Research On Effective Material Basis And Mechanism Action Of Qingfei Xiaoyan Wan

Airway inflammation is a central problem in allergic asthma and chronic obstructive pulmonary disease (COPD). Airway inflammation is a consequence of complex interactions between multiple cell types, cytokines, and mediators in an inflammatory network. A single-target approach is unlikely to be effective for the treatment of inflammatory lung diseases. Multiple-drug therapy, which maintains efficacy and diminishes the risk of toxicity from individual drugs, has become widely recognized in recent years. Traditional Chinese medicine (TCM) preparations have been used as an effective multi-target strategy for the treatment of complex diseases; however, their bioactive constituents are difficult to identify and the mechanisms are undefined.In this study, the global chemome of Qingfei Xiaoyan Wan (QFXY) was illustrated by using the ultra-performance liquid chromatography/quadrupole time-of-flight (UPLC/Q-TOF) mass spectrum, and55compounds were identified. Chemical constituents of QFXY were submit to Molinspiration, PharmMapper and KEGG bioinformatics softwares for predicting their absorption parameters, target proteins and related pathways, respectively. The predicted results showed that19of the27absorbable constituents, which are sulfur alkynes, lignan lactones, phenolic acids and steroid compounds, may be involved in inflammation through11protein targets such as HRAS and PDPK1.We investigate anti-inflammatory effects of QFXY in vivo and in vitro. QFXY was administered orally to KM mice twice per day for one week, followed by challenge with an intratracheal Pseudomonas aeruginosa suspension. Mortality, lung histological features, and inflammatory cytokines were evaluated" Cytokines were also evaluated in TNF-a stimulated bronchial epithelial cells (BEAS-2B) incubated with QFXY, and NF-κB activation was evaluated using luciferase assay and Western blot. QFXY treatment significantly reduced Pseudomonas aeruginosa-induced mortality and also reduced cytokine production in lung tissue and plasma (TNF-a, IL-6, IL-8, and RANTES) and in TNF-a stimulated BEAS-2B cells (IL-6and IL-8). Moreover, in bronchial epithelial cells, an NF-κB luciferase assay and Western blot analysis demonstrated that QFXY inhibited NF-κB activation and IκB-α degradation.A simple method based on UPLC/Q-TOF MS combined NF-κB dual-luciferase reporter assay systems was developed for the rapid determination of anti-inflammatory compounds of TCM preparations.8potential NF-κB inhibitors were characterized and they could be classified into four types according to their chemical structures:arctigenin derivatives (tracheloside and arctigenin), cholic acid derivatives (cholic acid, glycocholic acid, deoxycholic acid, and deoxyglycocholic acid), chlorogenic acid, and sinapic acid. Tracheloside was considered a new NF-κB inhibitor. Further cytokine and chemokine (IL-6and IL-8) detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors.TNF-a induced inflammation model in human bronchial epithelial cells BEAS-2B was used to investigate synergistic anti-inflammatory effects and mechanisms of the four NF-κB inhibitors in QFXY. NF-κB luciferase analysis revealed that paired combination with arctigenin showing stronger inhibition effect than the single use. Real-time PCR was used to detect the expression of key inflammatory genes for the study of synergistic anti-inflammatory mechanism of arctigenin. The results showed that paired combination with arctigenin not only affect the expression of p38, JNK, ERK1, FAK, but also significantly affect the expression of JAK and PKC. The results of ELISA test showed that synergistic anti-inflammatory mechanism of paired combination with arctigenin mainly focused on the intervention of the levels of p38, JNK and ERK proteins.We developed a simple method based on UPLC/Q-TOF MS combined β2-adrenergic receptor (β2-AR) dual-luciferase reporter assay systems for the rapid determination of spasmolytic constituents in TCM preparations. One β2-AR agonist ephedrine and four synergistic constituents (arctiin, arctigenin, descurainoside and descurainolide B) were characterized. The four synergistic compounds could be classified into one type according to their chemical structures:arctigenin derivatives. Ephedrine and arctigenin combination strongerly relax the isolated guinea pig trachea strip precontraction with acetylcholine (ACh) than the single use, and the β-AR antagonist propranolol (Pro) could block the synergistic effect. From the β2-AR dual luciferase reporter gene activity analysis, specific the β2-AR blocking agent ICI118551blocked the synergistic effect of ephedrine and arctigenin combination, and10-5mol/L of arctigenin can significantly enhance the effects of the AC activator forskolin. These results indicated that β2-AR signal is necessary for the synergistic effect, and it is not β2-AR dependent.In this study, we demonstrate the anti-inflammatory and anti-asthmatic ingredients, the synergistic effects of active compounds and their mechanism of action. These results reveal multiple active components, multiple targets and synergistic effects in the mechanism of action of a TCM.