Based On The Intestinal Flora And Immunity, The Mechanism Of Yinlai Decoction In The Treatment Of Gastrointestinal Fever With Pneumonia Was Explored

Gastrointestinal heat retention syndrome(GHRS)is a common syndrome in children.Pneumonia is a common pediatric lung disease.Those two often interact with each other and have a greater impact on pediatric growth and development.Yinlai Decoction(YL)was invented on theory of "lung and yangming being related",which had a remarkable efficacy in treating clinical pediatric pneumonia with GHRS,but still remained mechanism unclear.Our preliminary study found that YL may have a therapeutic effect by modulating the gut microbiota and thus immune function.In this study,a combination of in vivo and in vitro experiments were used to investigate the pathogenesis of GHRS with pneumonia and the therapeutic mechanism of YL on the methods of fecal microbiota transplantation and molecular biotechnology.This study aimed to enrich the theory of "lung and yangming being related" and provide an experimental basis for the treatment of GHRS with pneumonia.Experiment 1:Mechanism of Yinlai Decoction treating gastrointestinal heat retention syndrome with pneumonia based on gut-associated immune inflammation via gut microbiotaObjective:To explore the effects of Yinlai Decoction for gastrointestinal heat retention syndrome with pneumonia on gut microbiota and gut-associated immune inflammationMethods:Male SPF-grade SD rats(4-week-old)were used.GHRS model was established by feeding a specially formulated high-calorie diet.Pneumonia model was established by LPS neutralization.Yinlai Decoction was used as intervention and acid dexamethasone acetateas a positive control.Grouped as NC group,pneumonia group(P),GHRS group(G),GHRS with pneumonia group(GP),treatment of pneumonia group(PT),treatment of GHRS with pneumonia group(GPT),positive group(DEX).Body mass,abdominal circumference,organ index,Lee's index,feeding volume,body temperature,lung and colon pathology and leukocyte classification of BALF were observed.Inflammatory factor levels in serum and lung tissue were measured by ELISA.Proliferation and differentiation of T cells in the mesenteric lymph nodes was measured by flow cytometry.Expression of TLR4/NF-?B pathway proteins in dendritic cells,lung tissue and colon tissue.Expression of M1/M2 polarization-related proteins in macrophage cells were measured by western blot.Gut microbiota was evaluated by 16S rDNA.The efficacy and mechanism of Yinlai Decoction were studied.Results:?YL's treatment increased abdominal circumference(P<0.05).?YL remodeled the structure of alveolar,causing thinning of the alveolar wall and reduction of inflammatory infiltration.As shown in colon pathology,YL improved the arrangement of goblet cells and epithelial cells and reduce the number of goblet cells.?Leukocyte classification of bronchial alveolar lavage fluid(BALF):YL reduced NE%and increased MONO%(P<0.05).?Inflammatory factor levels in serum and lung tissue:YL reduced the levels of pro inflammatory factors IL-1?,IL-6,and TNF-? in serum,and IL-1? and TNF-? in lung tissue(P<0.01).?YL reduced Th1 level and ratio of Th1/Th2 in the mesenteric lymph nodes.? YL down-regulated NF-?B expression in dendritic cells(P<0.05),resulting in a downward trend in TLR4,MD2,and MyD8 8 expression.?YL down-regulated TLR4,MD2,MyD88 and NF-?B expression in colon tissue(P<0.05).? YL down-regulated iNOS expression(P<0.05).? YL down-regulated MD2 and NF-?B expression in lung tissue(P<0.05).?The relative abundance of microbiota,microecological diversity,proportions of beneficial gut microbiota and microbiota function after treatment were close to the NC group.YL maintained the mucosal barrier via enhancing beneficial gut microbiota such as Bacteroidales and S24-7.YL further reduced the LPS-induced immune inflammation via removing conditioned pathogenic microbiota such as Campylobacterales.Conclusion:Yinlai Decoction increased the relative abundance of microbiota,microecological diversity,proportions of beneficial gut microbiota and microbiota function,remodeled the structure of alveolar,reduced inflammatory infiltration,corrected the Th1/Th2 imbalance,down-regulated the protein expressions of TLR4/NF-?B signaling pathway in dendritic cells,colon tissue and lung tissue.The mechanism of alleviating lung inflammatory response may based on correcting dysregulation of immune linked to gut microbiota disordersExperiment 2:Effects of GHRS's gut microbiota on gut-associated immunity and its role in pneumonia development using fecal microbiota transplantationPart 1:Study on the effect of gastrointestinal heat retention on gut-associated immunity based on fecal microbiota transplantationObjective:Pseudo-sterile rat model was established for fecal bacterial transplantation.To verify whether FMT influence gut microbiota in SD rats,and provides basis for the further study on its role in pneumonia development.Methods:(1)Male SPF-grade SD rats(15-day-old)were used for pseudo germ-free model(antibiotic group,AB)and GHRS model establishment.High-calorie diet for the GHRS model and "moxifloxacin hydrochloride+metronidazole" method for the antibiotic group.Gut microbiota was detected by 16S rDNA for evaluation of pseudo germ-free model.(2)FMT:Pseudo germ-free rats were transplanted with normal fecal microbiota and GHRS fecal microbiota respectively.Grouped as normal control group(NC),GHRS group(G),FMT with NC fecal microbiota(FNC),FMT with GHRS fecal microbiota(FG).Body mass,abdominal circumference,organ index,Lee's index,feeding volume,body temperature and colon pathology were observed.Pathology score was evaluated.Inflammatory factor level in serum was detected using ELISA.Proliferation and differentiation of T cells in the mesenteric lymph nodes was measured by flow cytometry.Expression of TLR4/NF-?B pathway proteins in dendritic cells,lung tissue and colon tissue.Expression of M1/M2 polarization-related proteins in macrophage cells were measured by western blot.Gut microbiota was evaluated by 16S rDNA.Results:(1)Pseudo germ-free model:? Body mass:Pseudo germ-free rats had slower body mass growth than the NC group.? Gut microbiota:Compared with the NC group,abundance and diversities(both ? and ? index)in the AB group decreased showing a simple,nearly clean gut microbiota with stable microbiota function.The relative abundance of the Bacteroidetes,Bifidobacterium,Proteobacteria and etc increased.Shannon index,Simpson index,ace index and chao1 index decreased,with differences in the microbiota composition of the NC group.(2)FMT with GHRS fecal bacteria:? Body mass and abdominal circumference:The FG group were significantly decreased(P<0.01).? Organ index:Compared with the NC group,liver index and thymus index were significantly higher in the FNC group.The liver index and thymus index were significantly higher in the FG group and the spleen index was higher in the FG group than in the FNC group(P<0.01).? Lee's index;The FG group was significantly lower(P<0.01).?Feeding volume:The FG group had lower feeding volume(P<0.01).?Body temperature:Body temperature was higher in the FG group than in the FNC group(P<0.05).? Pathology of colon tissue:The G group was lack of structural integrity,with more goblet cells.Both FMT groups had inflammatory cells and higher pathology score.Structure in the FG group was more neat but with more visible goblet cells than in the FNC group.?Inflammatory factor levels in serum:IL-1?,IL-6 level in the G group,the FG group and the FNC group had a upward trend,nevertheless IL-4 level had a downward trend.?Gut microbiota:Species number,ratio of Firmicutes/Bacteroidetes increased,relative abundance of Actinobacteria,Erysipelotrichales decreased in the G group.Shannon index,Simpson index,ace index and chao1 index increased,beta diversity decreased in the G group.The FMT groups had changes in gut microbiota relative abundance,structure,energy metabolism and immunomodulatory functions.Relative abundance,structure and function in the FG group was similar to the G group.Enterobacterales and Lachnospiraceae of the FG group irregularity changed.? Th1 and Th2 in the FG group presented an upward trend.?MD2 expression of colon tissue increased in the FG group.MD2 and NF-?B expression of lung tissue in the FG group presented an upward trend.Conclusion:Gastrointestinal heat retention syndrome may altered the structure,energy metabolism and immune changes of gut microbiota.Transplantation of fecal microbiota with gastrointestinal heat retention can cause gastrointestinal heat retention syndrome..The unbalance of GHRS's gut microbiota may caused gut-associated immunity via T cells proliferation and TLR4/NF-?B signaling pathway.Part 2:The role of gastrointestinal heat retention syndrome in pneumonia development based on gut microbiotaObjective:To explore effects of GHRS in pneumonia based on gut microbiota,and to clarify the role of GHRS mechanisms in pneumonia development.Methods:The FNC group and the FG group were neutralized with LPS.Grouped as NC group,neutralization? group and neutralization? group.Lung pathology and leukocyte classification of BALF was observed.Pathology score was evaluated.Inflammatory factor levels in serum and lung tissue were measured by ELISA.Results:? Lung pathology:The Neutralization? group was more severe than the neutralization? group in inflammatory infiltration.? Inflammatory factor levels in serum and lung tissue:IL-1? and TNF-? levels were elevated in the neutralization? group compared with the neutralization? group.? Leukocyte classification of BALF:The Neutralization?group had increased NE%compared to the neutralization? group.Conclusion:Gastrointestinal heat retention syndrome may change the structure and function of the gut microbiota,thereby altering gut-associated immune and affecting the overall immunity of the body,which in turn exacerbates pneumonia.Experiment 3:Yinlai Decoction for LPS-induced inflammation RAW264.7 in vitro based on TLR4/NF-?B signaling pathway and M1/M2 polarizationObjective:To explore anti-inflammatory effect and treating mechanism of Yinlai Decoction in vitro,aiming to validate the founding in vivo.Methods:The LPS-induced inflammation RAW264.7 model was used to intervene with Yinlai Decoction.Cellular proliferation was detected by CCK8.NF-?B gene activation was detected by dual luciferase reporter gene technology.The result of NF-?B gene and IL-1?mRNA,TNF-?mRNA expression predicted and verified the best time of inflammation model.Grouped as blank control group(control),LPS group(model),low dose YL group(L-YL),medium dose YL group(M-YL),high dose YL group(H-YL)and dexamethasone group(DEX).Inflammatory factor was detected by ELISA.Inflammatory factor mRNA expression was detected by RT-qPCR..Expressions of TLR4/NF-?B pathway-related proteins and M1/M2 polarization-related proteins were detected by WBResults:0-80?g/mL Yinlai Decoction were the safe intervention range for RAW264.7.NF-?B activity was strongest with IL-1? mRNA and TNF-? mRNA expression was highest after 6h-LPS stimulation.YL down-regulated IL-1? mRNA and TNF-? mRNA expression(P<0.01).YL reduced levels of proinflammatory factors IL-10,IL-6,TNF-? and IL-12,at the same time increased levels of anti-inflammatory factors IL-4 and IL-10(P<0.01).YL down-regulated iNOSmRNA expression and up-regulated Arg-1mRNA expression(P<0.01).YL down-regulated TLR4 and NF-?B expression and up-regulated I?B expression(P<0.01).Conclusion:Yinlai Decoction might inhibit LPS-induced inflammatory response in RAW264.7 cell.The mechanism might be based on TLR4/NF-?B pathway signaling and M1/M2 polarization.