (C-C motif) ligand 2) and inflammatory cytokines (interleukin-6, tumor necrosis factor-α) in vitro.
CONCLUSION: SP inhibits neutrophilic inflammation in NA, with evidence implicating NR1D1 as a key regulator. Pharmacological modulation of NR1D1 may represent a potential therapeutic approach for NA, although further genetic validation is required.
