Yi-Fei-Tong-Bi Decoction Alleviates Bleomycin Induced Pulmonary Fibrosis in Mice

Abstract

Background: Fei-Bi decoction, a Chinese ancient experience decoction collected in the book of Bianzhenglu (Syndrome Differentiation Record). Based on Fei-Bi Decoction, Yi-Fei-Tong-Bi decoction (YFTBD) is developed and has a significant effect in the treatment of pulmonary fibrosis. However, the underlying mechanisms of YFTBD affects pulmonary fibrosis remain to be elucidated.

Purpose: To investigate the protective effect and the underlying mechanism of YFTBD on bleomycin-induced pulmonary fibrosis in mice.

Methods: The chemical components of water extract of YFTBD were analyzed by combining the high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). A mouse model was established by intratracheal injection of bleomycin, and the effects of YFTBD were evaluated through pathological staining, immunohistochemistry analyses, and Enzyme-Linked Immunosorbent Assay (ELISA). Subsequently, the effect of YFTBD on the gut microbiota of mice was analyzed by 16S rRNA high-throughput gene sequencing.

Results: Compared with the model group, the survival rate and lung coefficient of mice with pulmonary fibrosis were increased after the intervention of YFTBD, the pathological morphology of lung tissue was improved, and the expression of the inflammatory factor levels were decreased. The expression of α-SMA, TGF-β1, p21, and p16 senescence-related proteins was significantly down-regulated. The expression of Smad7 and PGC-1α senescence-related proteins was significantly up-regulated. Meanwhile, gut microbiota analysis showed that YFTBD could induce changes in the abundance of Alloprevotella, unclassified Muribaculaceae, and Lachnospiraceae NK4A136 group.

Conclusion: Our findings suggest that YFTBD could alleviate the bleomycin-induced pulmonary fibrosis in mice via regulating TGF-β1/Smad signaling pathway, inflammation and gut microbiota. It provides experimental evidence and a theoretical basis for the application of YFTBD in pulmonary fibrosis.

Keywords: Yi-Fei-Tong-Bi decoction; cell senescence; collagen; gut microbiota; pulmonary fibrosis.

Figure 1

Total ion chromatogram of YFTBD by HPLC-MS. (A) Positive ion mode total ion chromatogram. (B) Negative ion mode total ion chromatogram.

Figure 2

Effects of YFTBD on lung injury and collagen deposition in mice. (A) H&E staining of lung tissue. (B) Masson staining of lung tissue. (C) Collagen volume fraction. n=5. Compared to control, ^##P<0.01. Compared to model, **P<0.01, ***P<0.001. (D) Content of hydroxyproline in lung tissue. n=5. Compared to control, ^###P<0.001. Compared to model, *P<0.05, **P<0.01.

Figure 3

Effects of YFTBD on α-SMA, Smad7, and TGF-β1. (A) Immunohistochemistry of α-SMA. (B) AOD of α-SMA. n=5. Compared to control, ^##P<0.01. Compared to model, *P<0.05, **P<0.01. (C) Immunohistochemistry of TGF-β1. (D) AOD of TGF-β1. n=5. Compared to control, ^###P<0.001. Compared to model, *P<0.05, **P<0.01. (E) Immunohistochemistry of Smad7. (F) AOD of Smad7. n=5. Compared to control, ^##P<0.01. Compared to model, *P<0.05, **P<0.01.

Figure 4

Effects of YFTBD on cell senescence in the lung tissue of mice. (A) Immunohistochemistry of p16. (B) AOD of p16. n=5. Compared to control, ^##P<0.01. Compared to model, *P<0.05. (C) Immunohistochemistry of p21. (D) AOD of p21. n=5. Compared to control, ^##P<0.01. Compared to model, *P<0.05, **P<0.01. (E) Immunohistochemistry of PGC-1α. (F) AOD of PGC-1α. n=5. Compared to control, ^#P<0.05. Compared to model, *P<0.05, **P<0.01.

Figure 5

Effects of YFTBD on inflammatory factor levels in the serum of mice. (A) Concentration of IL-1β. n=8. Compared to control, ^###P<0.001. Compared to model, *P<0.05, **P<0.01. (B) Concentration of IL-6. n=8. Compared to control, ^###P<0.001. Compared to model, **P<0.01. (C) Concentration of TNF-α. n=8. Compared to control, ^###P<0.001. Compared to model, **P<0.01.

Figure 6

Effects of YFTBD on diversity of gut microbiota in mice. (A) OUTs. (B) Shannon index. n=5. *P<0.05, **P<0.01, ***P<0.001. (C) Chao1 index. n=5. *P<0.05, **P<0.01, ***P<0.001. (D) Principal component analysis (PC1 vs PC2). (E) Principal component analysis (PC1 vs PC3).

Figure 7

The relative abundance of gut microbiota at the phylum level. (A) The histogram of species distribution at the phylum level (n=5). (B) Relative abundances of Firmicutes (%). n=5. Compared to control, ^##P<0.01. Compared to model, ***P<0.001. (C) Relative abundances of Bacteroidota (%). n=5. Compared to control, ^###P<0.001. Compared to model, ***P<0.001. (D) The ratio of Firmicutes to Bacteroidota. n=5. Compared to model, *P<0.05, ***P<0.001. (E) Relative abundances of Proteobacteria (%). n=5. Compared to control, ^##P<0.01. Compared to model, ***P<0.001.

Figure 8

Lefse biomarkers cladogram less strict of the gut microbiota (LDA > 4).