Study On The Mechanism Of Yifei Sanjie Decoction Regulating "Lung-gut" Axis To Relieve Airway Inflammation In COPD Rats

ObjectChronic obstructive pulmonary disease（COPD）is classified into the categories of"lung distension"and"cough"in traditional Chinese medicine（TCM）.Based on the core pathogenesis of"qi deficiency,phlegm turbidity with blood stasis",Professor Li Qingsheng,a famous practitioner in Yunnan Province,adopted the treatment principle of"tonifying lung and spleen,purging phlegm and removing blood stasis",and formulated the"Yifei Sanjie Decoction（YSD）".The clinical application has been more than 30years,and the curative effect is significant and accurate.The main pathological feature of COPD is persistent airflow limitation,and the pathological features are airway inflammation and airway remodeling.Meanwhile,in recent years,the discovery of homology between lung and large intestine in embryonic development has confirmed the connection between lung-intestinal mucosal immunity,and it has become a research hotspot to explore the pathogenesis of COPD from the perspective of mucosal immunity.In this study,the theory of"the lung and the large intestine coexist"and the common mucosal immunity of the lung and intestine were taken as the starting point.The aim of this study is to investigate whether YSD can regulate the"lung-gut"axis balance of COPD,alleviate airway inflammation,and block the development of COPD by regulating the structure and abundance of gut microbiota,reducing the levels of inflammatory factors,and improving the pathological morphology of lung and colon tissues by replicating COPD model rats.In order to clarify the biological basis of YSD"nourishing qi,purifying phlegm and removing blood stasis".Methods1.The COPD rat model was replicated by intratracheal instillation of Lipopolysaccharide（LPS）combined with cigarette smoking,and the model was evaluated by observing the changes of pulmonary ventilation function and pulmonary pathological morphology.2.YSD was prepared and the clinical equivalent dose was set as the group of YSD.The COPD animal model was successfully replicated at the 8th week of the experiment.After 8 weeks,COPD rats were treated with YSD at the clinical equivalent dose of11.61g/kg by gavage,2ml once time.The control（CT）group and the model（COPD）group were given the same amount of sterile distilled H₂O by gavage for a total of 4weeks of drug intervention.3.⑴To evaluate the degree of airflow limitation,the body plethysmography system was used to detect pulmonary function indicators.For example,the Expiratory flow at50%（EF50）,Tidal volume（TV）and Minute volume（MV）.Hematoxylin-eosin（HE）staining and Masson staining were used to observe the structure of small airways and alveoli under microscope.⑵Hematoxylin eosin（HE）staining and Masson staining were used to observe the structure of small airways and alveoli under microscope,and HE staining was used to observe the pathological morphology of colon under microscope to determine the effect of YSD on the pathological morphology of lung and colon tissue in COPD rats.⑶16S r RNA gene high-throughput sequencing of gut microbiota structure and abundance,to clarify the regulatory effect of YSD on gut microbiota.⑷Secretory immunoglobulin A（SIg A）in lung and colon tissues was detected by Enzyme linked immunosorbent assay（ELISA）.Meanwhile,spleen and thymus indexes were analyzed.To clarify the regulatory effect of YSD on respiratory and intestinal mucosal immunity.⑸ELISA was used to detect the inflammatory levels in lung tissue,colon tissue and serum,mainly Interleukin-6（IL-6）and Tumor necrosis factor-α（TNF-α）,to determine the regulatory effect of YSD on inflammation.⑹Western blot（WB）was used to detect the expression of Transforming growth factorβ1（TGFβ1）in lung tissue,the protein levels of Ras and Raf1 and the phosphorylation levels of p-MEK and p-ERK in lung tissue.To clarify the mechanism of YSD in alleviating airway inflammation and airway remodeling.In order to clarify the relationship between the"lung-gut"axis and the biological basis of"nourishing qi,removing phlegm and removing blood stasis".Results1.YSD can improve pulmonary ventilation function and small airway collagen deposition in COPD ratsAfter 4 weeks of YSD treatment,EF50,TV and MV of COPD rats were significantly increased（P<0.05）.The lung tissue pathology showed that the airway mucosal epithelial cells in the YSD group were arranged neatly,and the alveolar structure was relatively complete,which was significantly improved compared with the COPD group.Compared with the COPD group,the YSD group had significantly decreased collagen deposition in the small airway wall and TGF-β1 in the lung tissue（P<0.05）.The results showed that YSD could improve the pulmonary ventilation function of COPD rats and reduce the collagen deposition in the inner wall of small airways,thereby improving airway remodeling.2.YSD can enhance respiratory mucosal immunity and body immunity in COPD ratsAfter YSD treatment,the spleen and thymus index and SIg A expression in lung tissue of rats were significantly higher than those of the COPD group（P<0.05）.These results indicated that YSD could significantly enhance both respiratory mucosal immunity and body immunity.3.YSD can down-regulate the expression of inflammatory factors in COPD rats and regulate the Ras/Raf/MEK/ERK pathwayAfter YSD intervention,the expression levels of inflammatory factors IL-6 and TNF-αin lung tissue were significantly lower than those in COPD group（P<0.05）.At the same time,the protein levels of Ras and Raf1 and the phosphorylation levels of p-MEK and p-ERK in lung tissues were also significantly decreased（P<0.05）.The results showed that YSD could regulate the Ras/Raf/MEK/ERK pathway,reduce the expression level of inflammatory factors,and alleviate airway inflammation.4.YSD can improve colonic pathology and enhance intestinal mucosal immunity in COPD ratsAfter YSD intervention,compared with the model group,HE staining of colon pathology showed that the arrangement of villi,glandular structure and muscle layer loose in COPD rats were significantly improved.The expression level of colonic SIg A was also increased significantly（P<0.05）,suggesting that YSD could improve the damage of intestinal mucosal barrier and enhance mucosal immune defense function in COPD model rats.5.YSD can reduce local and systemic inflammation levels in COPD ratsThe inflammatory factors IL-6 and TNF-αin colon tissue and serum of COPD rats were significantly up-regulated（P<0.05）.After YSD treatment,the inflammatory factors IL-6 and TNF-αin the colon and serum of rats were significantly down-regulated（P<0.05）.These suggesting that YSD can reduce local and systemic inflammation levels and improve COPD inflammation.6.YSD can regulate the structure and abundance of gut microbiota in COPD rats6.1 YSD can regulate the structure and abundance of gut microbiota at the genus level in COPD ratsAt the genus level,unidentified＿Lachnospiraceae（3.23%-9.09%）and unidentified＿Ruminococcaceae（8.64%-10.64%）were the dominant genera of gut microbiota in rats.After YSD treatment,the relative abundance of intestinal beneficial bacteria such as Lactobacillus showed an upward trend（P>0.05）.The relative abundance of pathogenic bacteria such as Allobaculum showed a decreasing trend（P>0.05）.6.2 YSD can regulate the structure and abundance of gut microbiota at species level in COPD ratsAt the species level,Lactobacillus＿reuteri（0.72%-2.39%）and rumen＿bacterium＿NK4A214（0.91%-1.47%）were the dominant bacteria in COPD rat.After YSD intervention,the relative abundance of intestinal beneficial bacteria Lactobacillus＿reuteri showed an upward trend（P>0.05）.The relative abundance of Ruminococcus＿flavefaciens and Trichinella＿pseudospiralis decreased significantly（P<0.05）.6.3 The characteristic bacteria associated with COPD rats and YSDLEf Se analysis showed that Desulfovibrio was more significant in the CT group（P<0.05）,Cloacibacillus and Synergistaceae were more significant in the COPD group（P<0.05）.Lactobacillus and Lactobacillus＿reuteri were more significant in YSD group（P<0.05）.Conclusions1.YSD has the effect of"tonifying lung and spleen"in the treatment of COPD by regulating the common mucosal immunity of lung and intestine and alleviating airway inflammation.2.YSD"purging phlegm and removing blood stasis"can reduce lung and intestinal inflammation,regulate the structure and abundance of intestinal flora,and improve the continuous decline of COPD lung function.One of the mechanisms is to down-regulate Ras/Raf/MEK/ERK inflammatory signaling pathway.