Effect And Mechanism Of Qingre Huatan Tongfu Decoction On Stroke-associated Pneumonia In Rats Based On Metabonomics

objectiveTo study the intervention effect of Qingre Huatan Tongfu Formula(HTF)on stroke-associated pneumonia(SAP)rats,and to explore the mechanism of HTF enema in the treatment of SAP based on the metabolomic technology of ultra-high performance liquid chromatography-four-stage rod time-of-flight tandem mass spectrometry(UPLC-QTOF/MS).MethodTwenty-one SPF male SD rats were randomly divided into sham-operated(J),model(M)and treatment(Z)groups(HTF,lg/ml,7.6ml/d).There were 7 rats in each group.Except group J,SAP models were established by modified suture Embolism Combined with tracheal injection in the other two groups.Group Z was treated with HTF,while the other two groups were treated with normal saline for 7 days.After modeling,one rat in each group was randomly selected to observe cerebral infarction by TTC staining,and the general conditions(state,hair,activity,body temperature,nasal secretions)of the remaining groups were observed.The neurological function scores were made on the first day and the seventh day of modeling.Pseudomonas aeruginosa was identified from lung tissues of three groups of rats;HE staining was used to observe the pathological changes of brain and lung tissues of rats in each group;serum ET-1,IL-1beta and IL-1beta in lung tissues were detected by enzyme-linked immunosorbent assay(ELISA);and metabolites in serum of three groups of rats were detected by UPLC-QTOF/MS analysis.Principal Component Analysis(PCA),Orthogonal Partial Least Squares(OPLS-DA)and t-test were used to screen metabolites in serum.Potential biomarkers were searched for.Related metabolic pathways were obtained through potential biomarkers.Abnormal metabolism and regulation of HTF on abnormal metabolism were found.The mechanism of HTF in treating SAP rats was elucidated.Result1.General situation: In the course of the experiment,rats in group J were in good condition with smooth and lustrous coat,sensitive movement,normal body temperature and moist nose without asthma;rats in group M were in poor condition,with chaotic and lustrous coat,listlessness,high fever,a large number of secretions around the nose and asthma,while rats in group Z were in general condition,lacking lustrous coat,poor activity,low fever and a little secretions around the nose.2.Neurological function score: In group J,the neurological function score was 0 on the first day and the seventh day of modeling.There was no significant difference in neurological function score between group M and group Z on the first day of modeling(P > 0.05).On the 7th day,the neurological function score of group Z was significantly lower than that of group M.Compared with the first day,the score of nerve function in group M increased on the 7th day.Compared with the first day,the score of nerve function in group Z decreased significantly on the 7th day.3.HE staining of brain and lung tissueHE staining of brain tissue: In group J,there was almost no change in brain tissue,no significant swelling,orderly arrangement,homogeneous stroma and no infiltration of inflammatory cells.In group M,the cells were disorderly arranged,a large number of inflammatory cells infiltrated,and microglia increased.Under light microscopy,neurons,glia and microvascular periphery space were widened,nerve fiber network was loose,showing spongy or vacuole-like.Inflammatory infiltration of brain tissue in group Z was significantly less than that in group M.HE staining of lung tissue: There was no inflammatory cell infiltration around alveolar duct and alveolar sac in group J;A large number of inflammatory cell infiltration was observed in group M,with large consolidation of lung tissue and obscure alveolar contour;Inflammatory cell infiltration in group Z was less than that in group M,lung tissue consolidation was better than that in group M,alveolar contour was visible,but inflammatory infiltration was more significant than that in group J.4.ELISA results showed that serum IL-1 beta concentration in the model group was significantly higher than that in the sham operation group(P < 0.01).Compared with the model group,the serum IL-1 beta concentration in the treatment group was significantly lower than that in the model group(P < 0.01);compared with the sham operation group,the serum ET-1 concentration in the model group was significantly higher(P < 0.01),and the serum ET-1 concentration in the treatment group was significantly lower than that in the model group(P < 0.01).Compared with the model group,the concentration of IL-1beta in the lung tissue of the operation group was significantly higher(P < 0.01),and the concentration of IL-1beta in the lung tissue of the treatment group was significantly lower than that of the model group(P < 0.01).5.Metabolomic analysis of rat serum: 22 potential biomarkers were screened out.Compared with group J,serum concentrations of 3-methoxy-L-tyrosine,leukotriene B4,phenylacetylglycine,serine phosphate,prostaglandin E2,prostaglandin F2 alpha,thromboxane B2,11-deoxycorticosterone,19-oxtestosterone,prostaglandin F1 alpha,oleic acid and palmitic acid increased significantly in group M(P < 0.01).The concentration of prostaglandin E3 increased(P < 0.05),while the concentration of estrogen,sebacic acid,deoxycytidine,tyrosine,shikimic acid and cis-aconitic acid decreased significantly(P < 0.01).Concentrations of N-acetylglutamate acid,cytidine and vitamin E decreased(P < 0.05).Compared with group M,concentrations of N-acetylglutamate acid,estrogen,sebacic acid,deoxycytidine,cytidine,tyrosine,shikimic acid,cis-aconitic acid and vitamin E increased significantly in group Z(P < 0.01).Concentrations of 3-methoxy-L-tyrosine,leukotriene B4,phenylacetylglycine,serine phosphate and prostaglandin E2 increased significantly(P < 0.01).The concentrations of prostaglandin F2 alpha,thromboxane B2,11-deoxycorticosterone,19-oxytestosterone,prostaglandin E3,prostaglandin F1 alpha,oleic acid and palmitic acid decreased significantly(P < 0.01).There are 15 related metabolic pathways,including arachidonic acid metabolism,fatty acid biosynthesis,pyrimidine metabolism,citric acid cycle(TCA cycle),steroid hormone biosynthesis,mitochondrial fatty acid elongation,phenylalanine,tyrosine and tryptophan biosynthesis,panquinone and other terpenoids quinone biosynthesis,phenylalanine metabolism,glycine,serine and threonine generation.Metabolism of metabolism,acetaldehyde and dicarboxylic acid,fatty acid metabolism,cysteine and methionine metabolism,aminoacyl tRNA biosynthesis,arginine and proline metabolism.Seven metabolic pathways were closely related according to the magnitude of the impact value,which were in turn: biosynthesis of phenylalanine,tyrosine and tryptophan,citric acid cycle(TCA cycle),arachidonic acid metabolism,steroid hormone biosynthesis,fatty acid metabolism,glycine,serine and threonine metabolism,pyrimidine metabolism.The abnormal metabolisms involved are lipid metabolism(arachidonic acid metabolism,steroid metabolism,stearic acid metabolism,other lipid metabolism),energy metabolism,and others(ribonucleic acid and deoxyribonucleic acid synthesis,pyrimidine metabolism).conclusion1.HTF has a good intervention effect on SAP rats induced by impro ved suture embolization combined with tracheal injection,which can reduc e the neurological function score of SAP rats,reduce the inflammation of brain and lung tissues of SAP rats,reduce the contents of IL-1beta,ET-1 and IL-1beta in serum and lung tissues of SAP rats,and HTF has antiinflammation and improvement of ischemia and hypoxia in brain and lung tissues of SAP rats.2.The serum metabolic profiles of SAP rat model induced by modifi ed suture Embolism Combined with tracheal injection changed significantly.The main manifestations were that the serum levels of 22 biomarkers we re up-regulated or down-regulated.There were 15 disturbed metabolic path ways,7 of which were closely related,including lipid metabolism and en ergy metabolism.The mechanism of anti-inflammation and improvement o f hypoxic-ischemic effects of HTF on brain and lung tissues of SAP rats may be related to the recovery of abnormal metabolism by regulating the above related pathways and reversing the level of serum metabolic mark ers in SAP rats.