| Stroke is one of three leading causes of mortality throughout the world.Few drugs have been approved for the treatment of stroke nowadays,thus there is an urgent need to develop new drugs for stroke treatments.Previous researches have proved the protective effects of isosteviol sodium(STV-Na)against stroke.However,the protective mechanism of STV-Na against stroke was still unclear.In this work,the metabolomics study via the ultra-high performance liquid chromatography coupling with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)and the lipidomics study by the ultra-high performance supercritical fluid chromatography coupling with ion-trap and time-of-flight tandem mass spectrometry(UHSFC-IT-TOF/MS)were established to investigate the metabolite profiles and the lipid profiles of stroke rats with STV-Na treatment for the first time.The possible mechanism of STV-Na was further elucidated.Moreover,a robust and direct endogenous quantitative method using background subtracting calibration curves(BSCC)by liquid chromatography-tandem mass spectrometry was firstly developed for the determination of endogenous lipid mediators in ischemic stroke rats.The main contents and results of the study were as follows:1.The middle cerebral artery occlusion/reperfusion(MCAO/R)rat model was established to imitate ischemic stroke.The evaluation of rat models included the monitoring of regional cerebral blood flow(rCBF),the neurobehavioral abnormality evaluation and the 2,3,5-Triphenyte-trazoliumchloride(TTC)staining of brain slices.The results illustrated the success of the model construction,and further indicated that STV-Na as well as EDA had protective effects on stroke and STV-Na showed the better effect on reducing the infarction.2.The metabolomics study based on UHPLC-Q-TOF/MS was established and employed to investigate the protective effects of STV-Na.The metabolomics study indicated the protective effects of STV-Na against stroke and further explored the related pathways associated with the possible mechanism of STV-Na.The reliability of the analytical method was demonstrated.Based on the multivariate analysis and identification of the differential metabolites,18 differential metabolites that presented significant differences between the sham group and the model group were found and identified,including lipids and nucleosides.With the treatment of STV-Na,18 differential metabolites in stroke rats could return towards the normal level.Among them,12 metabolites were significantly reversed to the normal levels by STV-Na.Pathway analysis indicated that altered lipid metabolisms play an essential role in stroke process,the protective effects of STV-Na might be related to the regulation of several metabolic pathways including glycerophospholipid metabolism,arachidonic acid metabolism,linolenic acid metabolism and nucleotide metabolism.3.The lipidomics study based on UHSFC-IT-TOF/MS was estavlished and employed to investigate the protective effects of STV-Na.The lipidomics study indicated the protective effects of STV-Na against stroke and further explored the related pathways associated with the possible mechanism of STV-Na.The UHSFC-IT-TOF/MS-based method with a qualified repeatability achieved a faster separation of various lipids than the LC-MS-based method.Based on the multivariate analysis and identification of the differential lipids,15 differential lipids that presented significant differences between the sham group and the model group were found and identified.With the treatment of STV-Na,15 differential lipids in stroke rats could return towards the normal level.Among them,6 lipids were significantly reversed to the normal levels by STV-Na.Pathway analysis indicated that the protective effects of STV-Na might be related to the regulation of several metabolic pathways including glycerophospholipid metabolism,arachidonic acid metabolism and sphingolipid metabolism.4.Besides,a direct and robust method using BSCC for absolute quantification of endogenous lipids by LC-MS/MS was developed.The precision with the relative standard deviation(RSD)was less than 13.8%,the accuracy with the relative error(RE)was within ± 15.0%,the recoveries of this method were in the range of 50.3%–98.3%,and the matrix effects were ranged from 91.1% to 114.9%,which were accorded with relevant regulations of guidance for bioanalytical method validation.In addition,BSCC were further verified by validation factors ranged from 90.3% to 110.9%.Absolute quantification without surrogate matrix and surrogate analyte could be achieved by using BSCC.The method was successfully applied to the determination of seven endogenous lipid mediators in ischemic stroke rats.The results indicated that the levels of inflammation-related lipid mediators were up-regulated in stroke and STV-Na could significantly reduce the levels of inflammation-related lipid mediators of stroke rats to inhibit inflammation. |
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