#METABOLOMICS WORKBENCH shanpingwang_20200929_230106_mwtab.txt DATATRACK_ID:2196 STUDY_ID:ST001503 ANALYSIS_ID:AN002490 PROJECT_ID:000000
VERSION             	1
CREATED_ON             	October 9, 2020, 12:00 pm
#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 - Kidney
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.
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	Treatment:Control	RAW_FILE_NAME=Ctrl-W-1
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-2	Treatment:Control	RAW_FILE_NAME=Ctrl-W-2
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-3	Treatment:Control	RAW_FILE_NAME=Ctrl-W-3
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-4	Treatment:Control	RAW_FILE_NAME=Ctrl-W-4
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-5	Treatment:Control	RAW_FILE_NAME=Ctrl-W-5
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-6	Treatment:Control	RAW_FILE_NAME=Ctrl-W-6
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-7	Treatment:Control	RAW_FILE_NAME=Ctrl-W-7
SUBJECT_SAMPLE_FACTORS           	wild-type	Ctrl-8	Treatment:Control	RAW_FILE_NAME=Ctrl-W-8
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-1	Treatment:Model	RAW_FILE_NAME=LPS-W-1
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-2	Treatment:Model	RAW_FILE_NAME=LPS-W-2
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-3	Treatment:Model	RAW_FILE_NAME=LPS-W-3
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-4	Treatment:Model	RAW_FILE_NAME=LPS-W-4
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-5	Treatment:Model	RAW_FILE_NAME=LPS-W-5
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-6	Treatment:Model	RAW_FILE_NAME=LPS-W-6
SUBJECT_SAMPLE_FACTORS           	wild-type	LPS-7	Treatment:Model	RAW_FILE_NAME=LPS-W-7
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-1	Treatment:Treatment	RAW_FILE_NAME=STV-W-1
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-2	Treatment:Treatment	RAW_FILE_NAME=STV-W-2
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-3	Treatment:Treatment	RAW_FILE_NAME=STV-W-3
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-4	Treatment:Treatment	RAW_FILE_NAME=STV-W-4
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-5	Treatment:Treatment	RAW_FILE_NAME=STV-W-5
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-6	Treatment:Treatment	RAW_FILE_NAME=STV-W-6
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-7	Treatment:Treatment	RAW_FILE_NAME=STV-W-7
SUBJECT_SAMPLE_FACTORS           	wild-type	STV-8	Treatment:Treatment	RAW_FILE_NAME=STV-W-8
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-1	Treatment:Positive	RAW_FILE_NAME=Dex-W-1
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-2	Treatment:Positive	RAW_FILE_NAME=Dex-W-2
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-3	Treatment:Positive	RAW_FILE_NAME=Dex-W-3
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-4	Treatment:Positive	RAW_FILE_NAME=Dex-W-4
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-5	Treatment:Positive	RAW_FILE_NAME=Dex-W-5
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-6	Treatment:Positive	RAW_FILE_NAME=Dex-W-6
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-7	Treatment:Positive	RAW_FILE_NAME=Dex-W-7
SUBJECT_SAMPLE_FACTORS           	wild-type	Dex-8	Treatment:Positive	RAW_FILE_NAME=Dex-W-8
#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:COLLECTION_PROTOCOL_FILENAME  	shanpingwang_Collection_Protocol.docx
CO:SAMPLE_TYPE                   	Kidney
#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.
TR:TREATMENT_PROTOCOL_FILENAME   	shanpingwang_Treatment_Protocol.docx
#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.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	shanpingwang_Sampleprep_protocol.docx
#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)
CH:METHODS_FILENAME              	shanpingwang_Chromatography_Methods.docx
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:ANALYSIS_PROTOCOL_FILE        	shanpingwang_Analysis_Protocol.docx
#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               	ST001503_AN002490_Results.txt	UNITS:Intensity	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END