To Explore The Efficacy And Mechanism Of Zhuyang Bufei Chubi Decoction In The Treatment Of Interstitial Lung Disease Based On Multi-omics Analysis

Objective: The common TCM syndromes and prescription rules of interstitial lung diseases were summarized by retrospective study.Clinical and animal studies to evaluate the effect of Zhuyang Bufei Chubi Decoction on interstitial lung diseases;To explore the mechanism of Zhuyang Bufei Chubi Decoction in treating interstitial lung disease by intestinal flora and non-targeted metabolomics analysis.Methods:1.Retrospective study The medical records of Professor Wang Tan’s clinical treatment of interstitial lung disease were collected and sorted out,and the TCM syndromes and prescription rules of interstitial lung disease were summarized.2.Clinical studyPatients with interstitial lung disease who met the syndrome of slow obstruction of pathogenic and minor obstruction,deficiency and cold in the lung,and phlegm and blood stasis were treated with Zhuyang Bufei Chubi decoction for 4 weeks.The TCM symptom score and St.George’s respiratory questionnaire were collected and analyzed before and after taking medicine,and the improvement of symptoms and quality of life were evaluated before and after taking medicine,so as to determine the clinical efficacy of Zhuyang Bufei Chubi decoction in the treatment of pulmonary fibrosis.Feces of patients before and after treatment and feces of healthy volunteers who came to the hospital during the same period were collected,and 16 S r DNA high-throughput sequencing was performed to screen out the key strains that were related to the treatment of interstitial lung disease with Zhuyang Bufei Chubi decoction.3.Animal studiesTo study the effect of Zhuyang Bufei Chubi decoction on bleomycin-induced pulmonary fibrosis in mice.Mice were randomly divided into 6 groups: control group,model group,low-dose,medium-dose,and high-dose Zhuyang Bufei Chubi decoction group,and pirfenidone positive drug group.Mice with bleomycin-induced pulmonary fibrosis were treated with Zhuyang Bufei Chubi decoction and pirfenidone for 4 weeks.After 4 weeks,pathological staining of lung tissue was performed to observe the degree of fibrosis.The serum levels of alanine aminotransferase,aspartate aminotransferase,urea and creatinine were detected to evaluate the liver and kidney function of mice.The above experiments were used to verify the efficacy and safety of Zhuyang Bufei Chubi decoction in the treatment of bleomycin-induced pulmonary fibrosis in mice.The feces of mice in each group were collected for 16 S r DNA high-throughput sequencing of intestinal flora.The key strains of Zhuyang Bufei Chubi Decoction in the treatment of pulmonary fibrosis in mice were screened,and the possible pathways related to the differential flora were predicted and verified.The serum of mice in each group was collected for metabolomics detection,and the differential metabolites in serum were screened,and the possible metabolic pathways were predicted and verified.To analyze the correlation between 16 S r DNA high-throughput sequencing results of intestinal flora and serum metabolomics,and further clarify the mechanism of Zhuyang Bufei Chubi decoction in the treatment of pulmonary fibrosis.Results:1.Retrospective studyThe medical records of Professor Wang Tan’s clinical treatment of interstitial lung disease were collected and sorted out,and the TCM syndromes and prescription rules of interstitial lung disease were summarized.2.Clinical ResearchTCM symptom scale showed that TCM symptoms were significantly improved after taking Zhuyang Bufei Chubi Decoction for 4 weeks(P<0.05);The score of SGRQ after treatment was significantly lower than that before treatment(P < 0.01),and the symptoms,daily activity ability and disease impact were significantly improved(P < 0.01).The results clarified the clinical efficacy of Zhuyang Bufei Chubi Decoction in the treatment of pulmonary fibrosis.The results of the study on the intestinal flora of patients with fibrosis suggest that,compared with healthy people,the intestinal microbial community diversity of patients with fibrosis decreases,and the microbial species composition changes significantly.After4 weeks of treatment with Zhuyang Bufei Chubi Decoction,the decrease of intestinal microbial community diversity can be improved to a certain extent.Moreover,at the level of phylum,class,order,family,genus and species,the intestinal flora of fibrosis patients can be positively regulated.The bifidobacterium was significantly enriched in the intestine after taking the drug.3.Animal ResearchPathological staining of lung tissue of mice showed that the damage of lung tissue structure,inflammatory cell infiltration and collagen fiber deposition of mice in Zhuyang Bufei Chubi Tang group were significantly reduced compared with model group,and the histopathological improvement was dose-dependent.The serum levels of alanine aminotransferase,aspartic acid aminotransferase,urea and creatinine in Zhuyang Bufei Chubi Decoction group were similar to those in the control group.Similar to clinical studies,microbial community diversity in mice with fibrosis model decreased and microbial species composition changed significantly.Application of Zhuyang Bufei Chubi decoction group could ameliorate the decline of intestinal microbial community diversity to a certain extent,and had positive regulation on intestinal microbial community changes in mice with fibrosis at the level of phylum,class,order,family,genus and species.After taking the drug,the intestinal flora was significantly enriched in Bacteroidetes,Bacteroidetes,Bacteroidetes,S24＿7 and Ruminococcus.Metagenome Seq analysis suggested that Zhuyang Bufei Chubi Decoction may play an anti-pulmonary fibrosis role by affecting the intestinal flora abundance and regulating the ECM receptor interaction pathway.Immunohistochemical staining was used to detect the main components of ECM in mouse lung tissue.The expression levels of collagen,fibronectin and α smooth muscle actin were quantitatively analyzed.The results showed that the expression intensity of collagen,fibronectin and α smooth muscle actin in lung tissue of mice in the control group was weak,while the positive expressions of the above three proteins in mice in the model group were significantly increased.The quantitative analysis showed that the content of model histone was significantly increased(p < 0.01).The positive staining of Zhuyang Bufei Chubi decoction in low,medium and high dose groups and pirfenidone groups was reduced,and had statistical significance.The results of serum metabolomics showed that the metabolites of mice in the control group and mice in the fibrosis model had significant differences(P < 0.05).After treatment with Zhuyang Bufei Chubi Decoction,the abnormal changes of the following 9 metabolites could be adjusted.These include 5-methylcytosine,7-methyl-3-methylidene-6-(3-oxobutyl)-4,7,8,8a-tetrahydro-3ah-cyclohepta[b]furan-2-one,Vernolate,Eicosapentaenoic acid,Prostaglandin f1.alpha,Glycolate,L-(+)-erythrulose,Riluzole,Taurine.The results of differential metabolites predicted that Zhuyang Bufei Chubi Decoction might affect the metabolic pathway of arachidonic acid,alanine,aspartic acid and glutamic acid.ELISA was used to detect the expression level of arachidonic acid in the serum of mice,and quantitative analysis was conducted.The results showed that compared with the control group,the content of arachidonic acid in the serum of mice in the model group was increased,with statistical significance(p < 0.05).Serum arachidonic acid content in Zhuyang Bufei Chubi Decoction group and Bleomycin group was decreased,and the decrease of serum arachidonic acid was most significant in Zhuyang Bufei Chubi decoction high-dose group(p < 0.001).The results of heat map showed that there was a correlation between gut flora and metabolites,for example,Vernolate and g-unidentified-Erysipelotrichaceae were negatively correlated(P < 0.05).Riluzole was positively correlated with g-Akkermansia,g-Prevotella and g-unidentified Bacteroidales(P < 0.05).It was negatively correlated with g-[Ruminococcus](P < 0.05).L-(+)-erythrulose was associated with g-Sutterella(P < 0.01),G-sutterella(P < 0.05),g-Prevotella(P < 0.05)0.01),g-Parabacteroides(P < 0.05),g-unidentified-Rikenellaceae(P < 0.01),g-unidentified-Rikenellaceae(P < 0.01),g-unidentified-Bacteroidales(P < 0.05)were positively correlated with UNIDENTIFIED UNIDENTIFIED L-(+)-erythrulose was associated with g-unidentified-S24-7(P < 0.01),g-Sutterella(P < 0.05),g-Prevotella(P < 0.05),Unidentified-S24-7(P < 0.01)0.01),g-Parabacteroides(P < 0.05),g-unidentified unidentified Rikenellaceae(P < 0.01),g-unidentified unidentified Bacteroidales(P < 0.01)and G-Parabacteroides(P < 0.05)<0.05)were positively,and negatively correlated with g-unidentified F16.The association network diagram shows that multiple metabolites are closely related to microflora,of which L-(+)-erythrulose,Ectoine,Octaethylene＿glycol and other metabolites are associated with more than 5 microflora.g-[Ruminococcus],g-Prevotella,g – unclassified＿Clostridiales)associated with a variety of metabolites,such as flora g-Pediococcus G-pediococcus is positively correlated with fibrosis model differential metabolites L-(+)-erythrulose,Vernolate and Riluzole.Conclusion:1.The most common clinical TCM syndromes of interstitial lung disease are lung and kidney deficiency and phlegm and blood stasis blocking collaterals.The most frequently used prescription is Zhuyang Bufei Chubi Decoction.2.Zhuyang Bufei Chubi Decoction can improve clinical symptoms and improve quality of life of patients;It alleviates the inflammatory response of mouse lung tissue and inhibits collagen fiber deposition.It has definite therapeutic effect on interstitial lung disease.3.Interstitial lung disease can affect the composition and abundance of intestinal flora.Taking Zhuyang Bufei Chubi Decoction can regulate the composition and abundance of intestinal microorganisms in human body and mice,and increase the abundance of bifidobacterium in human body,and the abundance of Bacteroidetes,Bacteroidetes,Bacteroidetes,S24＿7 and Ruminococcus in mice.4.Interstitial lung disease can lead to changes in serum metabolites,and abnormal products can be improved after treatment with Zhuyang Bufei Chubi Decoction.These include 5-methylcytosine,7-methyl-3-methylidene-6-(3-oxobutyl)-4,7,8,8a-tetrahydro-3ah-cyclohepta[b]furan-2-one,Vernolate,Eicosapentaenoic acid,Prostaglandin f1.alpha,Glycolate,L-(+)-erythrulose,Riluzole,Taurine.5.Zhuyang Bufei Chubi Decoction may correct metabolic disorders by regulating the abundance of intestinal flora,so as to regulate the ECM receptor interaction pathway,arachidonic acid metabolic pathway and alanine,aspartic acid and glutamic acid metabolic pathway to play an anti-pulmonary fibrosis role.