You-Gui-Wan ameliorates house dust mite-induced allergic asthma via modulating amino acid metabolic disorder and gut dysbiosis

Introduction Allergic asthma is a worldwide health problem, and its etiology remains incompletely understood. Besides, as current therapies for allergic asthma mainly rely on administration of glucocorticoids and have many side effects, new therapy is needed. You-Gui-Wan (YGW), a traditional Chinese herbal remedy, has been used for boosting Yang, enhancing immunity and treating allergic asthma. Objectives This study aims to explore the molecular changes during the development of allergic asthma and investigate the potential bio-signatures and the effect of YGW on house dust mite (HDM)-induced chronic allergic asthma in mice. Methods Dermatophagoides pteronyssinus (Der p), one of HDMs, was intratracheally administered once a week for a total of 7 treatments over 6 consecutive weeks to induce allergic asthma in mice. Serum metabolomics was analyzed by LC-QTOF-MS/MS. 16S rRNA-based microbiome profiling was used to analyze gut microbiota, and the correlation between metabolomic signatures and microbial community profiling was explored by Spearman correlation analysis. Results Serum metabolomic analysis revealed that 10 identified metabolites — acetylcarnitine, carnitine, hypoxanthine, tryptophan, phenylalanine, norleucine, isoleucine, betaine, methionine, and valine, were markedly elevated by Der p. These metabolites are mainly related to branch-chain amino acid (BCAA) metabolism, aromatic amino acid (AAA) biosynthesis, and phenylalanine metabolism. YGW administration reversed 7 of the 10 identified metabolites and chiefly affected BCAA metabolism. 16S DNA sequencing revealed that YGW profoundly changed Der p-induced gut microbiota composition. Multiple correlation analysis indicated 10 selected metabolites have a good correlation with gut microbiota. Conclusion Der p induced BCAA metabolic deviation in allergic asthma mice, and YGW administration effectively ameliorated the AA metabolic disorder, and improved gut dysbiosis. This study paves the way towards the interactions of Der-p on microbiome and gut microbiota, and the effects of YGW treatment as well as provides a support for YGW administration with potential benefits for allergic asthma.

137 Der p-induced allergic asthma in mice mainly followed a previous method [10]. Briefly, 138 mice were intratracheally administered 40 μl of Der p (2.5 μg/μl). Der p (in PBS) once a 139 week for a total of 7 treatments over 6 consecutive weeks to induce chronic allergic 140 asthma. YGW and dexamethasone were orally administered daily and once a week, 141 respectively. Both YGW and dexamethasone were orally administered 30 min prior to 142 Der p stimulation once a week. After the last treatment, mice were injected with xylazine 143 (200 μg/mouse) and ketamine (2 mg/mouse) in the abdominal cavity and sacrificed [10].
144 Blood serum samples were collected from brachial artery after anesthesia and stored at 145 −80°C for QTOF-MS analysis. After blood collected, mouse feces were collected from 146 the rectum as soon as possible and kept immediately at -80 ° C.

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148 Measurement of airway hyperresponsitivity 149 The airway resistance of mice was measured by using a single-chamber, whole-body  (Fig. 1B).

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256 Effect of YGW on metabolic pathway 257 IPA was used to identify the biochemical pathways which are responsible for the 258 observed metabolic abnormalities. In the network analysis, Der p-induced serum 259 metabolites related to allergic asthma tended to gather in a single network. Among the 260 several "hub" molecules at the center of this network, TNF-α was the most important 261 marker ( Fig. 2B and S3 Fig.).

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To further evaluate the underlying implication of the metabolites with changed 263 expression, we analysed the metabolic pathways by using MetaboAnalyst 4.0 264 (www.metaboanalyst.ca/). To define the relationships among the metabolites, we 265 generated pathway analysis for the 10 potential biomarkers by using "mouse" as the 282 of the microbial community (alpha diversity). The average microbiota diversity was 283 slightly lower in Der p only than control group, but the low microbiota diversity in Der p 284 group was ameliorated by YGW treatment (Fig. 3A).

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Furthermore, to intuitively assess the specific changes in the microbial community 286 in the gut microbiota between groups, we analyzed the relative abundance of the dominant 287 taxa identified by sequencing in each group. The top 15 taxa generated the relative 288 abundance superposition histogram at the genus level (Fig. 3B) 3B and S4 Fig.). LEfSe 294 analysis, a biomarker discovery tool for high-dimensional data, was used to explore the 295 differences by analysis of taxon abundance in the gut microbiota (from phylum to species) 296 between groups. LDA revealed distinct taxa in the gut microbiota of the groups. A