Abstract
Gubenfangxiao decoction (GBFXD) is a traditional Chinese medicine based on a combination of Yu-Ping-Feng-San and Erchen decoctions. GBFXD has been widely used for decades in treating asthma at the Affiliated Hospital of Nanjing University of Chinese Medicine. Previously, GBFXD was found to reduce lung inflammation and airway remodeling; however, the underlying mechanism remains unknown. In this study, the effects of GBFXD on asthmatic mice were evaluated based on pathology and lung function; airway hyperresponsiveness (AHR) and pathology were compared among the two different mouse models utilized. Furthermore, the mechanism of action of GBFXD on asthmatic mice was analyzed using iTRAQ labeling technology combined with ingenuity pathway analysis (IPA). Modeling analysis of pre- and post-treatment proteins identified 75 differentially expressed proteins. These proteins were related to B-cell development, calcium-induced lymphocyte apoptosis, antigen presentation, and Th1 and Th2 activation pathways. Moreover, 68 differentially expressed proteins were identified in the GBFXD treatment group compared with the model group. Upstream regulatory factors predicted that interleukin (IL)-4 (necessary for inducing polarization of type 2 [M2] macrophages), cyclooxygenase, and prostaglandin E2 are significantly elevated in the model group. Based on IPA analysis, it was concluded that several pathways, including mitochondrial dysfunction and oxidative phosphorylation, are closely associated with the therapeutic effects of GBFXD in asthma. Moreover, the differential expression of several proteins, including the M2 markers, MRC1, ARG1, Retnla, Chil3, and CHIA, were validated by western blotting, confirming that GBFXD can reduce airway inflammation, which fits the pattern of an alternative M2 activation state, and attenuate AHR. Overall, our findings indicate that GBFXD significantly suppresses M2 macrophage polarization, providing further insights into the mechanism underlying the protective effects of GBFXD.
Highlights
MATERIALS AND METHODSAsthma is one of the most common chronic, non-infectious airway diseases in children, with a global prevalence of 1–18% (Ebmeier et al, 2017; Papi et al, 2018)
Gubenfangxiao decoction (GBFXD) Alleviates Airway Inflammation and Airway Hyperresponsiveness (AHR) Caused by Persistent Challenge
Mucus expression in the airways was evaluated by quantifying and comparing periodic acid Schiff (PAS)-positive cells between the two models; the chronic persistent asthmatic (CPA) model presented with more prominent goblet cell hyperplasia and mucus overproduction
Summary
Asthma is one of the most common chronic, non-infectious airway diseases in children, with a global prevalence of 1–18% (Ebmeier et al, 2017; Papi et al, 2018). Subsequent studies have shown that GBFXD can reduce lung inflammation in OVA-sensitized mice, inhibit the expression of asthma susceptibility genes ORMDL3 and ADAM33, and reduce endoplasmic reticulum stress (ERS) (Huang et al, 2016; Lu et al, 2016). As a model of chronic persistant asthma, RSV-OVA-sensitized mice was established. ITRAQ-based proteomics studies and ingenuity pathway analysis (IPA) were performed to reveal the protein features of RSV-OVA-sensitized mice and investigate the underlying mechanisms of GBFXD. The iTRAQ-labeled peptide mixtures were reconstituted with 100 μL of high pH RP buffer A (98% H2O, 2% acetonitrile; pH 10.0) and loaded onto a C18 column with a particle size of 1.7 μm (2.1 mm × 100 mm; Waters Corporation, Milford, MA, United States). Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis Peptides were re-dissolved in buffer A (2% acetonitrile, 0.1% formate) and centrifuged at 4◦C and 20,000 × g for 10 min.
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