| Purpose:The prototype components and metabolites of Qi-Yu-San-longdecoctio(QYSLD)in rat urine were analyzed by ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)combined with targeted screening strategy,and their dynamic changes in rat urine were observed.To establish a rat model of lung adenocarcinoma,using metabonomics technology to explore the endogenous small molecular metabolites involved in the inhibition of lung adenocarcinoma by QYSLD,and systematically explain the mechanism of QYSLD inhibition of lung adenocarcinoma.Method:1.Twelve male SD rats were randomly divided into blank group(n=6)and QYSLD group(n=6).The rats in blank group were given normal saline and the rats in QYSLD group were given intragastric administration according to the dose of 25m L/kg.After continuous intragastric administration for 3 days,urine samples were collected at 0,2,4,4,8,12and 12 hours,and the urine of rats in the blank group was collected as blank control.The urine of rats collected at different time was detected by UPLC-QTOF-MS~E.The chemical composition database of QYSLD was established to quickly screen the prototype components of QYSLD in rat urine,and then the exact molecular weights of the potential metabolites of these prototype components were calculated by Excel according to their possible metabolic pathways.The ion flow was extracted by the predicted m/z value,and the metabolites of QYSLD in rat urine were identified by retention time,excimer ions and fragment ions.2.The rat model of lung adenocarcinoma was established by intraperitoneal injection of uratan in male Wistar rats.After successful modeling,the rats were divided into two groups:model group(n=10)and QYSLD group(n=10).The QYSLD group was given intragastric administration according to the dose of 14.07g/kg for 14 days.Before sampling,the rats were anesthetized with uratan,the blood was taken from the abdominal aorta,the supernatant was collected,the lung tissue was stripped off,the appearance of the lung tissue was observed,the morphological changes were investigated,and the left lobe of the same part of the lung was stained with HE staining to observe the effect of QYSLD on the histopathological changes of lung adenocarcinoma rats.The serum and lung tissue of rats were taken as the research objects,and the differential metabolites among control group,model group and QYSLD group were detected by UPLC-QTOF-MS metabonomics technique to reveal the endogenous metabolites involved in the inhibition of non-small cell lung cancer by QYSLD and analyze their biological significance.Results:1.After administration of QYSLD,20 prototype components and 65metabolites were identified from rat urine.The main metabolic reactions of QYSLD in vivo include oxidation,reduction,decarboxylation,hydrolysis,demethylation,glucuronylation,sulfation,methylation,deglycosylation and acetylation.Among the identified chemical components,14 prototype components and 58 metabolites were eliminated for a long time,5prototype components and 2 metabolites existed in a short interval,and 1prototype component and 5 metabolites showed the"appearance-disappearance-reappearance"phenomenon in rat urine.2.The morphological observation results of lung tissue appearance showed that compared with the normal control rats,both lungs of rats in the model group were swollen and large white tumor nodules were visible on the tissue surface.After QYSLD administration,lung swelling was improved and tumor nodules were reduced.HE staining results showed that compared with normal control group,normal alveolar structure disappeared,alveolar wall thickened and inflammatory cells infiltrated in model group.Compared with the model group,after QYSLD intervention,the alveolar wall was less thickened and inflammatory infiltration was significantly reduced3.In the metabolomics study results,the principal component analysis diagram showed that there was a certain degree of dispersion between the model group and the normal control group,and between the QYSLD administration group and the model group.Compared with the control group,a total of 30 different metabolites were identified in serum and lung tissue samples of lung adenocarcinoma model rats,23 of which were regulated by QYSLD,including 7 metabolites up regulated and 16metabolites down regulated.The differential metabolites of QYSLD intervention are mainly involved in the metabolism of glycerophospholipids,sphingolipids and arachidonic acid.Conclusion:In this study,UPLC-QTOF-MS technology combined with targeted screening strategy was used to identify 20 prototype components and 65metabolites of QYSLD in rat urine and identify their dynamic changes in urine.QYSLD has an inhibitory effect on lung adenocarcinoma,which may be related to its regulation of glycerolphospholipid metabolism,sphingolipid metabolism and arachidonic acid metabolism. |
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