#METABOLOMICS WORKBENCH XIAODAN9979_20250914_212838 DATATRACK_ID:6427 STUDY_ID:ST004196 ANALYSIS_ID:AN006974 PROJECT_ID:PR002645
VERSION             	1
CREATED_ON             	September 17, 2025, 8:41 am
#PROJECT
PR:PROJECT_TITLE                 	High Fluoride Exposure Disrupts Gut Microbiota and Induces Intestinal Barrier
PR:PROJECT_TITLE                 	Damage via RhoA/ROCK-Mediated Cytoskeletal Remodeling
PR:PROJECT_SUMMARY               	Fluoride is an environmental pollutant that causes intestinal barrier damage,
PR:PROJECT_SUMMARY               	but the role of the "gut microbiota-RhoA/ROCK-cytoskeleton" axis in this process
PR:PROJECT_SUMMARY               	remains unclear. The aim of this project is to investigate the damage mechanism
PR:PROJECT_SUMMARY               	of high fluoride exposure on the intestinal barrier in mice. Focusing on the
PR:PROJECT_SUMMARY               	core objective of how high fluoride affects the intestinal barrier through the
PR:PROJECT_SUMMARY               	gut microbiota-mediated "RhoA/ROCK - cytoskeleton remodeling" axis, the study
PR:PROJECT_SUMMARY               	combines metagenomic approaches and leverages untargeted metabolomic analysis to
PR:PROJECT_SUMMARY               	reveal the gut microbiota dysbiosis and significant changes in intestinal
PR:PROJECT_SUMMARY               	metabolites induced by high fluoride. It clarifies the variation of
PR:PROJECT_SUMMARY               	cytoskeleton-related differential metabolites and screens out the key bacterial
PR:PROJECT_SUMMARY               	species that are strongly associated with the phenotypic characteristics of
PR:PROJECT_SUMMARY               	intestinal barrier damage, cytoskeletal core proteins, key tight junction
PR:PROJECT_SUMMARY               	proteins, and RhoA/ROCK pathway molecules in mice. In doing so, this research
PR:PROJECT_SUMMARY               	provides a new perspective for studying the mechanism of fluoride-induced
PR:PROJECT_SUMMARY               	intestinal barrier damage and lays a foundation for the prevention of
PR:PROJECT_SUMMARY               	fluoride-related intestinal diseases.
PR:INSTITUTE                     	Sichuan Agricultural University
PR:DEPARTMENT                    	College Of Veterinary Medicine
PR:LABORATORY                    	Animal Microecology Institute, Sichuan Agricultural University
PR:LAST_NAME                     	DanDan
PR:FIRST_NAME                    	Wang
PR:ADDRESS                       	Huimin Road, Wenjiang District, Chengdu, Sichuan, 611100, China
PR:EMAIL                         	1150784177@qq.com
PR:PHONE                         	17656691293
#STUDY
ST:STUDY_TITLE                   	High fluoride exposure disrupts the intestinal microbiota and induces intestinal
ST:STUDY_TITLE                   	barrier damage
ST:STUDY_SUMMARY                 	Our study establishes an acute high-fluoride exposure model by gavaging
ST:STUDY_SUMMARY                 	3-week-old C57BL/6J mice with 0 (control, C), 12 (low fluoride, FL), or 24 mg/kg
ST:STUDY_SUMMARY                 	(high fluoride, FH) sodium fluoride (NaF) for 8 weeks (n=10 per group), with
ST:STUDY_SUMMARY                 	standardized housing conditions (22±2℃, 12 h light/dark cycle). After
ST:STUDY_SUMMARY                 	euthanasia, ileal tissues, ileal contents, and serum were collected for
ST:STUDY_SUMMARY                 	histological, molecular, metagenomic, and non-targeted metabolomic analyses.
ST:STUDY_SUMMARY                 	Histological and biochemical results showed that high fluoride (FH group)
ST:STUDY_SUMMARY                 	increased ileal crypt depth, elevated serum intestinal permeability indicators
ST:STUDY_SUMMARY                 	(LPS, DAO, D-LA), and reduced ileal antioxidant capacity (decreased T-AOC, GSH,
ST:STUDY_SUMMARY                 	SOD; increased H₂O₂). Immunofluorescence, Western blotting, and transmission
ST:STUDY_SUMMARY                 	electron microscopy revealed that high fluoride activated the RhoA/ROCK pathway
ST:STUDY_SUMMARY                 	(upregulated RhoA, ROCK1/2, p-MLC), induced abnormal rearrangement and
ST:STUDY_SUMMARY                 	aggregation of F-actin (P<0.001 vs C), and disrupted the apical junctional
ST:STUDY_SUMMARY                 	complex (AJC)—manifested by downregulated expression and cytoplasmic
ST:STUDY_SUMMARY                 	translocation of ZO-1, Claudin-1, and β-catenin. Metagenomic analysis of ileal
ST:STUDY_SUMMARY                 	contents indicated high fluoride-induced gut microbiota dysbiosis, with reduced
ST:STUDY_SUMMARY                 	abundance of Akkermansia muciniphila and enrichment of Lactobacillus johnsonii;
ST:STUDY_SUMMARY                 	two key species (Bifidobacterium sp. SO1 and Schaalia turicensis) were screened,
ST:STUDY_SUMMARY                 	showing strong correlations with intestinal barrier phenotypes, RhoA/ROCK
ST:STUDY_SUMMARY                 	pathway molecules, and cytoskeleton-related metabolites. Non-targeted
ST:STUDY_SUMMARY                 	metabolomics identified 620 differential metabolites, with 11 linked to
ST:STUDY_SUMMARY                 	cytoskeleton function; high fluoride enriched linoleic acid metabolism
ST:STUDY_SUMMARY                 	(upregulated 9-HODE, 13-KODE; P<0.05 vs C) and sphingolipid metabolism
ST:STUDY_SUMMARY                 	(downregulated sphingosine) pathways. These findings confirm that high fluoride
ST:STUDY_SUMMARY                 	impairs the mouse intestinal barrier via the gut microbiota-mediated
ST:STUDY_SUMMARY                 	"RhoA/ROCK-cytoskeleton remodeling" axis, providing novel insights into
ST:STUDY_SUMMARY                 	fluoride-induced intestinal toxicity mechanisms and a basis for preventing
ST:STUDY_SUMMARY                 	fluoride-related intestinal diseases.
ST:INSTITUTE                     	Animal Microecology Institute, Sichuan Agricultural University
ST:DEPARTMENT                    	College Of Veterinary Medicine
ST:LABORATORY                    	Animal Microecology Institute, Sichuan Agricultural University
ST:LAST_NAME                     	DanDan
ST:FIRST_NAME                    	Wang
ST:ADDRESS                       	Huimin Road, Wenjiang District, Chengdu, Sichuan, 611100, China
ST:EMAIL                         	1150784177@qq.com
ST:PHONE                         	17656691293
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#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           	-	C-1	experimental factor(s):gavage with normal saline | Sample source:mouse ileum	RAW_FILE_NAME(POS)=C-1.raw; RAW_FILE_NAME(NEG)=C-1.raw
SUBJECT_SAMPLE_FACTORS           	-	C-2	experimental factor(s):gavage with normal saline | Sample source:mouse ileum	RAW_FILE_NAME(POS)=C-2.raw; RAW_FILE_NAME(NEG)=C-2.raw
SUBJECT_SAMPLE_FACTORS           	-	C-3	experimental factor(s):gavage with normal saline | Sample source:mouse ileum	RAW_FILE_NAME(POS)=C-3.raw; RAW_FILE_NAME(NEG)=C-3.raw
SUBJECT_SAMPLE_FACTORS           	-	C-4	experimental factor(s):gavage with normal saline | Sample source:mouse ileum	RAW_FILE_NAME(POS)=C-4.raw; RAW_FILE_NAME(NEG)=C-4.raw
SUBJECT_SAMPLE_FACTORS           	-	C-5	experimental factor(s):gavage with normal saline | Sample source:mouse ileum	RAW_FILE_NAME(POS)=C-5.raw; RAW_FILE_NAME(NEG)=C-5.raw
SUBJECT_SAMPLE_FACTORS           	-	C-6	experimental factor(s):gavage with normal saline | Sample source:mouse ileum	RAW_FILE_NAME(POS)=C-6.raw; RAW_FILE_NAME(NEG)=C-6.raw
SUBJECT_SAMPLE_FACTORS           	-	FL-1	experimental factor(s):gavage with low-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FL-1.raw; RAW_FILE_NAME(NEG)=FL-1.raw
SUBJECT_SAMPLE_FACTORS           	-	FL-2	experimental factor(s):gavage with low-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FL-2.raw; RAW_FILE_NAME(NEG)=FL-2.raw
SUBJECT_SAMPLE_FACTORS           	-	FL-3	experimental factor(s):gavage with low-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FL-3.raw; RAW_FILE_NAME(NEG)=FL-3.raw
SUBJECT_SAMPLE_FACTORS           	-	FL-4	experimental factor(s):gavage with low-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FL-4.raw; RAW_FILE_NAME(NEG)=FL-4.raw
SUBJECT_SAMPLE_FACTORS           	-	FL-5	experimental factor(s):gavage with low-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FL-5.raw; RAW_FILE_NAME(NEG)=FL-5.raw
SUBJECT_SAMPLE_FACTORS           	-	FL-6	experimental factor(s):gavage with low-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FL-6.raw; RAW_FILE_NAME(NEG)=FL-6.raw
SUBJECT_SAMPLE_FACTORS           	-	FH-1	experimental factor(s):gavage with high-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FH-1.raw; RAW_FILE_NAME(NEG)=FH-1.raw
SUBJECT_SAMPLE_FACTORS           	-	FH-2	experimental factor(s):gavage with high-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FH-2.raw; RAW_FILE_NAME(NEG)=FH-2.raw
SUBJECT_SAMPLE_FACTORS           	-	FH-3	experimental factor(s):gavage with high-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FH-3.raw; RAW_FILE_NAME(NEG)=FH-3.raw
SUBJECT_SAMPLE_FACTORS           	-	FH-4	experimental factor(s):gavage with high-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FH-4.raw; RAW_FILE_NAME(NEG)=FH-4.raw
SUBJECT_SAMPLE_FACTORS           	-	FH-5	experimental factor(s):gavage with high-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FH-5.raw; RAW_FILE_NAME(NEG)=FH-5.raw
SUBJECT_SAMPLE_FACTORS           	-	FH-6	experimental factor(s):gavage with high-concentration sodium fluoride | Sample source:mouse ileum	RAW_FILE_NAME(POS)=FH-6.raw; RAW_FILE_NAME(NEG)=FH-6.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	After the mice are euthanized, the intestinal tissues are immediately removed.
CO:COLLECTION_SUMMARY            	The ileal segments are separated, cut longitudinally to collect the contents of
CO:COLLECTION_SUMMARY            	the mouse ileum. The samples are stored in a -80℃ refrigerator.
CO:SAMPLE_TYPE                   	Ileum
#TREATMENT
TR:TREATMENT_SUMMARY             	The control group (C) was given 0.2 ml of normal saline by gavage; the
TR:TREATMENT_SUMMARY             	low-concentration sodium fluoride group (FL) was given an equal volume of 12
TR:TREATMENT_SUMMARY             	mg/kg sodium fluoride solution by gavage; and the high-concentration sodium
TR:TREATMENT_SUMMARY             	fluoride group (FH) was given an equal volume of 24 mg/kg sodium fluoride
TR:TREATMENT_SUMMARY             	solution by gavage.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	After collecting the mouse ileal contents, they were stored at -80°C.
SP:SAMPLEPREP_SUMMARY            	Subsequently, the methanol-water extraction method was adopted: using different
SP:SAMPLEPREP_SUMMARY            	proportions of methanol and water (e.g., 80% methanol aqueous solution), the
SP:SAMPLEPREP_SUMMARY            	mouse ileal content samples were subjected to operations such as vortex
SP:SAMPLEPREP_SUMMARY            	oscillation and ultrasonic treatment at low temperature to promote the release
SP:SAMPLEPREP_SUMMARY            	of polar and moderately polar metabolites in the samples into the extract.
SP:SAMPLEPREP_SUMMARY            	Methanol can precipitate proteins, avoiding protein interference with subsequent
SP:SAMPLEPREP_SUMMARY            	metabolite analysis, and effectively extract various polar metabolites such as
SP:SAMPLEPREP_SUMMARY            	amino acids, organic acids, and carbohydrates. The composition of metabolites
SP:SAMPLEPREP_SUMMARY            	was analyzed, and a comprehensive analysis was performed.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Normal phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 μm)
CH:SOLVENT_A                     	100% Water; 0.1% Formic acid
CH:SOLVENT_B                     	100% Acetonitrile; 0.1% Formic acid
CH:FLOW_GRADIENT                 	0-1 min:5%B; 1-4.7 min:5%-95%B; 4.7-6 min:95%B; 6-6.1 min:95%-5%B; 6.1-8.5 min:5%B.
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	40℃
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Exploris 120
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	A Thermo Orbitrap Exploris 120 mass spectrometer was used to collect DDA mass
MS:MS_COMMENTS                   	spectrometry data in positive and negative ion modes respectively under the
MS:MS_COMMENTS                   	control of software Xcalibur (version: 4.7, Thermo). The HESI source was
MS:MS_COMMENTS                   	employed, with a spray voltage of 3.5 kV/-3.0 kV, sheath gas of 40 arb,
MS:MS_COMMENTS                   	auxiliary gas of 10 arb, capillary temperature of 320°C, auxiliary gas
MS:MS_COMMENTS                   	temperature of 300°C, first - order resolution of 60,000, scanning range of 70
MS:MS_COMMENTS                   	- 1000 m/z, AGC Target Standard, Max IT 100 ms. The top 4 ions in response were
MS:MS_COMMENTS                   	selected for secondary fragmentation, with a dynamic exclusion time of 4 s,
MS:MS_COMMENTS                   	secondary resolution of 15,000, HCD collision energy of 30%, AGC Target
MS:MS_COMMENTS                   	Standard, and Max IT Auto. All formal samples and QC samples were analyzed on
MS:MS_COMMENTS                   	the instrument according to the above - mentioned chromatography and mass
MS:MS_COMMENTS                   	spectrometry methods. Before formal injection, 2 - 4 needles of QC samples were
MS:MS_COMMENTS                   	injected for system equilibration. During the injection process, one needle of
MS:MS_COMMENTS                   	QC sample was injected every 5 - 10 samples. If there were 5 or fewer samples,
MS:MS_COMMENTS                   	no QC was performed, which was used for subsequent data evaluation and quality
MS:MS_COMMENTS                   	control.
MS:MS_RESULTS_FILE               	ST004196_AN006974_Results.txt	UNITS:peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END