#METABOLOMICS WORKBENCH shanpingwang_20200929_175706_mwtab.txt DATATRACK_ID:2190 STUDY_ID:ST001499 ANALYSIS_ID:AN002482 PROJECT_ID:PR001015 VERSION 1 CREATED_ON October 7, 2020, 9:47 am #PROJECT PR:PROJECT_TITLE Metabolomics reveals the protective effect of isosteviol sodium against multiple PR:PROJECT_TITLE organ injury in septic mice PR:PROJECT_SUMMARY Sepsis is a severe inflammatory disorder that can lead to multiple organ injury. PR:PROJECT_SUMMARY Isosteviol sodium (STV-Na) is a terpenoid derived from stevioside that exerts PR:PROJECT_SUMMARY anti-inflammatory, antioxidant and anticancer activities. However, the influence PR:PROJECT_SUMMARY of STV-Na on sepsis remains unknown. Here, we assessed the potential effects of PR:PROJECT_SUMMARY STV-Na on sepsis and multiple organ injury induced by lipopolysaccharide (LPS). PR:PROJECT_SUMMARY We found that STV-Na increased the survival rate of mice treat with LPS, PR:PROJECT_SUMMARY significantly improved the functions of the heart, lung, liver, and kidney, and PR:PROJECT_SUMMARY reduced the production of inflammatory cytokines. Moreover, Multiorgan PR:PROJECT_SUMMARY metabolomics analysis demonstrated that glutathione metabolism, purine PR:PROJECT_SUMMARY metabolism, glycerophospholipid metabolism and pantothenate and CoA PR:PROJECT_SUMMARY biosynthesis, were significantly altered by STV-Na. This study provides novel PR:PROJECT_SUMMARY insights into the metabolite changes of multiple organ injury in septic mice, PR:PROJECT_SUMMARY which may help characterize the underlying mechanism and provide an improved PR:PROJECT_SUMMARY understanding of the therapeutic effects of STV-Na on sepsis. PR:INSTITUTE Guangdong University of Technology PR:LAST_NAME Wang PR:FIRST_NAME Shanping PR:ADDRESS No. 100, Waihuan Xilu, Guangzhou Higher Education Mega Center, Panyu District, PR:EMAIL shanpingwang@outlook.com PR:PHONE 15521002792 #STUDY ST:STUDY_TITLE Metabolomics reveals the protective effect of isosteviol sodium against multiple ST:STUDY_TITLE organ injury in septic mice - Sample Type: Heart, Ion Mode: Pos/Neg (part- ST:STUDY_TITLE Heart_ Pos/Neg) ST:STUDY_SUMMARY Sepsis is a severe inflammatory disorder that can lead to multiple organ injury. ST:STUDY_SUMMARY Isosteviol sodium (STV-Na) is a terpenoid derived from stevioside that exerts ST:STUDY_SUMMARY anti-inflammatory, antioxidant and anticancer activities. However, the influence ST:STUDY_SUMMARY of STV-Na on sepsis remains unknown. Here, we assessed the potential effects of ST:STUDY_SUMMARY STV-Na on sepsis and multiple organ injury induced by lipopolysaccharide (LPS). ST:STUDY_SUMMARY We found that STV-Na increased the survival rate of mice treat with LPS, ST:STUDY_SUMMARY significantly improved the functions of the heart, lung, liver, and kidney, and ST:STUDY_SUMMARY reduced the production of inflammatory cytokines. Moreover, Multiorgan ST:STUDY_SUMMARY metabolomics analysis demonstrated that glutathione metabolism, purine ST:STUDY_SUMMARY metabolism, glycerophospholipid metabolism and pantothenate and CoA ST:STUDY_SUMMARY biosynthesis, were significantly altered by STV-Na. This study provides novel ST:STUDY_SUMMARY insights into the metabolite changes of multiple organ injury in septic mice, ST:STUDY_SUMMARY which may help characterize the underlying mechanism and provide an improved ST:STUDY_SUMMARY understanding of the therapeutic effects of STV-Na on sepsis. Mice are randomly ST:STUDY_SUMMARY assigned to 4 groups in study design. Control: saline + saline Model: saline + ST:STUDY_SUMMARY LPS; Treatment: STV-Na + LPS; Positive: dexamethasone (Dex) + LPS. Drugs were ST:STUDY_SUMMARY administered i.p. Six hours after LPS injection, mice were sacrificed. And blood ST:STUDY_SUMMARY and tissues (heart, lung, liver, spleen and kidney) were subjected to UHPLC-TIMS ST:STUDY_SUMMARY TOF MS/MS-based metabolomics analyses. ST:INSTITUTE Guangdong University of Technology ST:LAST_NAME Wang ST:FIRST_NAME Shanping ST:ADDRESS No. 100, Waihuan Xilu, Guangzhou Higher Education Mega Center, Panyu District, ST:EMAIL shanpingwang@outlook.com ST:PHONE 15521002792 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS wild-type Ctrl-1 Groups:Control RAW_FILE_NAME=Ctrl-W-1 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-2 Groups:Control RAW_FILE_NAME=Ctrl-W-2 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-3 Groups:Control RAW_FILE_NAME=Ctrl-W-3 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-4 Groups:Control RAW_FILE_NAME=Ctrl-W-4 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-5 Groups:Control RAW_FILE_NAME=Ctrl-W-5 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-6 Groups:Control RAW_FILE_NAME=Ctrl-W-6 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-7 Groups:Control RAW_FILE_NAME=Ctrl-W-7 SUBJECT_SAMPLE_FACTORS wild-type Ctrl-8 Groups:Control RAW_FILE_NAME=Ctrl-W-8 SUBJECT_SAMPLE_FACTORS wild-type LPS-1 Groups:Model RAW_FILE_NAME=LPS-W-1 SUBJECT_SAMPLE_FACTORS wild-type LPS-2 Groups:Model RAW_FILE_NAME=LPS-W-2 SUBJECT_SAMPLE_FACTORS wild-type LPS-3 Groups:Model RAW_FILE_NAME=LPS-W-3 SUBJECT_SAMPLE_FACTORS wild-type LPS-4 Groups:Model RAW_FILE_NAME=LPS-W-4 SUBJECT_SAMPLE_FACTORS wild-type LPS-5 Groups:Model RAW_FILE_NAME=LPS-W-5 SUBJECT_SAMPLE_FACTORS wild-type LPS-6 Groups:Model RAW_FILE_NAME=LPS-W-6 SUBJECT_SAMPLE_FACTORS wild-type LPS-7 Groups:Model RAW_FILE_NAME=LPS-W-7 SUBJECT_SAMPLE_FACTORS wild-type LPS-8 Groups:Model RAW_FILE_NAME=LPS-W-8 SUBJECT_SAMPLE_FACTORS wild-type STV-1 Groups:Treatment RAW_FILE_NAME=STV-W-1 SUBJECT_SAMPLE_FACTORS wild-type STV-2 Groups:Treatment RAW_FILE_NAME=STV-W-2 SUBJECT_SAMPLE_FACTORS wild-type STV-3 Groups:Treatment RAW_FILE_NAME=STV-W-3 SUBJECT_SAMPLE_FACTORS wild-type STV-4 Groups:Treatment RAW_FILE_NAME=STV-W-4 SUBJECT_SAMPLE_FACTORS wild-type STV-5 Groups:Treatment RAW_FILE_NAME=STV-W-5 SUBJECT_SAMPLE_FACTORS wild-type STV-6 Groups:Treatment RAW_FILE_NAME=STV-W-6 SUBJECT_SAMPLE_FACTORS wild-type STV-7 Groups:Treatment RAW_FILE_NAME=STV-W-7 SUBJECT_SAMPLE_FACTORS wild-type STV-8 Groups:Treatment RAW_FILE_NAME=STV-W-8 SUBJECT_SAMPLE_FACTORS wild-type STV-9 Groups:Treatment RAW_FILE_NAME=STV-W-9 SUBJECT_SAMPLE_FACTORS wild-type Dex-1 Groups:Positive RAW_FILE_NAME=Dex-W-1 SUBJECT_SAMPLE_FACTORS wild-type Dex-2 Groups:Positive RAW_FILE_NAME=Dex-W-2 SUBJECT_SAMPLE_FACTORS wild-type Dex-3 Groups:Positive RAW_FILE_NAME=Dex-W-3 SUBJECT_SAMPLE_FACTORS wild-type Dex-4 Groups:Positive RAW_FILE_NAME=Dex-W-4 SUBJECT_SAMPLE_FACTORS wild-type Dex-5 Groups:Positive RAW_FILE_NAME=Dex-W-5 SUBJECT_SAMPLE_FACTORS wild-type Dex-6 Groups:Positive RAW_FILE_NAME=Dex-W-6 SUBJECT_SAMPLE_FACTORS wild-type Dex-7 Groups:Positive RAW_FILE_NAME=Dex-W-7 SUBJECT_SAMPLE_FACTORS wild-type Dex-8 Groups:Positive RAW_FILE_NAME=Dex-W-8 SUBJECT_SAMPLE_FACTORS wild-type Dex-9 Groups:Positive RAW_FILE_NAME=Dex-W-9 #COLLECTION CO:COLLECTION_SUMMARY Samples of blood were gathered in heparinized tubes and then spun down at 3000 CO:COLLECTION_SUMMARY rpm at 4 °C for 10 min to obtain supernatant of plasma samples for subsequent CO:COLLECTION_SUMMARY preparation. Heart, lung, liver, spleen and kidney samples were homogenized in a CO:COLLECTION_SUMMARY five-fold volume of normal saline, respectively, and spun down at 12,000 rpm at CO:COLLECTION_SUMMARY 4 °C for 20 min to acquire supernatants for further preparation. CO:SAMPLE_TYPE Heart #TREATMENT TR:TREATMENT_SUMMARY After being acclimatized for 1 week, the mice were separated, at random, into TR:TREATMENT_SUMMARY four groups: 1) saline + saline; 2) saline + LPS; 3) STV-Na+ LPS; and 4) TR:TREATMENT_SUMMARY dexamethasone (Dex) + LPS. Mice were intraperitoneally administered saline (0.1 TR:TREATMENT_SUMMARY mL/10 g) or STV-Na (5, 10, 20 mg/kg) and Dex (10 mg/kg) two times per day every TR:TREATMENT_SUMMARY 12 h for 3 consecutive days, and one hour after the first intraperitoneal TR:TREATMENT_SUMMARY injection on day 3, saline (0.1 mL/g body weight) or LPS from E. coli (0111: B4, TR:TREATMENT_SUMMARY 20 mg/kg) was intraperitoneally administered. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A total of 160 µL of MTBE solution (methyl-T-butyl-ether: methanol: water, SP:SAMPLEPREP_SUMMARY 6/3/1, v/v/v) was applied to 40 µL of the plasma or tissue homogenate SP:SAMPLEPREP_SUMMARY supernatant, vortexed for 30 min at 4°C and spun at 12,000 rpm for 30 min. Two SP:SAMPLEPREP_SUMMARY extract components were produced: an organic hydrophobic layer and a hydrophilic SP:SAMPLEPREP_SUMMARY layer. These two extracts were vacuum-dried and dissolved in 0.1% (v/v) formic SP:SAMPLEPREP_SUMMARY acid in water (45 µL), followed by analysis. The pooled quality control (QC) SP:SAMPLEPREP_SUMMARY samples including whole plasma and tissues were utilized for monitoring data SP:SAMPLEPREP_SUMMARY acquisition performance throughout the analysis. Finally, 6 duplicate QC samples SP:SAMPLEPREP_SUMMARY were prepared and injected at the start of the sequence, and after each of the SP:SAMPLEPREP_SUMMARY six tissue samples was inserted, the QC samples were added to determine system SP:SAMPLEPREP_SUMMARY stability. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separations were conducted utilizing a Waters BEH C18, 2.1 CH:CHROMATOGRAPHY_SUMMARY mm×50 mm 1.7 µm particle column with a Dionex Ultimate 3000 UHPLC system from CH:CHROMATOGRAPHY_SUMMARY Thermo Fisher Scientific (CA, USA). The mobile phase encompassed water with 0.1% CH:CHROMATOGRAPHY_SUMMARY v/v formic acid (A) and acetonitrile with 0.1% v/v formic acid (B). Columns were CH:CHROMATOGRAPHY_SUMMARY kept at 40°C and eluted using a linear gradient: 2-30% B at 0-4 min, 30-40% B CH:CHROMATOGRAPHY_SUMMARY at 4-5 min, 40% B at 5-8 min, 40-60% B at 8-10 min, 60-100% B at 10-17 min, 100% CH:CHROMATOGRAPHY_SUMMARY B at 17-19 min, 100-2% B at 19-19.1 min, and 2% B at 19.1-25 min. To increase CH:CHROMATOGRAPHY_SUMMARY the amount of metabolites and save experimental time, a new sampling method was CH:CHROMATOGRAPHY_SUMMARY used to detect both the organic phase and the aqueous phase extracts (Qiuhui CH:CHROMATOGRAPHY_SUMMARY Xuan et al., 2018; Shanping Wang et al., 2019): 5 µL of organic phase extracts CH:CHROMATOGRAPHY_SUMMARY were first loaded without running the elution gradient, which lasted for one CH:CHROMATOGRAPHY_SUMMARY minute at the initial mobile phase, and then 5 µL of the aqueous phase extracts CH:CHROMATOGRAPHY_SUMMARY were added to the same column in order to start running the elution gradient CH:CHROMATOGRAPHY_SUMMARY using a 0.4 mL/min flow rate. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker TIMS TOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MSMS Progenesis QI 2.1 software EZinfo 3.0 software MS:MS_RESULTS_FILE ST001499_AN002482_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END