Yifei Sanjie Formula Treats Chronic Obstructive Pulmonary Disease by Regulating Lung Microbiota Dysbiosis

Background Chronic obstructive pulmonary disease (COPD) is one of the most common pulmonary diseases. There is evidence to suggest that dysbiosis of pulmonary microbiota participates in COPD development. Yifei Sanjie Formula (YS) is widely used to treat diseases in respiratory systems, yet its mechanisms are little known. Methods In the present study, the ecacy of YS was evaluated by analyzing its effects on the severity of pulmonary pathological damage, pulmonary function, pro-inammation cytokines, the activation of NLRP3/caspase-1/IL-1β signaling pathway, and changes of lung microbiota. Results YS improved animal behaviors, prevented declines in pulmonary ventilatory function and lung injury in a rat model of COPD. Administration of YS signicantly suppressed the release of proinammatory cytokines and collagen deposition and downregulated NLRP3/caspase-1/IL-1β signaling in vivo. YS changed the relative abundance of specic pulmonary microbiota and modulated bacterial ora in the rat model. Conclusions These results suggest that the effects of YS involved lung microbes and anti-inammatory mechanisms. These results suggest that the effects of YS involved lung microbes and anti-inammatory mechanisms.


Introduction
Respiratory symptoms and air ow limitation characterize chronic obstructive pulmonary disease (COPD); it has become the third most common life-threatening disease worldwide [1] . The pathogenesis of COPD includes in ammation, oxidative stress, and protease/antiprotease imbalance. Regulation of the pulmonary in ammatory microenvironment to remove the pathological products produced by the in ammatory response might reduce the level of in ammation, restoring the balance of the lung microenvironment and delaying the development of COPD [2] . Nod-like receptor protein 3(NLRP3) is a potent inducer of in ammation that, when overactive, may be targeted therapeutically in in ammatory lung diseases [3] . Studies [4] showed that acute exacerbations of COPD are related to NLRP3 activation, and its activation may be involved in initiating the in ammatory response by binding to cytoplasmic pathogen-associated molecular patterns or damage-associated molecular patterns.
Bacteria in the respiratory tract resist colonization by foreign pathogens. Cigarette smoking alters the composition of the lung microbiota [5] . Lung microbiota dysbiosis is related to in ammation, pathological airway alterations, immune responses, and the aggravation of clinical symptoms in patients with COPD [6] . Pathogens stimulate in ammatory cells to produce in ammatory media that often destroy the immune function of the airway and mucosa, leading to chronic in ammation and lung microbiota dysbiosis, further aggravating COPD [7] . Our understanding of pulmonary microorganisms is limited, and many questions remain. How does the lung microbiota dysbiosis in COPD patients, and how do these changes affect disease development? How does the NLRP3 signal pathway interact with lung microbiota dysbiosis?
A great deal of attention has been paid to the development of Chinese herbal medicines to treat COPD [8] .
Yifei Sanjie Formula (YS), a traditional Chinese medicine, comprises eight medicinal herbs and has been shown to possess extensive pharmacological effects against COPD, including reduction of lung injury, in ammatory responses, and pulmonary brosis in a COPD animal model. Nevertheless, the pharmacological mechanisms of action of YS remain poorly understood and warrant further investigation [9][10] . Therefore, we determined whether the regulation of lung microbiota by YS would alleviate lung injury, ventilatory function, in ammatory reactions, and collagen deposition in a rat model of COPD. We aimed to provide theoretical support for the e cacy of YS in correcting lung microbiota dysbiosis (Fig.

Groups and treatments
According to the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health, all rats experiments were approved by the Animal Ethics Experiment Committee of Yunnan University of Traditional Chinese Medicine and were conducted by the guidelines of the committee. SPF Wistar rats (n=18; 200±20g; male) were provided by Chengdu Dashuo Laboratory Animal Co., Ltd.(Chengdu, China; license no. SCXK(Chuan)-2015-030). The rats were randomly divided into Control (CT), COPD, COPD+YS. Except for CT group, the rat of the COPD model was replicated by using cigarette smoke exposure (Hongyun cigarette, Tar11mg, Nicotine1.1mg, CO12mg) combined with airway instillation of lipopolysaccharide(LPS, Sigma) [11] . 11.6g·kg -1 ·d -1 of YS was administered daily by intragastric administration from day 57 to day 84(The same dosage as is used clinically). Animal weight and behaviorism were monitored regularly and recorded. The composition of YS is shown in Table 1.

Histological evaluation
The upper lobe of the right lung of the rat was xed with 4% formalin for 48 hours and then embedded in para n, stained with hematoxylin and eosin (H&E) and Masson, and nally evaluated histopathologically under an optical microscope (OLYMPUS VS200, magni cation×200).

Western blotting
Protein was extracted from lung tissue, protein quanti cation was performed using the BCA protein quanti cation kit (P0012, Beyotime, Jiangsu, China) to calculate loading capacity, samples were added to a precast SDS polyacrylamide gel for electrophoretic separation, and proteins in the gel were electrotransferred to a PVDF membrane using water bath electroblotting. the primary antibody was added, which was anti-TGF-β1 (rabbit polyclonal antibody, Abcam, ab179695), anti-NLRP3 (rabbit polyclonal antibody, Bioss, bs-10021R) overnight at 4℃; after washing the membrane added secondary antibody, shaker at 4℃, incubated for 1h; PVDF was dropped ECL luminescence liquid, developed, photographed (GeneGnome). Using gene tools software, the grayscale values of individual bands were analyzed.

Enzyme-linked immunosorbent assay
ELISA kit (Jiangsu Enzyme-Linked Biotechnology Co., Ltd.) was used to detect the concentration of TNFα, IL-6 in serum as well as the concentration of IL-1β, IL-18 and SIgA in the lung. The method and procedure were completed according to the kit operating instructions. Finally, read the absorbance at 450nm after adding stop solution and within 15min.
2.6 16S rRNA gene high-throughput sequencing to detect the lung microbiota Total genome DNA from samples was extracted using CTAB/SDS method. DNA concentration and purity were monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1ng/µL using sterile water.16S rRNA genes of distinct regions(16S V4/16S V3/16S V3-V4) were ampli ed used a speci c primer(16S V4:515F-806R) with the barcode. PCR products were mixed in equidensity ratios. Then, mixture PCR products were puri ed with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scienti c) and AgilentBioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.

Statistical analysis
Data were expressed as means ± standard deviation(`x ± s). The normal distribution of the data was assessed with the Shapiro-Wilk test. Signi cant differences in the variance of parameters were evaluated either with ANOVA or Kruskal-Wallis test, depending on the data normality distribution. All statistical tests were two-sided, and a P value of <0.05, or FDR adjusted Padj value < 0.05 was considered as statistically signi cant.

YS improves pulmonary function in COPD rats
Pulmonary function is an important index in evaluating COPD, including TV, MV and EF50, re ecting small airway obstruction. Studies [12] found that COPD patients showed decreased TV and MV. We found that EF50, TV and MV were signi cantly lower in COPD rats (P < 0.05), while EF50 (P < 0.05), TV and MV (P > 0.05) were higher after YS intervention ( Fig. 1C-1E). COPD rats displayed hair withering, slow action, and shortness of breath. There were signi cant improvements in animal behaviors after the YS intervention. The body weights of COPD rats signi cantly decreased (P < 0.05). Weights in the YS+COPD group increased (P > 0.05) (Fig. 1B). These ndings suggest that YS improves animal behaviors and pulmonary ventilatory function in COPD rats.

YS improved immune function in COPD rats
The spleen and thymus are critical immune organs, and their organ indexes re ect the strength of immune function to a certain extent [13] . Results show that thymus index and spleen index were decreased signi cantly in COPD rats (P > 0.05); after YS intervention, thymus index and spleen index were increased (P > 0.05) ( Fig. 2A-2B). SIgA is a major effector molecule of the mucosal immune defense system against the colonization and adhesion of pathogenic microorganisms on mucosal surfaces [14] .
The amount of SIgA in the sputum and alveolar lavage uid of patients with COPD was signi cantly lower than in the control group. In the present study, we have found that SIgA was decreased in COPD rats (P > 0.05), while the SIgA of the COPD+YS group was signi cantly increased (P > 0.05) (Fig. 2C). All these suggested that YS could enhance the local mucosal immunity and improve immune function in COPD rats.

3.YS relieves in ammation in COPD rats
The in ammatory response of COPD primarily involves neutrophil in ltration. TNF-activates neutrophils, and IL-6 inhibits apoptosis [15] . HE staining showed that substantial amounts of neutrophil in ltration plugged the bronchi. The lumens were signi cantly narrowed, the alveolar walls become thinner and fused in COPD rats. There were fewer of these pathological changes in the YS intervention group (Fig.  3A). Levels of TNF-and IL-6 were signi cantly higher in the serum of COPD rats (P > 0.05) with YS intervention (Fig. 3C-3D). TGF-β1 is a powerful pro brotic cytokine that participates in in ammatory repair [16] . Masson staining showed that much collagen ber deposition occurred around the trachea and pulmonary interstitium in COPD rats. Collagen deposition was less severe in YS rats (Fig. 3B). TGF-β1 protein expression was signi cantly elevated in lung tissue of COPD rats (P > 0.05). TGF-β1 levels in the COPD+YS group were signi cantly lower (P > 0.05) (Fig. 3F). These ndings suggest that YS ameliorates lung tissue damage, attenuates in ammatory responses, and reduces collagen deposition in COPD rats.

Effect of YS on lung microbiota in COPD rats
The lung microbiota is strongly associated with COPD. To explore the mechanisms, 16s rRNA highthroughput sequencing was employed to analyze the lung microbiota. The chao1 index re ects the relative abundance of ora, and the Shannon index re ects the diversity of ora. The results are represented in Figure 5A. Compared with the CT group, the relative abundance of ora in the COPD group decreased. After YS intervention, the relative abundance of the ora showed no signi cant change.
Compared with the CT group, the diversity of the ora in the COPD group was signi cantly decreased (P > 0.05). The diversity of the ora was signi cantly greater after YS intervention (P > 0.05).
On the phylum level, the lung ora of rats in each group was dominated by Proteobacteria, Firmicutes, and Bacteroidota. Compared with the CT group, the COPD group showed an increased relative abundance of Proteobacteria and Firmicutes, with a decreased relative abundance of Bacteroidota; after YS intervention, the relative abundance of Bacteroidota increased those of Proteobacteria and Firmicutes decreased (Fig. 5B). At the genus level, the lung ora of rats in each group was dominated by Ralstonia, Mycoplasma, Halomonas, Lactobacillus, Dizetzia, and Bacteroides. Compared with the CT group, the COPD group showed an increased relative abundance of Ralstonia, Mycoplasma, Halomonas, and Dizetzia, while there was a decreased relative abundance of Lactobacillus and Bacteroides. The relative abundance of Halomonas, Lactobacillus, Dizetzia, and Bacteroides were increased, and there was a lower relative abundance of Ralstonia and Mycoplasma after YS intervention (Fig. 5C).
Linear discriminant analysis of effect size shows the iconic microorganisms in each group that contributed signi cantly to differences in microbial structure. As shown in Figure 5D, Mycoplasma was more abundant in the gut microbiota of the COPD group. Mycoplasma was signi cantly increased in COPD patients, while Halomonas, Dietzia, and Nesterenkonia were enriched after YS intervention. These results suggest that YS changes the relative abundance of speci c bacteria and modulates the bacterial ora in COPD rats.

Discussion
Airway in ammation and remodeling are the primary pathological features of COPD and are causes of air ow limitation and remodeling [18] . Smoking cessation, bronchodilators, and hormones have not been effective in halting the progressive deterioration of pulmonary function and the progression of the disease in patients with COPD, although they may improve symptoms to some extent [19] . Traditional Chinese medicine is characterized by the effects of multiple components, targets, and pathways, and these are used to treat COPD [20] . YS preserves the pathological morphology of lung tissue and reduces collagen deposition. It also promotes the expression of SIgA to strengthen local mucosal immunity and regulate the in ammatory microenvironment in COPD rats. Pharmacological studies [21] suggested that astragalus polysaccharide regulates the in ammatory response and exerts anti-in ammatory effects by inhibiting the TLR4/NF-κB pathway. Huangqi-Fangfeng protected against allergic airway remodeling by inhibiting the epithelial-mesenchymal transition process in mice via regulating epithelial-derived TGF-β1 [22] . Total avonoids of cortex mori reduced airway in ammation and had a signi cant effect on asthma in mice [23] . Sinapine acted as an antiasthmatic by dilating airway smooth muscle and increasing lung and tracheal volumes [24] . Quercetin-3-O-β-D-glucopyranosyl-7-O-β-D-gentiobioside reduced apoptosis in lung tissues, repaired damaged tissue, and maintained the integrity of the organ [25] . In summary, the components of YS relieve COPD by improving autoimmune function, alleviating in ammatory response, inhibiting apoptosis, and reducing collagen deposition. In the present study, the COPD group was reproduced by two intratracheal injections of LPS combined with cigarette smoke. A substantial amount of neutrophil in ltration in the lung tissue of COPD rats plugged the bronchi. The alveolar walls were thinned and fused. The lung interstitium displayed substantial in ammatory cell in ltration and collagen ber deposition around the trachea and pulmonary interstitium.
TGF-β1 is a pro brotic cytokine that stimulates collagen deposition and aggravates local in ammatory responses [26] . Its expression is elevated in acute and chronic lung diseases. Our previous study [27] found that the Yuping Fengsan Jiawei formula downregulated TGF-β1 levels, suggesting that this is an intervention that prevents airway remodeling. In the present study, expression levels of TGF-β1, TNF-, and IL-6 were reduced in COPD rats after YS intervention. These ndings suggest that YS ameliorates the pathological changes of lung tissue, reduces collagen deposition, and attenuates in ammatory responses in COPD rats.
The NLRP3 in ammasome is involved in developing diseases by promoting the release of downstream in ammatory cytokines, inducing acute and chronic in ammatory responses [28] . It comprises NLRP3, apoptosis-associated speck-like protein containing a CARDl(ASC), and cysteine-requiring aspartate protease-1 (caspase-1) [29] . In response to endogenous or exogenous stimuli, NLRP3 is activated, and ASC acts as an adaptor protein to recruit the precursor caspase-1, which has an enzymatic activity after cleaving pro-interleukin-1β and IL-18 precursor, producing mature IL-1β and IL-18, exerting proin ammatory effects [30] . In the present study, we found that levels of NLRP3, caspase-1, ASC, IL-18, and IL-1β were decreased.
Respiratory mucosal surfaces are colonized by a community of commensal bacteria and are also the primary site of entry for pathogenic agents [31] . Mucosal B cells release large amounts of immunoglobulin (Ig) molecules via both follicular and extrafollicular routes to prevent microbial invasion [32] . IgA and IgG are the most abundant antibodies in mucosal secretions, and SIgA is the predominant antibody secreted by the respiratory mucosa. When SIgA levels secreted in the respiratory mucosa are reduced, the organism has a greater probability of developing an upper respiratory tract infection, and SIgA levels directly correlate with the organism's ability to ght an upper respiratory tract infection. We found that SIgA levels were signi cantly decreased in lung. These ndings suggest YS decreases NLRP3/caspase-1/ IL-1β signaling pathway expression, alleviates in ammation, and enhances local mucosal immune function in the lungs of COPD rats.
Bacterial colonization has been found in the lower respiratory tract of patients with stable COPD [33] .
Analysis of bronchoalveolar lavage uid from patients with COPD revealed that airway bacterial load is associated with in ammatory factors. Studies found that cigarette smoke and inhalation of contaminated air can affect the composition of the lung microbiota [34] . When the mucosal immune system is defective, bacteria can cross the epithelial barrier and stimulate in ammatory responses. Typically, this response is a feature of the protective immune defense response. Persistent airway in ammatory response damages normal lung tissue and defense functions. Reduced defense function allows other infections and in ammatory reactions, creating an infection-in ammation-injury vicious cycle. We observed lower microbial alpha diversity in the COPD group than in the Control group. Lung microbial alpha diversity of mice in the COPD+YS groups differed from that of the COPD group. Analysis of ora diversity showed that YS prevented lung microbiota dysbiosis in COPD rats. Consistent with this nding, linear discriminant analysis of effect size showed different lung microbial compositions in these groups. Mycoplasma belongs to Firmicutes, which accelerates disease processes by promoting chronic in ammation [35] . Halomonas are gram-positive bacteria. Various oligotypes of Christensenella and Clostridium were detected in lung tissue and a few oligotypes of Halomonas in both airway uid and lung tissue from pulmonary brosis and lung cancer patients, suggesting the involvement of these microbial communities in brotic diseases [36] . Nesterenkonia belongs to Actinobacteria and Lactobacillaceae. The microbiome might play a signi cant role in maintaining normal lung function, including structural and immune barriers [38] . These ndings suggest that, in COPD treatment, the relative abundance of Halomonas and Nesterenkonia are increased by decreasing the relative abundance of Mycoplasma, which might inhibit the in ammatory factors IL-1β, TNF-α, IL-6, IL-1β, and TGF-β1, increasing SIgA expression, enhancing immunity, alleviating collagen deposition, and improving lung function.

Summary and prospects
What role does the lung microbiota dysbiosis play in COPD formation? We suggest that lung microbiota dysbiosis promotes the progression of COPD. The colonization of pathogens in the lower respiratory tract due to mucociliary clearance dysfunction caused by inhalation of smoke particles impairs mucociliary clearance, which leads to increased mucus production, airway epithelial damage, downregulation of IgA levels, and disrupts phagocytic function. Bacteria-related products and bacterial-induced epithelial damage impair host immunity, further allowing microbes to enter the lower respiratory tract and ultimately leading to persistent chronic in ammation and microbial colonization of the lungs. Ultimately, this generates a vicious cycle. In conclusion, we found altered lung microbiota in COPD rats, and the imbalance of lung microbiota was associated with lung in ammation. The mechanism of action by which YS prevents this process is not fully elucidated and should be studied in the future.

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These results suggest that the effects of YS involved lung microbes and anti-in ammatory mechanisms.

Declarations
Ethics approval and consent to participate The experimental protocol was approved by the Experimental Animal Care and Ethics Committees of Yunnan University of Chinese Medicine (Approval No.: R-06202023). Consent for participation was not applicable to this study because it does not involve the use of any human data.  Dates are expressed as means ± SEM of three independent experiments (ns non-signi cant, *P < 0.05).  Heatmap of the correlation between the lung microbiota of the rats and environmental factors (*P < 0.05, **P < 0.01; blue represents negative correlation; red represents positive correlation).

Supplementary Files
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