Study On The Antitussive Mechanism Of Farfarae Flos Based On Gut Microbiota

Topic basis:"Lung and Large Intestine" was first published in the"Huang Di Nei Jing",which mainly clarified that the lung and the large intestine externally and internally.In the clinical treatment of traditional Chinese medicine,"the lung is treated from the intestine".In recent years,studies have found that the two-way regulation of the "lung-gut" axis was closely related to respiratory diseases.Gut microbiota could have a therapeutic effect on respiratory diseases by the "lung-gut" axis directly or indirectly.Gut microbiota could regulate the lung immune response through lipopolysaccharides,short-chain fatty acids,and immune cells.Therefore,the regulation of gut microbiota based on the "lung-gut" axis might play an important role in the "gut treatment of lung disease".Farfarae Flos(FF),dried flower bud of Tussilago farfara L.was used in traditional Chinese medicine for the treatment of cough,bronchitis and asthmatic disorders.Previous studies showed that caffeoylquinic acids(CQAs)were closely related to the antitussive and expectorant effects through metabolomics and pharmacodynamic analysis.Most of CQAs need to be transformed and absorbed through gut microbiota metabolism.However,the relationship between the antitussive effect of Farfarae Flos and gut microbiota is still unclear.Objective:The gut microbiota and metabolites after the FF treatment will be analyzed to explain the antitussive mechanism of FF by regulating gut microbiota and microbial metabolism.Methods:(1)The influence of FF treatment on the structure of gut microbiota was analyzed by 16S rRNA,and the antibiotic-induced gut microbiota disorder experiment and fecal microbiota transplantation(FMT)experiment were used to analyze whether the antitussive effect of FF depends on the gut microbiota.(2)The level of short-chain fatty acids(SCFAs)was analyzed by the GC-MS to demonstrate the correlation between the antitussive effect of FF and the content of SCFAs in feces,and the relationship between antitussive and SCFAs was further analyzed by the SCFAs supplement experiment.(3)LC-MS metabolomics technology and multivariate statistical analysis were used to confirm the polar and non-polar metabolites.Results:(1)The FF could significantly reduce the cough times and prolong the latent period,and reduce the levels of IL-6 and TNF-α in the serum;the antitussive effect of FF decreased,and the decreasing effect on IL-6 and TNF-α was weakened after antibiotic pretreatment.FF could regulate the abundance of some gut microbiota by 16S rRNA sequencing,including Prevotella,Bacteroides,Butyricimonas,and so on.Fecal microbiota suspension could significantly reduce the cough times and prolong the latent period.(2)The established method for quantitative detection of the three SCFAs had methodological verification parameters such as resolution,linear relationship(r>0.9995),repeatability,stability and precision,which meet the requirements of content determination.The FF could significantly increase the content of acetic acid,propionic acid and butyric acid in feces,and the regulating effect of FF on acetic acid and butyric acid was significantly weakened after antibiotic pretreatment.SCFAs supplement experiments showed that sodium acetate and sodium butyrate could significantly reduce the cough times and prolong the latent period.(3)A total of 45 different metabolites were identified from the polar and non-polar parts from feces about FF.Among them,the level of 30 different metabolites was decreased significantly after the administration of FF,and the level of 15 different metabolites was increased significantly.But after antibiotic pretreatment,the regulation tendency of 35 metabolites was weakened.Correlation analysis results showed that the gut microbiota was closely related to the changes of metabolites.Conclusion:This study found that the antitussive mechanism of FF might be through regulating the structure of the gut microbiota,related to SCFAs(such as Prevotella,Bacteroides,Butyricimonas,and so on.),thereby increasing the content of acetic acid and butyric acid in feces.In addition,non-targeted metabolomics and correlation analysis results demonstrated that the antitussive mechanism of FF might be related to intestinal metabolites.