Study On Metabolic Pattern Of Bacterial Pneumonia (Pneumonia And Cough·Wind Heat Stagnation Lung Syndrome) And Intervention Of Qingfei Yin Based On LC-MS Technology

Background:The main clinical manifestations of pneumonia are fever,cough,phlegm obstruction,dyspnea and pulmonary rales.It belongs to the category of"pneumonia and cough"in traditional Chinese medicine.It is a common respiratory disease in children.The main pathogens causing pneumonia are bacteria,viruses,mycoplasma and so on.Bacterial pneumonia accounted for about 80%,among which Streptococcus pneumoniae（S.pn）accounted for about 29.9%.S.pn pneumonia is often seen as heat syndrome,and clinically wind heat stagnation lung syndrome is the most common.However,an important problem that needs to be solved urgently in modern Chinese medicine is how to expound the essence and connotation of TCM syndromes scientifically and comprehensively.The World Health Organization lists pneumonia as one of the world’s three most important pediatric diseases.Infantile bacterial pneumonia is second only to cardio-cerebrovascular disease,which is one of the causes of infant death.The main treatment methods for pediatric bacterial pneumonia are antibacterial drugs,but due to the low positive rate of etiological culture,most of the treatment is based on the experience of clinicians.The irrationality rate of the use of antibacterial drugs is high,and the abuse of antibacterial drugs often occurs,resulting in the increase of the resistance rate of pneumonia pathogens,which increases the difficulty of clinical treatment.In TCM treatment,the side effects of western medicine can be avoided through the differentiation analysis of symptoms and signs of the children,combined with the four TCM diagnoses,and personalized treatment.Therefore,the TCM treatment of pneumonia and cough has higher research significance.Under the guidance of traditional Chinese medicine master Professor Wang Lie’s innovative theory--"heat toxicity"theory,with the treatment principle of TCM"strengthen the normal qi and dispelling evil",after 60 years of clinical application of pediatric pneumonia prescription--Qingfei Yin（QFY）,has cured thousands of pneumonia patients,and was developed into a widely used clinical hospital preparation in the 1980s.In order to further study QFY,through a series of preliminary studies,our research group found clear anti-inflammatory and antibacterial mechanism of QFY from the aspects of immunity,inflammation,flora,etc.,but the mechanism of QFY on the metabolic level remains to be explored.This topic explores the metabolomics characteristics of QFY in alleviating lung inflammation,in order to enrich the scientific connotation of treating S.pn pneumonia in children based on the theory of"heat toxicity"in traditional Chinese medicine.Metabolomics is a new subject,which studies the type,quantity and change rule of metabolites,and is an important part of systems biology technology.Since the change of metabolites is the end point of all the functional activities of genes and proteins in the body,it avoids the disadvantages of a single or few indicators in the previous study of certain physiological and pathological changes and is regarded as a"biochemical phenotype"that can immediately,sensitively and truly show the overall functional state of organisms under the stimulation of various external factors.Therefore,the metabolomics method was adopted in this study to comprehensively characterize the changes of metabolites caused by S.pn pneumonia,wind heat stagnation lung syndrome,in an attempt to elucidate the nature of S.pn pneumonia,wind heat stagnation lung syndrome and the therapeutic effect of QFY on S.pn pneumonia infection in mice.The characteristics of plasma metabolism in children with bacterial pneumonia（pneumonia and cough,wind heat stagnation lung syndrome）were studied,and the essence of syndrome of pneumonia and cough,wind heat stagnation lung syndrome was discussed.The plasma metabolic characteristics of BALB/C mice with S.pn pneumonia were studied,and the intervention effect of QFY on BALB/C mice with S.pn pneumonia and the potential mechanism of metabolic regulation were explored.Methods:Metabolomics Clinical trial of S.pn pneumonia,wind heat stagnation lung syndrome:According to the diagnostic standard of S.pn pneumonia and the syndrome differentiation standard of traditional Chinese medicine,11 cases of children with S.pn pneumonia,wind heat stagnation lung syndrome were selected,and 11 healthy volunteers were selected as the normal group.According to the diagnostic criteria of S.pn pneumonia and the Guidelines for the Diagnosis and Treatment of Common Diseases in Pediatrics of Traditional Chinese Medicine of the Chinese Association of Traditional Chinese Medicine,11 children with S.pn pneumonia,wind heat stagnation lung syndrome were selected for the experiment,and 11 healthy volunteers were identified as the normal group.The plasma of the two groups of children was collected,and the metabolites of plasma of the two groups were detected by Q Exactive HF mass spectrometer and Vanquish UHPLC ultra-high pressure liquid chromatograph,and the syndrome related metabolic model of S.pn pneumonia was established.Principal component analysis（PCA）and orthogonal partial least squares（OPLS-DA）were used to statistically analyze the detected matrix data,screen potential plasma biomarkers,analyze the metabolic characteristics of S.pn pneumonia,wind heat stagnation lung syndrome,and analyze its related metabolic pathways.Animal experiment of QFY intervention on BALB/c mice with S.pn pneumonia:S.pn nasal drops infected BALB/C mice were treated with QFY,and ceftriaxone was set as positive control.48h later,serum and lung tissues of mice were collected,and morphological analysis of pathological sections of lung tissues was performed to evaluate lung lesions.Based on PCA and OPLS-DA analysis and detection data,the overall metabolic changes of serum and lung tissues of mice in each group were studied.Potential serum biomarkers were screened and related metabolic pathways were analyzed to clarify the metabolic regulation characteristics of QFY.Results:Metabolomics Clinical trial of S.pn pneumonia,wind heat stagnation lung syndrome:The results of plasma metabolomics pattern recognition analysis showed that,in the ellipse of the scatter plot（95%confidence region）,the scores of main components of most children with S.pn pneumonia,wind heat stagnation lung syndrome and healthy children in the normal group were concentrated,and although there was a certain separation trend,there was still crossover and overlap.The parameters of OPLS-DA model were 0.408 for positive ion mode R²X,0.989 for R²Y and 0.969 for Q².The negative ion mode of R²X is 0.603,R²Y is 0.986,and Q² is 0.925.The normal group and the S.pn pneumonia,wind heat stagnation lung syndrome group could be significantly separated along the t[1]axis,indicating significant differences in metabolic characteristics between the two groups.In this project,679 metabolites were identified after combining positive and negative ion patterns,among which 358 positive ion patterns（POS）and 321 negative ion patterns（NEG）were identified.Potential biomarkers were screened in bacterial pneumonia（pneumonia and cough,wind heat stagnation lung syndrome）group,and the metabolic pattern was found to be reduced expression of L-carnitine,cholesterol,L-histidine,pyruvate,etc.The expression levels of testosterone,choline,L-phenylalanine,L-arginine,prostaglandin A2,leukotriene B4,L-lactic acid,palmitic acid,arachidonic acid,butyric acid and myristic acid were increased.The expression levels of bacterial pneumonia（pneumonia and cough,wind heat stagnation lung syndrome）group were changed by basal cell carcinoma pathway,D-arginine and D-ornithine metabolic pathway,central carbon metabolic pathway,choline metabolic pathway,amoebiasis pathway,HIF-1signaling pathway,ovarian steroid production pathway,fatty acid biosynthesis pathway,glucagon signaling pathway,protein digestion and absorption Adduction pathway,glycine-serine-threonine metabolic pathway,glycolysis/gluconeogenesis pathway,unsaturated fatty acid biosynthesis pathway,aminoacyl-t RNA biosynthesis pathway,amino acid biosynthesis pathway,arginine and proline metabolic pathway,etc.Animal experiment of QFY intervention on BALB/c mice with S.pn pneumonia:1.The pathological changes of lung tissue in S.pn infected BALB/C mice were mainly manifested as monocyte macrophage and neutrophilic granulocyte infiltration,accompanied by bronchitis and vasculitis,alveolar wall congestion and thickening,and lung tissue color darkening.QFY alleviated pulmonary congestion edema and inflammation to varying degrees.Pathological score results showed that compared with the model group,the pathological changes in the treatment group were significantly reduced.2.48h after S.pn infection,plasma data of BALB/C model mice and normal mice based on PCA model could be completely separated.Under positive and negative conditions,R²X and Q² were 0.794 and 0.833,0.681 and 0.644,respectively,indicating successful modeling.The characteristics of plasma metabolism of mice were changed by S.pn infection.There were226 different metabolites between the S.pn group and the blank group,169 different metabolites between the S.pn group and the QFY group,and 313 different metabolites between the S.pn group and the ceftriaxone group.Compared with the blank group,134metabolites were up-regulated and 90 were down-regulated in S.pn group.Compared with QFY group,there were 60 up-regulated metabolites and 101 down-regulated metabolites in S.pn group.Compared with the ceftriaxone group,231 up-regulated metabolites and 95 down-regulated metabolites were found in the S.pn group.115 metabolites(molecular weight error<30E^-06).The changes of metabolites in S.pn group compared with blank group,QFY group and ceftriaxone group were analyzed,and the trend of increasing and decreasing metabolites was found.The data of S.pn,QFY and ceftriaxone were further interpreted by analyzing the changes of metabolic pathways.S.pn group had a total of 30 metabolites involved in 5pathways;There are 25 metabolites in QFY group,which are involved in 8 pathways.A total of 46 metabolites in ceftriaxone group were involved in 6 pathways.The metabolites involved in more than two metabolic pathways were arachidonic acid in S.pn group;Four metabolites in the QFY group（palmitic acid,phosphatidylcholine,linoleic acid and arachidonic acid）;And linoleic acid in ceftriaxone group.Based on the selected biomarkers,we evaluated the efficacy of QFY,and found that QFY could regulate five of the biomarkers to varying degrees,mainly through regulating the metabolic pathway of linoleic acid.Conclusion:1.Q Exactive HF/Vanquish UHPLC analysis of metabolomics can initially interpret and distinguish the different metabolic patterns of S.pn pneumonia,wind heat stagnation lung syndrome from healthy children.2.The metabolic disorders of fatty acid biosynthesis pathway,unsaturated fatty acid biosynthesis pathway and cancer center carbon pathway are the main signs in the plasma of children with pneumococcal wind-heat pneumonic syndrome.3.QFY can significantly reduce the lung inflammation of S.pn infected mice.4.QFY can treat metabolic disorders caused by S.pn infection mainly by regulating arachidonic acid,palmitic acid,linoleic acid,oleic acid and phosphatidylcholine.