Analysis Of Mechanism Of Lung Injury Induced By Siegesbeckia Pubescens Water Elution Section On Rats By ~1H-NMR Metabolomics

Objective:¹H-NMR Metabonomics and multivariate statistical analysis methods were used to study the dynamic changes of endogenous metabolites in serum and lung tissue of rats by Siegesbeckia pubescens water elution section?SWES?,to explore the possible mechanism of lung injury and to find the sensitive markers in serum and lung tissue.It is provide an experimental basis for the rapid diagnosis,prognosis judgment and safe use of drugs in the clinical treatment of lung damage caused by the SWES.Methods:1.36 male SPF male SD rats were randomly divided into 3 groups:normal group,SWES high-dose administration group(0.500 g·kg^-1·d^-1),low-dose administration group(0.125 g·kg^-1·d^-1),every group have 12 rats.The administration period is 4 weeks,and it is administered by intragastric administration.The normal group is given an equal volume of saline.During the administration period,observe the general condition of rats in each group,measure body weight every week and calculate lung index.After 4 weeks of administration,6 rats in each group were killed and lung tissue was collected to observe the morphology and pathological changes of the lung tissue.Two weeks after drug withdrawal,the above test was repeated for each group with drug withdrawal.2.Collect the serum and lung tissues of rats in the normal group and the drug-administered group,check their metabolic fingerprints by ¹H-NMR and compare them;Use multivariate statistical analysis methods:partial least squares discriminant analysis?PLS-DA?And Orthogonal Partial Least Squares Discriminant Analysis?OPLS-DA?to analyze the difference in metabolite changes between the normal group and the drug-administered group of rats;according to variable importance projection?VIP?and t-test screening for the different metabolites between the drug-administered group and the normal group;according to the changes in the relative content of the differential metabolites before and after administration,reference literature reports,combined with the disorder of the metabolic pathway caused by lung injury to speculate the mechanism of lung injury,and find sensitive markers of lung injury.Results:1.Measurement of body weight and lung index After 4 weeks of administration,body weight began to decrease significantly,and gradually recovered after 2 weeks of withdrawal.There were no obvious abnormalities in the low-dose group and the normal group.2.Pathological examination The lung specimens of each group showed no obvious swelling and bleeding points.After HE staining of the pathological section,the microscope showed that the capsule in the normal group was intact,the blood vessels of the septum were slightly expanded,and a small amount of lymphocyte infiltration was seen;there was no obvious abnormality in the low-dose group,and the rats in the high-dose group showed interstitial pneumonia,which showed an enlarged alveolar diaphragm In some areas,the interstitial and alveolar have a large number of red blood cells;after 2 weeks of withdrawal,the lung septum and alveolar epithelial proliferative lesions are reduced,but there is still a small amount of red blood cells exuded in the alveoli,suggesting that the lung injury is reversible.3.Metabolomics study The results of PLS-DA analysis showed that the metabolic profiles of the drug-administered group and the normal group were clearly separated in the two samples,indicating that the administration of SWES caused abnormalities of endogenous small molecule metabolites in rats.In the high-dose group of lung tissue,after2 weeks of drug withdrawal,the metabolic profile approached the normal group,suggesting that the degree of lung injury caused by the drug was partially reversed,which was consistent with pathological examination.?The two-sample permutation test shows that the intercept of the regression line where Q² is located on the Y axis is less than 0,proving that the model can be used in subsequent studies?.Select the most significantly separated high-dose group and normal group to establish the OPLS-DA score chart and S-plot chart,with VIP>1 as the standard,a total of 11 were identified in the serum and lung tissues related to the lung injury caused by this drug.4.Differential metabolites research Four differential metabolites were identified in the serum,namely acetate,trimethylamine oxide,glycine,and myo-inositol.Compared with the normal group,the acetate content of the high-dose group increased after 4 weeks of administration?P<0.05?,and the content of TMAO,glycine,and myo-inositol decreased?P<0.05,P<0.01?.After 2 weeks of drug withdrawal,all metabolic products had no significant callback?P>0.05?.Nine differential metabolites were identified in the lung tissue,namely acetate,glycine,lactate,pyruvate,dimethylamine,glutamate,PC,GPC and xanthine.After 4 weeks of administration,the content of lactate,acetate and PC in the high-dose group increased compared with the normal group?P<0.01?,pyruvate,dimethylamine,glutamate,GPC,glycine and xanthine content decreased?P<0.05,P<0.01?.After 2 weeks of drug withdrawal,except for acetate,PC,GPC,and glycine,most metabolite levels in lung tissue were significantly callback?P<0.05,P<0.01?.5.Lung injury mechanism study The lung injury caused by SWES are mainly related to the disorder of metabolic pathways such as pyruvate metabolism and glutamate metabolism.The main reason may be abnormal energy metabolism of the lung and oxidative stress damage in rats Rat lung inflammation occurs.Conclusion:In this experiment,metabolomics was used for the first time to analyze the changes of metabolites in the serum and lung tissues of SWES,to explore the mechanism of lung injury,and to find sensitive markers of lung injury.The experimental results suggest that lung tissue is one of the target organs of the drug's injury.Lung injury shows reversible changes after drug withdrawal,which is consistent with lung pathological changes,and lung injury is significantly correlated with differential metabolites.Through the study of the metabolic pathway disorder caused by lung injury,it is speculated that the lung injury caused by SWES is mainly related to the disorder of metabolic pathways such as pyruvate metabolism and glutamate metabolism,suggesting that the drug mainly caused early lung injury.Abnormal energy metabolism of the lung tissue and oxidative stress injury in rats.The differential metabolites measured in the lung tissue may become a potential lung injury sensitive substance,which needs further study.