Polydatin alleviates bleomycin-induced pulmonary fibrosis and alters the gut microbiota in a mouse model

Abstract

To investigate the effect and mechanism of polydatin on bleomycin (BLM)-induced pulmonary fibrosis in a mouse model. The lung fibrosis model was induced by BLM. The contents of TNF-α, LPS, IL-6 and IL-1β in lung tissue, intestine and serum were detected by ELISA. Gut microbiota diversity was detected by 16S rDNA sequencing; R language was used to analyse species composition, α-diversity, β-diversity, species differences and marker species. Mice were fed drinking water mixed with four antibiotics (ampicillin, neomycin, metronidazole, vancomycin; antibiotics, ABx) to build a mouse model of ABx-induced bacterial depletion; and faecal microbiota from different groups were transplanted into BLM-treated or untreated ABx mice. The histopathological changes and collagen I and α-SMA expression were determined. Polydatin effectively reduced the degree of fibrosis in a BLM-induced pulmonary fibrosis mouse model; BLM and/or polydatin affected the abundance of the dominant gut microbiota in mice. Moreover, faecal microbiota transplantation (FMT) from polydatin-treated BLM mice effectively alleviated lung fibrosis in BLM-treated ABx mice compared with FMT from BLM mice. Polydatin can reduce fibrosis and inflammation in a BLM-induced mouse pulmonary fibrosis model. The alteration of gut microbiota by polydatin may be involved in the therapeutic effect.

Keywords: bleomycin (BLM); gut microbiota; lung injury; polydatin; pulmonary fibrosis.

FIGURE 1

Pulmonary fibrosis modelling and functional investigation of polydatin. (A) Haematoxylin and eosin staining was performed to detect the pathological changes in lung tissue in each group; (B) Masson staining was performed to detect collagen deposition in the lung tissue of mice; (C) The expression levels of collagen I and α‐SMA in the lung tissue of mice were detected by IHC; (D) Western blotting was performed to detect the expression levels of collagen I and α‐SMA; (E) The W/D ratio of lung tissue in each group. **p < 0.01, compared to the control group; ##p < 0.01, compared to the BLM group.

FIGURE 2

Effects of polydatin on inflammatory factors in the BLM model. The inflammatory factors TNF‐α, LPS, IL‐6, and IL‐1β in lung tissue (A), intestine (B), and serum (C) of mice in each group were detected by ELISA. **p < 0.01, compared with the control group; ##p < 0.01, compared with the BLM group.

FIGURE 3

Quality control of 16S rRNA gene sequencing data. (A). Rarefaction curves (rank‐abundance) showing the richness (abundance) of species in the samples. (B). The α‐diversity analysis, including observed species, ACE, and Chao 1. (C). The β‐diversity analysis, including PCoA and NMDS analyses.

FIGURE 4

Dominant microflora species in different groups (A, B). Venn diagram shows the intersection of OTU abundances between the BLM group and the control group, the BLM group, and the BLM + PD group; (C, D). Top 20 species with the highest relative abundance between the BLM group and the control group, the BLM group and the BLM + PD group.

FIGURE 5

Specific effects of BLM and polydatin on the abundance of the dominant species of mouse gut microbiota. (A, B). Box plots show the abundance of the top 20 gut microbiota shared by different groups of mice.

FIGURE 6

Effects of polydatin‐treated mice's gut microbiota on PF. (A). The expression of 16S rRNA in mouse faeces was measured by qRT–PCR, **p < 0.01, compared with the control group. (B). Haematoxylin and eosin staining was performed to detect the pathological changes in the lung tissue of the mice in each group. (C). Masson staining was performed to detect collagen deposition in the lung tissue of mice in each group. (D). IHC was performed to detect the expression levels of collagen I and α‐SMA in each group. (E). Western blotting was performed to examine the protein levels of collagen I and α‐SMA in each group. (F). Analysis of the W/D ratio of lung tissue in each group. **p < 0.01 versus Control + Control‐FMT group; ##p < 0.01 versus BLM + BLM‐FMT group.