The Effect And Mechanism Of Jinhong Decoction Ameliorates Sepsis Lung Injury By Modulating Microbiota Composition

Background:Sepsis which caused by the pathogen infection,is a representative disease that threatens human health,and is often clinically performed as multiple organ dysfunction,among which ALI and ARDS are the most common ones.Its pathogenesis is related to inflammatory overreaction.immunosuppression and intestinal microenvironment.Thousands of diverse microbiome,mainly bacteria,inhabit in the intestinal tract and are collectively referred to as gut microbiome.Intestinal microbiome is the main medium of communication lung-gut axis,A large number of reports have shown that gut microbiome and pulmonary microbiome are significantly similar in severe infection,and this similarity is related to intestinal mucosal barrier function impairment.Jinhong Decoction(JHD)is a kind of traditional Chinese medicine(TCM)compound composed of Rheum palmatum L.,Sargentodoxa cuneata(Oliv.)Rehder&E.H.Wilson and araxacum mongolicum Hand.-Mazz.,which is developed according to the basic principle of lung are in contact with the gut.JHD has the function of purge endotoxin,and clinical can be used to treat sepsis,but its mechanism is still unclear.Objective:To investigate the therapeutic effect and mechanism of JHD on sepsis and lung injury induced by Cecum Ligation and Puncture(CLP),and explore its mechanism.Methods:Male C57BL/6 mice aged 6-8 weeks and weighing about 25g were randomly divided into Sham group,Sham+JHD group,CLP group and CLP+JHD group.CLP and CLP+JHD group were used to construct sepsis mouse model by CLP operation.Sham and Sham+JHD underwent sham operation by cutting open their skin and flesh,and CLP+JHD or Sham+JHD were given JHD intragastric administration 30 minutes after modeling,mice were sacrificed after 3 days.During this period,changes in body temperature,body weight,diet and drinking water of the mice were recorded.After the mice were killed,serum samples were taken to detect ALT and AST concentrations,and whole blood bacterial load,survival rate to evaluate whether JHD could relieve the symptoms of sepsis.The total cell number,total protein content in BALF,lung tissue MPO activity and pathological damage in lung tissue were detected,and the expression levels of pulmonary inflammatory factors were detected by q-PCR.The proportion of neutrophil.γδT and other immune cells was detected by flow cytometry.Gut microbiome was detected by 16S rRNA high-throughput sequencing.Fecal microbiome transfer(FMT)experiment was conducted to investigate the effect of fecal microbiome on sepsis.Then,in vivo imaging of small animals,WB,immunofluorescence and other experimental techniques were used to detect whether sepsis can damage the intestinal mucosal barrier function and whether JHD can play a protective role on the mucosal barrier injury.Results:Significant pathological symptoms of CLP induced sepsis were observed,including decreased body temperature and body weight,much lower diet and water intake,elevated serum ALT and AST concentration,expanded whole blood bacterial load and a sharp decline of survival rate.Furthermore,total protein in BALF,lung tissue MPO,inflammatory factor expression level and the proportion of neutrophil cells and γδT cells were all markedly increased.Lung tissue pathological changes including inflammatory cell infiltration,alveolar septum thickening and fibrin bundle hyaline membrane formation were observed as well.Notably,intestinal microbiome structure changed,and the abundance of Bacteroidetes and Proteobacteria was increased,E.coli was the most obvious,while the abundance of Firmicutes was decreased.Intestinal permeability was enhanced,and the protein level of intestinal mucosal integrity was down-regulated,and E.coli,which comes from the gut,can enter the lungs and further promote lung inflammation.After treatment with JHD,the symptoms and lung injury of CLP-induced sepsis mice were significantly alleviated:Body temperature and weight were significantly increased,the amount of diet and drinking water increased,the content of ALT AST in serum was significantly decreased,the bacterial load in whole blood was reduced,and the survival rate was increased.The content of total cell number and total protein in BALF decreased,neutrophils and γδT cells decreased.Lung tissue MPO activity enhanced,pathological changes significantly decreased,the expression level of inflammatory factors decreased.The imbalance of gut microbiome was significantly improved,the abundance of Bacteroidetes and Proteobacteria was significantly down-regulated,and the abundance of Firmicutes was up-regulated.FMT experiment suggested that intestinal microbiome had a significant intervention effect on the occurrence and development of sepsis.Further experiments showed that JHD could further reduce the intestinal permeability of sepsis mice,repair the intestinal mucosal barrier function(ZO-1,MUC1),and reduce opportunistic bacteria E.coli from the intestine to enter the distal lung and cause lung inflammation.Conclusion:JHD can significantly improve CLP-induced sepsis and lung injury by regulating intestinal microbiome,which may be mediated by repairing the intestinal mucosal barrier function and reducing the enteric-derived opportunistic pathogen E.coli entering the lung.