There were differences in intestinal and pulmonary microbiota expression in COPD model rats
The changes in richness, diversity, and community structure of the bacterial microbiome in the gut and lung with COPD model rats were observed using 16S rRNA high-throughput sequencing in this study. There were significant differences in the overall structure of intestinal and pulmonary microbiota between the control and model groups, and QBPF intervention could alleviate the trend of sample differences. It was suggested that intestinal and pulmonary microbiota changes occurred in COPD rats, and QBPF could optimize these changes. Based on the taxonomic composition analysis, the dominant microbiota phyla in the gut and lung include
Bacteroidetes,
Proteobacteria, and
Firmicutes in all groups.
Bacteroidetes of intestinal and pulmonary microbiota were lower in the model group than in the control group in this experiment, consistent with the previous facts in patients with COPD and mice exposed to diesel exhaust particles [
11,
38]. Consistent with previous clinical studies, the proportion of
Firmicutes in the lung was lower [
39], which was higher in the gut [
11] in the model group than in the control group. Nevertheless, interestingly, different from COPD and AECOPD patients, the proportion of
Proteobacteria in the gut and lung was lower in the model group than that of the control group in our study [
11,
40]. Many common human pathogens belong to the
Proteobacteria, and their proportions were negatively correlated with the FEV1/FVC value in stable COPD patients [
39]. However,
Proteobacteria deletion can lead to inflammation under the condition of dysbacteriosis [
41].
There is an imbalance of Th17, Treg cells and their related cytokines in the immune system in COPD model rats
The dynamic equilibrium of Th17 and Treg cells played an essential role in balancing COPD patients’ immune status [
4,
42]. We noticed that CCL20, CCR6 and Th17 cells and related cytokines were increased. In contrast, the expression of Treg and associated cytokines were reduced in the COPD model group compared with the control group. Th17 cells have the effect of inducing inflammatory response and increasing the expression of related cytokines IL-17A and transcription factor RORγt. While Treg cells antagonize the inflammatory response, their related cytokine IL-10 and transcription factor Foxp3 expression are decreased in the COPD group. CCL20 and its unique receptor CCR6 are mainly expressed on the surface of Thl7 and Treg cells, which can mediate and regulate the chemotaxis of Thl7 and Treg cells to inflammatory sites in humans [
43,
44]. Therefore, CCL20 and CCR6 may be involved in chronic inflammation and immune response of airways and thus may participate in the COPD pathogenesis process [
45].
The interactions between microbiota and immunity in the gut and lung are two-way [
19,
46‐
48].
On the one hand, intestinal microbiota affects the local immune system [
46]. Furthermore, intestinal microbiota and producing short-chain fatty acids (SCFAs) play an essential role in establishing and regulating the pulmonary immune system in mice and humans [
41,
49]. A crucial part of pulmonary microbiota in the maturation and homeostasis of lung immunity has also emerged in mice and humans [
50,
51]. On the other hand, Treg cell depletion from the intestinal lamina propria in mice influenced the intestinal microbiota composition [
48]. And reduced SCFA production is commonly associated with chronic autoimmune diseases in humans[
52]. A significant inflammation in the lung can also sickly transform the pulmonary microbiota composition in COPD patients [
53].
There is a significant relationship between gut-lung axis microecology and pulmonary function and immune function in COPD model rats
Traditional Chinese medicine theory suggests that “the lung stands in interior-exterior relationship with the large intestine” [
54], which expounds on the mutual dependence physiologically and influences pathologically between the organs [
55]. Modern research has confirmed that gastrointestinal and respiratory mucosal tracts share the same origin and aspects of physiology and structure [
56]. Consequently, as a specific axis with intensive dialogues, the GLA plays a vital role in functional structure, inflammatory response, and immunity between the intestine and lung [
56,
57].
We revealed nine genera simultaneously expressing in the gut and lung, including
Bacteroides,
Prevotella_9,
Lactobacillus,
Neisseria,
Lachnospiraceae_NK4A136_group,
Alloprevotella,
Prevotellaceae_UCG-001,
Prevotellaceae_NK3B31_group, and
Ruminococcaceae_UCG-014, among which connections exist. We noticed
Lactobacillus decreased in the model group compared with the control group and increased after QBPF treatment in the gut and lung. As a probiotic,
Lactobacillus has the protective effects of preventing asthma and anti-influenza in mice [
58,
59]. A mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells [
60]. Our study suggested that pulmonary
Neisseria decreased in the model group compared with the control group and increased after drug intervention. Commensal
Neisseria plays a part in humans' evolution and stability of the upper respiratory tract microbiome[
61].
Lachnospiraceae ferments different plant polysaccharides into SCFAs [
62], and its proportion increases in the gut and lung after QBPF treatment. SCFAs are believed to have anti-inflammatory and immunomodulatory effects [
63]. Our results showed that SCFAs-producing bacteria such as
Alloprevotella,
Prevotellaceae_NK3B31_group, and
Prevotellaceae UCG-001 decreased in the model group compared with the control group in the intestine and lung, suggesting disorders of fatty acids metabolism are involved in COPD. Studies have shown that
Ruminococcaceae can also produce SCFAs and maintain a healthy gastrointestinal tract in individuals [
64]. The relative abundance of
Ruminococcaceae_UCG-014 in the gut and lung increased after QBPF treatment compared with the model group.
In addition to the common bacteria, there were significant correlations between the different intestinal and pulmonary microbiota.
Mycoplasma of pulmonary microbiota is positively correlated with
Prevotella_9 and
Bacteroide and negatively correlated with
Prevotellaceae_NK3B31_group,
Prevotellaceae_UCG-001, and
Desulfovibrio of intestinal microbiota.
Mycoplasma belongs to the phylum
Tenericutes. Their members establish symbiotic or highly toxic relationships in animals and humans [
65].
Prevotella strains are associated with plant-rich diets in the gut, but they are also linked with chronic inflammatory conditions in mice and humans [
66]. The results suggested that the imbalance of COPD microbiota is related to the increase of harmful bacteria and affect the production of SCFAs. The correlations between the intestinal and pulmonary microbiota add pieces of evidence to the GLA [
67,
68].
Based on the changes in the community structure of intestinal and pulmonary microbiota, we performed COG and KEGG function prediction to analyse the differences in metabolic pathways among the three groups. The results showed that amino acid-related enzymes, aminoacyl-tRNA biosynthesis and purine metabolism pathway increased markedly in the model group compared with the control group but decreased observably after QBPF intervention. COPD patients are associated with amino acid metabolic deregulations [
69,
70]. Studies have shown that serum histidine levels are elevated in COPD patients with worse disease severity with emphysema, cachexia and increased systemic inflammation [
71,
72]. Cysteine, glycine, and glutamates increased in the lung of idiopathic pulmonary fibrosis compared with the control group [
73]. Uric acid is the end product of purine metabolism, and the increased level of serum uric acid is thought to be a consequence of increased purine catabolism in the presence of tissue hypoxia [
74]. Many patients with COPD have systemic hypoxia at rest or during acute exacerbation due to decreased oxygen diffusion capacity and alveolar hypoventilation. Therefore, serum uric acid is higher in patients with COPD [
75]. These results may indicate that the imbalance of intestinal and pulmonary microflora may lead to metabolic disorders, affecting the occurrence and development of COPD.
Spearman correlation analysis showed that
Mycoplasma in the lung was significantly positively associated with RORγt, IL-17A, and Th17/Treg, while dramatically negatively correlated with FEV 0.3/FVC, Foxp3 and IL-10. These further confirmed that
Mycoplasma was a common pathogen.
Acetatifactor in the intestine was notably negatively correlated with RORγt, IL-17A, and Th17/Treg and positively correlated with FEV 0.3/FVC, Foxp3, and IL-10.
Actatifactor strongly correlates with steroid hormone biosynthesis, unsaturated fatty acid biosynthesis, linoleic acid metabolism and other metabolic pathways [
76]. Similar to our results, the abundance of
Acetatifactor in the microbial community of the lung cancer mice was relatively lower than that of the healthy control group [
76].
Coprococcus_2 of intestinal microbiota was significantly positively correlated with RORγt, IL-17A, and Th17/Treg and negatively correlated with FEV 0.3/FVC, Foxp3, and IL-10. Similarly, lung cancer patients with a relatively higher abundance of
Coprococcus are prone to gastrointestinal reactions and disease progression after two cycles of chemotherapy [
77]. Previous studies have confirmed that the community changes of intestinal and pulmonary microbiota in COPD are related to the decline of pulmonary function and immune imbalance in COPD patients and mice [
39,
78,
79]. Consequently, our results further enrich the relationship between intestinal and pulmonary microbiota and pulmonary function and immune function in COPD model rats.
There exists regulation of QBPF in immune homeostasis and intestinal and pulmonary microbiota in COPD model rats
Consistent with previous studies, the expression of CCL20, CCR6, and Th17 cells and related cytokines were increased, whereas Tregs and associated cytokines were reduced in the COPD model group compared with the control group, and QBPF treatment could alleviate the changes in these expression levels. These results suggested that QBPF treatment is conducive to the new balance of Th17/Treg.
Our results showed that
Mycoplasma in the lung increased significantly in the COPD model group compared with the control group while decreased significantly after QBPF intervention.
Mycoplasma is a notable species traditionally associated with infection [
80], and
Mycoplasma pneumonia is a common respiratory pathogen [
81].
Rikenellaceae_RC9_gut_group in the lung decreased significantly in the COPD model group compared with the control group while increased significantly after QBPF intervention. Elevated gut microbiome abundance of
Rikenellaceae is associated with reduced visceral adipose tissue and a healthier metabolic profile in the Italian elderly [
82]. The relative abundance of
Coprococcus_2,
Prevotella_9, and
Blautia in the gut increased significantly in the COPD group compared with the control group and reduced after QBPF treatment.
Coprococcus is related to obese patients with Polycystic ovary syndrome is
Coprococcus_2 [
83]. Some species of
Prevotella have inflammatory properties in mice and humans [
84] and may be involved in COPD clinically [
11]. Specific operational taxonomic units in the
Blautia are associated with inflammatory indicators in obese Children [
85]. Therefore, these bacteria, which may be beneficial or harmful, are altered in the COPD model rats and modulated by QBPF. These results suggest that QBPF can regulate the composition of intestinal and pulmonary microbiota and improve community structure in COPD rats.
Many studies have shown that bioactive compounds or their metabolites from multiple herbs can inhibit COPD progression [
86]. Previous clinical experiments showed that QBPF combined with Montelukast could down-regulate Th17 expression and up-regulate CD4
+CD25
+Foxp3
+Treg expression in patients with AECOPD [
35]. As the main drugs of QBPF, Radix Astragali and sun-dried ginseng exhibit anti-inflammatory and immunomodulatory properties and improve lung function in mice and rats [
87‐
89]. Active ingredients isolated from other herbs in QBPF also have anti-inflammatory and immunomodulatory effects [
90‐
94]. Our experiment confirmed that QBPF could improve the imbalance of Th17/Treg cells and regulate immune function. Additionally, through this experiment, we believe that the imbalance of intestinal and pulmonary microbiota may be one of the pathological mechanisms of COPD rats, and traditional Chinese medicine has a regulatory effect on the microbiota of the intestines and lungs in humans [
95‐
97]. QBPF could treat COPD by regulating the intestinal and pulmonary microbiota, which provides a new idea and direction for exploring the potential biomarkers of COPD and the possible mechanism of QBPF in the prevention and treatment of COPD.
There are some limitations to our experiment. Firstly, we adopted a cross-sectional study to observe the changes in intestinal and pulmonary microbiota and immune homeostasis in control and model group rats. At the same time, we also confirmed the regulatory effect of QBPF on the microbiota of the intestine and lung and immune homeostasis in rats. However, the interaction mechanism between immune homeostasis and microbiota imbalance in COPD has not been specifically discussed. In addition, we should pay attention to the fact that the microbiota in the host is constantly changing under the influence of diet, environment, drugs and other factors, so we should control the uniqueness of variables to make the experimental results repeatable and comparable with the results of other similar studies. Our follow-up research direction is to carry out in vitro and in vivo colonization experiments on bacteria with significant differences between different groups, which is of great significance for developing new therapeutic approaches for COPD (Additional file
1).