Study On The Mechanism Of Yinlai Decoction In The Treatment Of Gastrointestinal Fever With Pneumonia Based On The Intestinal Flora-immune Network

Objective:Gastrointestinal heat retention refers to the syndrome of invisible heat evil or tangible heat obstructing in the gastrointestinal tract,which results in excessive heat in Qifen.The concept of gastrointestinal tract in traditional Chinese medical(TCM)includes stomach,large intestine and small intestine.It is mainly caused by dietary stagnation,especially in children.The gastrointestinal tract is the pivot of Qi movement.It can further affect the occurrence and development of multi-system diseases,once Qi blocked by invisible or tangible heat evil.Repeated and persistent respiratory infectious diseases are the most common.Yinlai Decoction has a remarkable therapeutic effect on gastrointestinal heat retention combined with pneumonia.In recent years,the correlation between respiratory diseases and gut microbiota has been gradually confirmed.Our previous studies have found that gastrointestinal heat retention can lead to gut microbiota disorder,intestinal immune and mechanical barriers damaged,and eventually the occurrence and aggravation of pulmonary inflammation in rats.However,it has not been confirmed that Yinlai Decoction can affect host immunity by regulating the balance of gut microbiota and inhibiting distal pulmonary inflammation.Based on the theory of "lung and yang-ming being related",high-throughput sequencing and molecular biology techniques were used to detect the changes of gut microbiota,pattern recognition receptor pathway protein expression related to antigen presenting cells,proinflammatory and anti-inflammatory factors in rats after Yinlai Decoction treatment.In this study,the rule of treating lung and yang-ming simultaneously for pediatric pneumonia was explained from the perspective of microbiota-immune-inflammation,so as to enrich the theory of "lung and yang-ming being related "and provide scientific basis for "rule of treating lung and yang-ming simultaneously" in the treatment of respiratory infectious diseases.Method:Gastrointestinal heat retention model was established by high calorie diet,and pneumonia model was prepared by aerosolized LPS inhalation.The body weight,temperature,food and water intake,visceral index,lung and intestinal histopathology were observed in rats of gastrointestinal gastrointestinal heat retention group(S),pneumonia group(P)and gastrointestinal heat retention combined with pneumonia group(SP).Toll-like pattern recognition receptor proteins related with antigen presenting cells in intestinal tissues of each group were detected by Western Blot.Aimplex was used to detect the levels of anti-inflammatory and pro-inflammatory factors in lung and intestinal tissues of each group.Yinlai Decoction was used to treat rats with gastrointestinal heat retention combined with pneumonia(SPT),and the general conditions of body weight,temperature,food and water intake,visceral index and pathology of lung and intestinal tissues were observed in rats of SP and SPT groups.Toll-like pattern recognition receptor proteins related with antigen presenting cells in intestinal tissues and the levels of anti-inflammatory and pro-inflammatory factors in lung and intestinal tissues were detected by Western Blot and Aimplex.16S rDNA technique was used to detect gut microbiota.Result:1.General status:The weight of rats in S group and SP group were lower(P<0.05)or significantly lower(P<0.01)than that in the normal group(N)and P group.After Yinlai decoction treatment,the weight of rats was significantly lower than that in SP group(P<0.01).High calorne diet reduced(P<0.05)or significantly reduced(P<0.01)the abdominal circumference of rats.There was no significant change in the abdominal circumference of pneumonia rats.There was no significant difference between the abdominal circumference of SP group and SPT group.Gastrointestinal heat retention or pneumonia could respectively increase(P<0.05)or significantly increase(P<0.01)toe temperature and axillary temperature of rats.The axillary and anal temperatures of rats in SP group decreased(P<0.05)or significantly decreased(P<0.01)after Yinlai decoction treatment-2.Visceral index:Compared with N group,the liver index of rats in S group and SP group decreased significantly(P<0.01).There was no statistical difference in the thymus and spleen index among all the groups.The liver,thymus and spleen index of rats treated with Yinlai Decoction had no statistical difference.3.Histopathology of lung and intestine:In normal group,alveolar structure of lung tissue was intact,the alveolar wall was thin,and no inflammatory infiltration was observed.The lung tissue of S group,P group and SP group showed inflammatory changes in varying degrees.There was no significant change in intestinal tissue in pneumonia group.Goblet cells in intestinal tissue of rats in S group and SP group enlarged slightly and arranged irregularly.Mild inflammatory changes were also observed.4.Pattern recognition receptor protein in intestine:The expression of TLR9 and NF-κB in intestine of rats in P group was higher than that in N group(P<0.05),the expression of NF-κB in intestine of rats in S group was higher than that in N group(P<0.05),and the expression of MYD88 and NF-κB in intestine of rats in SP group was significantly lower than that in P group(P<0.01).The expression of MYD88,NF-κB and IκBα was on the rise after Yinlai Decoction treatment.5.Expressions of inflammatory factors in lung and intestinal tissues:The levels of IL-4 in lung tissues of rats with high calorie diet were significantly increased(P<0.01),and IL-2 and IFN-γ levels were higher than those in P group(P<0.05).There was no significant difference in the levels of inflammatory factors in intestinal tissues.After infected with pneumonia,the levels of IL-2,IL-6,IL-17A,IFN-γ,IL-4 and IL-10 in lung tissues of rats with gastrointestinal heat retention decreased in varying degrees(P<0.05),and the levels of IL-2,IL-6 and IFN-y in intestinal tissue decreased(P<0.05).After Yinlai Decoction treatment,the levels of IL-2 and IFN-y in lung tissue increased(P<0.05)or significantly increased(P<0.01),while the levels of IL-2 in intestinal tissue increased(P<0.05).6.Intestinal content microflora:The intestinal content microflora of rats in N group,SP group and SPT group were detected.(1)Alpha diversity analysis:There was no significant difference in Chao and Ace indexes among the three groups.Shannon and Simpson indexes of intestinal contents microflora in rats of SP group were significantly lower than those in N group(P<0.01).There was no statistical difference between N group and SPT group.(2)Differential community of intestinal content microflora:Compared with SP group,the abundance of Planococcaceae,Bacillales,Aerococcus,Staphylococcaceae,Staphylococcus,Gemellales,Staphylococcus in intestinal content microflora of rats in N group and SPT group decreased significantly,and the abundance of Leuconostoceae was significantly higher than that in SP group.There was no significant difference in intestinal content flora between N group and SPT group.(3)Beta diversity analysis:Intra group difference of intestinal content microflora in rats of SP group was smaller than its difference between N group and SPT group.There was little difference in intestinal content flora between N group and SPT group.(4)Associated microflora network analysis:Lactobacillus was negatively correlated with Oscillospira,Ruminococcus,Roseburia and Dehalobacterium with a competitive relationship,and positively correlated with Jeotgalicoccus and Streptococcus with a collaborative relationship.Bifidobacterium was negatively correlated with Oscillospira with a competitive relationship,and positively correlated with Allobaculum with a competitive relationship.(5)Functional microflora analysis:In genetic information replication and repair,translation,immune system diseases,infectious diseases,metabolic disease,nucleotide metabolism,environmental adaptation,excretion and immune system pathways,there was difference or significant difference between SP group and SPT group,and there was no significant difference between the normal group and the treatment group.Conclusion:1.High calorie diet can cause mild inflammatory changes in the lungs.Yinlai Decoction treatment can alleviate the inflammation of lung in rats with gastrointestinal heat retention combined with pneumonia.2.Gastrointestinal heat retention can lead to two-way pre-excitation of proinflammatory and anti-inflammatory factors and two-way immune imbalance in the lung and intestinal tissues of rats.The proinflammatory and anti-inflammatory responses of the host can still be compensated,and the host is in a low inflammatory state.After infected with pneumonia on the basis of gastrointestinal heat retention,the compensatory state of proinflammatory and anti-inflammatory response of the host is gradually broken,leading to persistent inflammation and severe immunosuppression,which may be an important factor in the susceptibility and persistence of pneumonia caused by gastrointestinal heat retention.3.Yinlai Decoction can recover the diversity of gut microbiota structure in rats with gastrointestinal heat retention combined with pneumonia.Yinlai Decoction reduce the abundance of pathogenic bacteria such as Planococcaceae,Bacillales,Aerococcus,Gemellales and Staphylococcaceae,and increase the abundance of Leuconobacterium and Bifidobacterium,basically restoring the gut microbiota structure and function to a balanced state,and correct the immunosuppressive state of lung and intestinal tissues.Yinlai Decoction may activate host immunity and inhibit distal lung inflammation by adjusting gut microbiota balance.