The Bufei Jianpi Formula Improves Mucosal Immune Function by Remodeling Gut Microbiota Through the SCFAs/GPR43/NLRP3 Pathway in Chronic Obstructive Pulmonary Disease Rats

Abstract

Purpose: Bufei Jianpi formula (BJF), a traditional Chinese medicine, is an effective and safe therapeutic formula for chronic obstructive pulmonary disease (COPD). BJF treatment is known to reduce the incidence of loose stools in rats with COPD. It is unclear whether BJF regulates gut microbiota. This study examined whether BJF improved mucosal immune function by remodeling the gut microbiota and modulating metabolites in COPD rats.

Methods: Sixty Sprague Dawley (SD) rats were randomized into control, model, BJF, aminophylline (APL), and probiotics (PBT) groups. The stable COPD rat model was duplicated using repeated cigarette smoke inhalation and lipopolysaccharide (LPS) injection. Normal saline, BJF, APL, or PBT were intragastrically administered from weeks eight to twelve, and then the rats were sacrificed at week thirteen. Lung and colon tissues were removed; feces were collected. Pulmonary function, histopathology, levels of inflammatory factors, and activation of NF-κB in the lung tissues were evaluated. Gut microbiota were analyzed using 16S rRNA gene sequencing; fecal short-chain fatty acid (SCFA) concentrations were determined using gas chromatography/mass spectrometry. Mucosal immune response-related genes and proteins were determined using quantitative polymerase chain reaction and Western blotting.

Results: BJF improved pulmonary function and reduced lung inflammation. Further, BJF treatment altered the gut microbiota composition and significantly increased the abundance of Firmicutes and the ratio of Firmicutes to Bacteroides, raising SCFA levels, including acetate, butyrate, and propionate levels. However, the abundance of Bacteroidetes, Proteobacteria, Spirochaetes, Clostridiaceae, and Treponema decreased after BJF administration. BJF decreased the gene and protein expression of NLRP3, Caspase-1, IL-8, and IL-1β, and increased GPR43 expression.

Conclusion: Overall, BJF administration improved mucosal immune function by remodeling the gut microbiota and suppressing the SCFAs/GPR43/NLRP3 pathway in COPD rats. This study provides evidence for the mechanisms underlying BJF-induced improvements in COPD and supports clinical application of BJF.

Keywords: Bufei Jianpi formula; chronic obstructive pulmonary disease; gut microbiota; gut-lung axis; mucosal immune function; short-chain fatty acids.

Figure 1

Effects of Bufei Jianpi Formula (BJF) on respiratory function, lung and colon inflammation in COPD model rats. (A) Changes in Body weight in each group. (B–D) Changes in invasive respiratory function parameters FVC, FEV0.3, and FEV0.3/FVC in each group. (E) Histopathological changes of lung tissues in each group (HE staining, magnification, ×100, ×400). (F) Histopathological changes of colon tissues in each group (HE staining, magnification, ×100, ×400). (G) Histopathological changes of colon tissues in each group (PAS staining, magnification, ×400). Red arrows present the goblet cells. Data are presented as mean ± SD. N = 6 for each group. **p < 0.01, *p < 0.05 vs control group; ^##p < 0.01, ^#p < 0.05 vs model group.

Figure 2

Effect of BJF on the Inflammatory Responses in COPD rat lung tissue. (A) TNF-α, IL-6 and IL-10 were detected with immunohistochemistry (magnification, ×200). (B–D) Quantitative analysis for TNF-α, IL-6 and IL-10 expression was performed. (E–G) Level of TNF-α, IL-6 and IL-10 in lung tissue. (H) Changes of DNA binding activity of NF-κB p65 in the lung. (I) The protein levels of NLRP3 in lung tissue were quantified by Western blotting. Data are presented as mean ± SD. N = 6 for each group. **p < 0.01 vs control group; ^##p < 0.01 vs model group; ^ΔΔP < 0.01, ^ΔP < 0.05 vs aminophylline (APL) group; ^▲▲p < 0.01, ^▲p< 0.05 vs probiotics (PBT) group.

Figure 3

Modulatory effects of Bufei Jianpi Formula (BJF) on the gut microbiota composition in COPD model rats. (A) Chao and (B) Shannon diversity index. (C) Detailed relative abundance of the community at the phylum level. (D) The relative abundance of Firmicutes, Bacteroides, Proteobacteria, Spirochaetes, and TM7. (E) The ratio of Firmicutes/Bacteroides. (F) Detailed relative abundance of the community at the genus level. (G) The relative abundances of Oscillospira, Lactobacillus, Clostridiaceae, Treponema, and Allobaculum. Data are presented as the mean ± SD. N = 6 for each group. **p < 0.01, *p < 0.05 vs control group; ^##p < 0.01, ^#p < 0.05 vs model group; ^ΔΔP < 0.01, ^ΔP < 0.05 vs aminophylline (APL) group; ^▲▲p < 0.01, ^▲p < 0.05 vs probiotics (PBT) group.

Figure 4

Effects of Bufei Jianpi Formula (BJF) on the levels of fecal SCFAs and intestinal mucosal SIgA in COPD model rats. (A–C) Acetate, propionate, and butyrate concentrations in feces. (D) SIgA levels in the intestinal mucosa of each group. Data are presented as the mean ± SD. N = 6 for each group. **p < 0.01 vs control group; ^##p < 0.01, ^#p < 0.05 vs model group; ^ΔΔP < 0.01, ^ΔP < 0.05 vs aminophylline (APL) group; ^▲▲p < 0.01 vs probiotics (PBT) group.

Figure 5

Heatmap of Spearman correlation between the gut microbiota [at the phylum level (A) and genus level (B)] and the intestinal mucosal protein or fecal SCFA levels. Red grids indicate positive correlations, whereas blue grids indicate negative correlations. Significant correlations are indicated by ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 6

Effects of BJF on the SCFAs/GPR43/NLRP3 signaling pathway in COPD model rats. (A–E) The mRNA levels of GPR43, NLRP3, Caspase-1, IL-1β, and IL-8 in colon tissues were analyzed by real-time PCR. (F–J) The protein levels of GPR43, NLRP3, caspase-1, IL-1β, and IL-8 in colon tissues were quantified by Western blotting. Data are presented as mean ± SD. N = 6 for each group. **p < 0.01 vs control group; ^##p < 0.01, ^#p < 0.05 vs model group; ^ΔΔP < 0.01, ^ΔP < 0.05 vs aminophylline (APL) group; ^▲▲p < 0.01, ^▲p < 0.05 vs probiotics (PBT) group.