Bu-Fei-Huo-Xue capsule alleviates bleomycin-induced pulmonary fibrosis in mice through modulating gut microbiota

Abstract

Introduction: Bu-Fei-Huo-Xue capsule (BFHX) has been used to treat pulmonary fibrosis (PF) in clinic. However, the mechanism of Bu-Fei-Huo-Xue capsule on pulmonary fibrosis remains unclear. Recent studies have shown that the changes in gut microbiota were closely related to the progression of pulmonary fibrosis. Modulating gut microbiota provides new thoughts in the treatment of pulmonary fibrosis. Methods: In this study,a mouse model of pulmonary fibrosis was induced using bleomycin (BLM) and treated with Bu-Fei-Huo-Xue capsule. We firstly evaluated the therapeutic effects of Bu-Fei-Huo-Xue capsule on pulmonary fibrosis model mice. Besides,the anti-inflammatory and anti- oxidative effects of Bu-Fei-Huo-Xue capsule were evaluated. Furthermore, 16S rRNA sequencing was used to observe the changes in gut microbiota in pulmonary fibrosis model mice after Bu-Fei-Huo-Xue capsule treatment. Results: Our results showed that Bu-Fei-Huo-Xue capsule significantly reduced the collagen deposition in pulmonary fibrosis model mice. Bu-Fei-Huo-Xue capsule treatment also reduced the levels and mRNA expression of pro-inflammatory cytokines and inhibited the oxidative stress in lung. 16S rRNA sequencing showed that Bu-Fei-Huo-Xue capsule affected the diversity of gut microbiota and the relative abundances of gut microbiota such as Lactobacillus, Lachnospiraceae_NK4A136_group, and Romboutsia. Conclusion: Our study demonstrated the therapeutic effects of Bu-Fei-Huo-Xue capsule on pulmonary fibrosis. The mechanisms of Bu-Fei-Huo-Xue capsule on pulmonary fibrosis may be associated with regulating gut microbiota.

Keywords: Bu-Fei-Huo-Xue capsule; gut microbiota; inflammation; oxidative stress; pulmonary fibrosis.

FIGURE 1

Bu-Fei-Huo-Xue capsule (BFHX) treatment increased the survival rate, ameliorated the body weight loss and reduced the pathological changes in bleomycin (BLM)-induced pulmonary fibrosis (PF) mice. (A,B) The survival rate was increased (A) and the body weight loss was reduced (B) in PF model mice after treated with BFHX; (C) H&E staining indicated that BFHX ameliorated the damage of bronchial epithelial cells, the proliferation of fibrous tissue and the infiltration of inflammatory cells in lung (magnification: ×40 for full-field and 400× for bronchus and alveoli) ^## p < 0.01 compared with the Control group; *p < 0.05 compared with the Model group; **p < 0.01 compared with the Model group Control group (n = 15); Model group (n = 8); DXM (n = 8); LD-BFHX (n = 8); MD-BFHX (n = 9); HD-BFHX (n = 11).

FIGURE 2

BFHX treatment decreased the deposition of fibrotic contents in lung in PF model mice. (A,B) Masson staining showed that BFHX decreased the accumulation of fibrotic contents in lung (magnification: ×40 for full-field and 400× for bronchus and alveoli); (C) BFHX reduced the levels of hydroxyproline in lung tissue homogenates.

FIGURE 3

BFHX treatment decreased the expression of α-SMA and TGF-β1 in lung. (A,C) Immunohistochemistry showed that BFHX treatment reduced the expression of α-SMA in lung (magnification: ×40). (B,D) Immunohistochemistry showed that BFHX treatment reduced the expression of TGF-β1 in lung (magnification: ×40).

FIGURE 4

Treatment of BFHX alleviated inflammatory response in PF model mice. (A–C) BFHX treatment decreased the total cell counts (A), differential cell counts (B), and total protein concentration (C) in bronchoalveolar lavage fluid (BALF) in PF model mice. (D) The levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) levels in of lung tissue homogenate were decreased in PF model mice after BFHX treatment. (E) qPCR results showed that BFHX treatment downregulated the mRNA expression of IL-6, IL-1β and TNF-α in lung.

FIGURE 5

Treatment of high-dose HD-BFHX affected the gut microbiota community in mice with PF. (A,B) Shannon (A) and Simpson indexes (B) were higher in HD-BFHX group than that in the Model group. (C,D) Principal Co-ordinates Analysis (PCoA) score plot (C) and UPGMA cluster tree (D) indicated more similar beta diversity between HD-BFHX (B) and Control (C) groups than that between the Model (M) and Control groups. (E–L) At the phylum level, BFHX treatment decreased the F to B ratio (E,F); At the genus level, the relative abundances of Lactobacillus (G) , Candidatus_Saccharimonas (H) , Lachnospiraceae_NK4A136_group (I) , Bacteroides (J) , Helicobacter (K) , and Romboutsia (L) were changed in PF model mice, BFHX treatment affected the relative abundances of, Lachnospiraceae_NK4A136_group, and Romboutsia in mice with PF. Control group (n = 6); Model group (n = 6); HD-BFHX (n = 6).