Lingguizhugan Decoction Alleviates Lung Inflammatory in Obstructive Sleep Apnea by Modulating ROS and HIF-1α Signaling Pathway Based on UHPLC–MS, Network Pharmacology, Transcriptomics, and Experimental Verification

Abstract

Rationale

Lingguizhugan decoction (LGZG) is a traditional Chinese formula that has been commonly used in obstructive sleep apnea (OSA) for relieving lung inflammation. However, the active substance of LGZG and the specific mechanism remain unclear. This study aims to identify the bioactive components of LGZG and subsequently elucidate the underlying therapeutic mechanisms against OSA based on mass spectrometry analysis, network pharmacology, transcriptomics, and experimental verification.

Methods

Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC–HRMS) was used to identify the main ingredients of LGZG. The OSA animal model was induced by chronic intermittent hypoxia (CIH) for 5 weeks in C57BL/6 mice. Transcriptome sequencing and network pharmacology were used to analyze potential mechanisms, which were further validated by molecular docking. HE staining was used for detecting lung inflammation. Immunohistochemistry (IHC), ELISA, and Western blot were employed to investigate protein expression, while quantitative real-time PCR (RT-qPCR) was used to determine gene expressions. Tissue reactive oxygen species (ROS) levels were measured by the DCFH-DA probe method.

Results

LGZG inhibited CIH-induced pulmonary inflammatory infiltration, protein concentration in bronchoalveolar lavage fluid (BALF), and suppressed IL-17 and IL-1β gene expression. UHPLC–HRMS identified 482 compounds in the LGZG aqueous decoction. Network pharmacology analysis revealed that IL-6 and HIF-1α pathway were the major targets. Subsequently, transcriptomics analysis revealed that LGZG affected functions associated with ROS production and polymorphonuclear cells. LGZG suppressed the CIH-induced expression of neutrophil elastase and reduced MPO production in BALF. Furthermore, LGZG inhibited the IL-6 expression and secretion, and reduced CIH-induced ROS production. LGZG inhibited the CIH-induced activation of HIF-1α pathway. Moreover, molecular docking identified compounds in LGZG that could directly interact with the core targets IL-6, MPO, CYBB, and HIF-1α.

Conclusions

LGZG alleviates CIH-induced pulmonary inflammation, neutrophil infiltration, and IL-6 secretion in mice. These effects are associated with the suppression of ROS production and inhibition of the HIF-1α signaling pathway.