The therapeutic mechanism of Fuzheng-Qushi formula on lung injury in mice revealed by microbiomics, metabolomics and transcriptomics

Aim of the study: The objective of this study was to elucidate the protective effects of Fuzheng-Qushi formula (FZQS) against lung injury in mice, and to uncover its potential targets and key biological pathways for the treatment of lung injury in combination with transcriptomics, microbiomics, and untargeted metabolomics.Materials and methods: LPS was used to construct a mouse lung injury model by nasal inhalation. The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was used to analyze all the chemical constituents contained in FZQS. HE staining was used to analyze the histopathological changes in the lungs of mice. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect cytokines in mouse lung tissues. Macrophage polarization and neutrophil activation were detected by flow cytometry. RNA sequencing (RNA-seq) was deployed to screen for differentially expressed genes (DEGs) in lung tissue. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were utilized to validate key DEGs and target proteins in lung tissue. 16S rRNA sequencing was employed to characterize the gut microbiota of mice. Metabolites in the gut were analyzed using untargeted metabolomics.Results: FZQS treatment significantly ameliorated lung histopathological damage, decreased pro-inflammatory cytokine levels and increased anti-inflammatory cytokine levels. The level of M1 macrophages in the peripheral blood of lung-injured mice decreased, the level of M2 macrophages increased, and activated neutrophils were inhibited. Importantly, transcriptomic analysis showed that FZQS downregulated macrophage and neutrophil activation, migration and adhesion pathways by reversing 51 DEGs, which was further confirmed by RT-qPCR and Western blot. In addition, FZQS modulated the dysbiosis of the gut microbiota by reversing the abundance of Corynebacterium, Facklamia, Staphylococcus, Paenalcaligenes, Lachnoclostridium, norank__f__Muribaculaceae, and unclassified__f__ Lachnospiraceae. Meanwhile, metabolomics analysis revealed that FZQS significantly regulated tryptophan metabolism by reducing the levels of 3-Indoleacetonitrile and 5-Hydroxykynurenine.Conclusions: The present study demonstrated that FZQS ameliorated LPS-induced lung injury mainly by inhibiting the activation, migration and adhesion of immune cells, as well as modulating the disruption of the gut microbiota and its metabolites.

研究目的：本研究旨在阐明扶正祛湿方（Fuzheng-Qushi formula, FZQS）对小鼠肺损伤的保护作用，并结合转录组学（transcriptomics）、微生物组学（microbiomics）和非靶向代谢组学（untargeted metabolomics）技术，揭示其治疗肺损伤的潜在靶点与关键生物学通路。
材料与方法：采用鼻内吸入脂多糖（Lipopolysaccharide, LPS）构建小鼠肺损伤模型。运用超高效液相色谱-串联质谱法（Ultra-high performance liquid chromatography-tandem mass spectrometry, UHPLC-MS/MS）分析扶正祛湿方所含全部化学成分。通过苏木精-伊红（HE）染色观察小鼠肺部组织病理学变化。采用酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）检测小鼠肺组织中的细胞因子。利用流式细胞术检测巨噬细胞极化与中性粒细胞活化情况。通过RNA测序（RNA sequencing, RNA-seq）筛选肺组织中的差异表达基因（differentially expressed genes, DEGs）。采用实时荧光定量聚合酶链反应（Quantitative real-time polymerase chain reaction, RT-qPCR）与蛋白质印迹法（Western blot）验证肺组织中的关键差异表达基因与靶蛋白。通过16S rRNA测序表征小鼠肠道菌群结构。采用非靶向代谢组学分析肠道代谢物。
结果：扶正祛湿方治疗可显著改善肺组织病理学损伤，降低促炎细胞因子水平并升高抗炎细胞因子水平。肺损伤小鼠外周血中M1型巨噬细胞比例降低、M2型巨噬细胞比例升高，且中性粒细胞活化受到抑制。值得注意的是，转录组学分析表明，扶正祛湿方通过逆转51个差异表达基因，下调巨噬细胞与中性粒细胞活化、迁移及黏附通路，该结果进一步通过RT-qPCR与蛋白质印迹法得到验证。此外，扶正祛湿方通过纠正棒状杆菌属（Corynebacterium）、法克菌属（Facklamia）、葡萄球菌属（Staphylococcus）、产碱杆菌属（Paenalcaligenes）、Lachnoclostridium属、norank_f__Muribaculaceae及unclassified_f__Lachnospiraceae的丰度异常，调节肠道菌群失调。同时，代谢组学分析揭示，扶正祛湿方通过降低3-吲哚乙腈（3-Indoleacetonitrile）与5-羟基犬尿氨酸（5-Hydroxykynurenine）的水平，显著调控色氨酸代谢通路。
结论：本研究证实，扶正祛湿方主要通过抑制免疫细胞的活化、迁移与黏附，以及调节肠道菌群及其代谢物的紊乱，改善脂多糖诱导的小鼠肺损伤。