Yifei Sanjie Formula Treats Chronic Obstructive Pulmonary Disease by Remodeling Pulmonary Microbiota

Abstract

Chronic obstructive pulmonary disease (COPD) is one of the most common pulmonary diseases. Evidence suggests that dysbiosis of pulmonary microbiota leads to the COPD pathological process. Yifei Sanjie Formula (YS) is widely used to treat diseases in respiratory systems, yet little is known about its mechanisms. In the present study, we first established the fingerprint of YS as the background for UHPLC-QTOF-MS. Components were detected, including alkaloids, amino acid derivatives, phenylpropanoids, flavonoids, terpenoids, organic acids, phenols, and the like. The therapeutic effect of YS on COPD was evaluated, and the pulmonary function and ventilatory dysfunction (EF50, TV, and MV) were improved after the administration of YS. Further, the influx of lymphocytes was inhibited in pulmonary parenchyma, accompanied by down-regulation of inflammation cytokines via the NLRP3/caspase-1/IL-1β signaling pathway. The severity of pulmonary pathological damage was reversed. Disturbed pulmonary microbiota was discovered to involve an increased relative abundance of Ralstonia and Mycoplasma and a decreased relative abundance of Lactobacillus and Bacteroides in COPD animals. However, the subversive effect was shown. The abundance and diversity of pulmonary microflora were remodeled, especially increasing beneficial genua Lactobacillus and Bacteroides, as well as downregulating pathogenic genua Ralstonia and Mycoplasma in the YS group. Environmental factor correlation analysis showed that growing pulmonary microbiota was positively correlated with the inflammatory factor, referring to Ralstonia and Mycoplasma, as well as negatively correlated with the inflammatory factor, referring to Lactobacillus and Bacteroides. These results suggest that the effects of YS involved remodeling lung microbes and anti-inflammatory signal pathways, revealing that intervention microbiota and an anti-inflammatory may be a potential therapeutic strategy for COPD.

Keywords: NLRP3/caspase-1/IL-1β signaling pathway; Yifei Sanjie Formula; chronic obstructive pulmonary disease; inflammation; pulmonary microbiota.

Figure 1

The proposed mechanism by which YS treats COPD.

Figure 2

(A) Timeline diagrams of the experimental design (n = 6). (B) Body weight of the three groups including CT, COPD, and COPD + YS (n = 6). (C) EF50 of CT, COPD, and COPD + YS (n = 6). (D) TV of CT, COPD and COPD + YS (n = 6). (E) MV of CT, COPD and COPD+ YS (n = 6). The data are presented as the means ± SEM of three experiments (*P < 0.05).

Figure 3

(A) UHPLC-QTOF-MS analysis base peak intensity chromatograms of YS in positive mode. (B) UHPLC-QTOF-MS analysis base peak intensity chromatograms of YS in negative mode.

Figure 4

(A) Thymus index of CT, COPD, and COPD + YS (n = 6). (B) Spleen index of CT, COPD, and COPD + YS(n = 6). (C) The SIgA in the lung of CT, COPD, and COPD + YS (n = 6). The data are presented as the means ± SEM of three experiments (*P < 0.05).

Figure 5

(A) H&E staining of lung tissues of CT, COPD, and COPD + YS. (B) Masson staining of lung tissues of CT, COPD, and COPD + YS. (C) TNF-α in serum of CT, COPD, and COPD + YS (n = 6). (D) IL-6 in serum of CT, COPD, and COPD + YS (n = 6). (E) The relative expression of TGF-β1 in the lung of CT, COPD, and COPD + YS (n = 3). The data are presented as the means ± SEM of three experiments (*P < 0.05).

Figure 6

(A) NLRP3 in the lung of CT, COPD, and COPD + YS (n = 3). (B) Caspase1 in the lung of CT, COPD, and COPD + YS (n = 6). (C) ASC in the lung of CT, COPD, and COPD + YS (n = 6). (D) IL-1β in the serum of CT, COPD, and COPD+YS (n = 6). (E) IL-18 in the serum of CT, COPD, and COPD + YS (n=6). The data are presented as the means ± SEM of three experiments (ns, non-significant, *P < 0.05).

Figure 7

(A) Analysis of alpha diversity: Chao1 and Shannon index (n = 5). (B) The relative abundance of top ten pulmonary microbiota in the phylum (n = 5). (C) The advantage of pulmonary microbiota in the phylum (n = 5). (D) The relative abundance of the top twenty pulmonary microbiota in the genera (n = 5). (E) The advantage of pulmonary microbiota in the genera (n = 5). (F) Taxonomic differences of pulmonary microbiota among different groups. The taxonomic cladogram was obtained by LEfSe. Differences are represented by the color of the most abundant class. The diameter of each circle is proportional to the taxon's abundance (n = 5). (G) Linear discriminant analysis score of each group of gut microbiota (n = 5). The data are presented as the means ± SEM of three experiments (ns, non-significant, *P < 0.05). The analysis of pulmonary microbiota was performed using the online tool-NovoMagic at https://magic.novogene.com via a personal account.

Figure 8

Heatmap of the correlation among the pulmonary microbiota related to environmental factors (*P < 0.05, **P < 0.01; blue represents a negative correlation; red represents a positive correlation). The analysis of pulmonary microbiota was performed using the online tool of NovoMagic at https://magic.novogene.com via a personal account.