Summary of Study ST003817

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002388. The data can be accessed directly via it's Project DOI: 10.21228/M8ZK0F This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003817
Study Title	Irisin promotes bone tissue repair and intestinal protection in osteoporotic mice
Study Summary	In this study, we constructed a mouse model of osteoporosis by performing ovariectomies and administering irisin injections. The impact of ovariectomy was assessed based on changes in body and uterine weights. Bone microstructure analysis, HE staining, MASSON staining, and Goldner staining were employed to observe alterations in bone structure. Post-irisin injection, mice showed recovery in weight and restoration of bone structure, including the formation of new bone. The presence of bone-forming factors (ALP, BGP), bone resorption factors (CTX-1, TRACP-5b), and a bone protective protein (OPG) indicated that irisin could promote bone cell growth in bone tissue. Additionally, HE staining and immunohistochemical analysis on intestinal sections revealed that irisin restored the mucosal barrier of the intestinal wall and alleviated enlargement of intestinal epidermal cells. Analysis of IL-1β, IL-6, and TNF-α levels demonstrated irisin's role in reducing inflammation. Furthermore, sequencing and analysis of intestinal flora 16S microbial sequencing and blood metabolome in mice revealed that irisin mitigated the dysbiosis caused by osteoporosis and promoted flora growth, while enrichment analysis showed that irisin induced changes in basal metabolic levels. This study highlights the therapeutic potential of irisin in treating osteoporosis and its beneficial effects on the intestinal barrier and flora, suggesting that irisin enhances intestinal protection to foster bone tissue repair.
Institute	Huizhou Central People's Hospital
Department	Department of Traumatology and Orthopaedic Surgery, Orthopaedic Institute
Last Name	Zhang
First Name	Zhiwen
Address	No. 41, Eling North Road, Huicheng District, Huizhou City, Guangdong Province
Email	13794539802@163.com
Phone	13794539802
Submit Date	2025-03-03
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-04-21
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002388
Project DOI:	doi: 10.21228/M8ZK0F
Project Title:	Irisin promotes bone tissue repair and intestinal protection in osteoporotic mice
Project Summary:	In this study, we constructed a mouse model of osteoporosis by performing ovariectomies and administering irisin injections. The impact of ovariectomy was assessed based on changes in body and uterine weights. Bone microstructure analysis, HE staining, MASSON staining, and Goldner staining were employed to observe alterations in bone structure. Post-irisin injection, mice showed recovery in weight and restoration of bone structure, including the formation of new bone. The presence of bone-forming factors Alkaline phosphatase (ALP), Bone Gla Protein (BGP), bone resorption factors (CTX-1, TRACP-5b), and a bone protective protein Osteoprotegerin (OPG) indicated that irisin could promote bone cell growth in bone tissue. Additionally, HE staining and immunohistochemical analysis on intestinal sections revealed that irisin restored the mucosal barrier of the intestinal wall and alleviated enlargement of intestinal epidermal cells. Analysis of IL-1β, IL-6, and TNF-α levels demonstrated irisin's role in reducing inflammation. Furthermore, sequencing and analysis of intestinal flora 16S microbial sequencing and blood metabolome in mice revealed that irisin mitigated the dysbiosis caused by osteoporosis and promoted flora growth, while enrichment analysis showed that irisin induced changes in basal metabolic levels. This study highlights the therapeutic potential of irisin in treating osteoporosis and its beneficial effects on the intestinal barrier and flora, suggesting that irisin enhances intestinal protection to foster bone tissue repair.
Institute:	Huizhou Central People's Hospital
Department:	Department of Traumatology and Orthopaedic Surgery, Orthopaedic Institute
Last Name:	Zhang
First Name:	Zhiwen
Address:	No. 41, Eling North Road, Huicheng District, Huizhou City, Guangdong Province
Email:	13794539802@163.com
Phone:	13794539802

Subject:

Subject ID:	SU003951
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment
SA418786	OVX_2	feces	OVX + normal saline
SA418787	OVX_6	feces	OVX + normal saline
SA418788	OVX_5	feces	OVX + normal saline
SA418789	OVX_4	feces	OVX + normal saline
SA418790	OVX_3	feces	OVX + normal saline
SA418791	OVX_1	feces	OVX + normal saline
SA418792	r_irisin_2	feces	OVX + r-irisin
SA418793	r_irisin_6	feces	OVX + r-irisin
SA418794	r_irisin_5	feces	OVX + r-irisin
SA418795	r_irisin_4	feces	OVX + r-irisin
SA418796	r_irisin_3	feces	OVX + r-irisin
SA418797	r_irisin_1	feces	OVX + r-irisin
SA418798	Sham_1	feces	Sham + normal saline
SA418799	Sham_2	feces	Sham + normal saline
SA418800	Sham_3	feces	Sham + normal saline
SA418801	Sham_4	feces	Sham + normal saline
SA418802	Sham_5	feces	Sham + normal saline
SA418803	Sham_6	feces	Sham + normal saline
SA418780	P_QC1_240929222222	QC	Quality Control
SA418781	P_QC2_240930011652	QC	Quality Control
SA418782	P_QC3_240930035537	QC	Quality Control
SA418783	N_QC1_240930042723	QC	Quality Control
SA418784	N_QC2_240930072202	QC	Quality Control
SA418785	N_QC3_240930100044	QC	Quality Control

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO003944
Collection Summary:	Sample Collection Timing: Select an appropriate time for collection. For example, collect samples in the morning when mice are more active and have a relatively regular defecation pattern. Collection from cages: If using a clean cage, carefully transfer the mouse to a clean container. Then, using sterile forceps, pick up the fresh fecal pellets directly from the cage floor as soon as they are excreted. Collection from metabolic cages: In the case of metabolic cages, the fecal pellets will be collected in a separate compartment. Open the compartment and use sterile forceps to transfer the fecal samples to the pre - labeled collection tubes. Number of samples: Aim to collect at least 3 - 5 fecal pellets per mouse for each sample collection. If possible, collect samples from multiple time points for each mouse to increase the representativeness of the samples.
Sample Type:	Feces

Treatment:

Treatment ID:	TR003960
Treatment Summary:	In an environment with temperature control, 4 to 5 mice are housed in each cage, and the light/dark cycle is set at 12 hours. After a two-week adaptation period, 18 mice with a body weight ranging from 19.4 to 22.5 grams are selected and randomly divided into three groups: the OVX + r-irisin group, the OVX + normal saline group, and the Sham + normal saline group, with 6 mice in each group. Starting from the third day after the operation, a vaginal cell smear is taken from each animal once a day for 7 consecutive days. A successful bilateral ovariectomy is confirmed by screening the vaginal cell smear, which shows the absence of keratosis. OVX + r-irisin group: Ovariectomy is performed. One week after the operation, the mice start to receive intraperitoneal injections of 100 μg/kg irisin (MCE, HY-P70665, 100 μg) twice a week for 5 consecutive weeks. OVX + normal saline group: Ovariectomy is carried out. One week after the operation, the mice begin to receive intraperitoneal injections of an equal volume of normal saline twice a week for 5 consecutive weeks. Sham + normal saline group: A sham operation is performed. One week after the operation, the mice start to receive intraperitoneal injections of an equal volume of normal saline twice a week for 5 consecutive weeks. Five weeks after the treatment, the animals are euthanized.

Treatment ID:

TR003960

Treatment Summary:

In an environment with temperature control, 4 to 5 mice are housed in each cage, and the light/dark cycle is set at 12 hours. After a two-week adaptation period, 18 mice with a body weight ranging from 19.4 to 22.5 grams are selected and randomly divided into three groups: the OVX + r-irisin group, the OVX + normal saline group, and the Sham + normal saline group, with 6 mice in each group. Starting from the third day after the operation, a vaginal cell smear is taken from each animal once a day for 7 consecutive days. A successful bilateral ovariectomy is confirmed by screening the vaginal cell smear, which shows the absence of keratosis. OVX + r-irisin group: Ovariectomy is performed. One week after the operation, the mice start to receive intraperitoneal injections of 100 μg/kg irisin (MCE, HY-P70665, 100 μg) twice a week for 5 consecutive weeks. OVX + normal saline group: Ovariectomy is carried out. One week after the operation, the mice begin to receive intraperitoneal injections of an equal volume of normal saline twice a week for 5 consecutive weeks. Sham + normal saline group: A sham operation is performed. One week after the operation, the mice start to receive intraperitoneal injections of an equal volume of normal saline twice a week for 5 consecutive weeks. Five weeks after the treatment, the animals are euthanized.

Sample Preparation:

Sampleprep ID:	SP003957
Sampleprep Summary:	1. Thawing the Samples Take the frozen fecal samples stored at - 80°C out of the freezer. Place them on ice in a cold room or a cold - box to thaw slowly. Avoid rapid thawing at room temperature, as this may cause chemical changes in the metabolites due to temperature shock. Ensure that the samples are completely thawed but still kept cold throughout the process. 2. Homogenization Weighing: Transfer the thawed fecal sample into a pre - weighed sterile tube. Weigh the tube with the sample to determine the weight of the fecal material. Record this value accurately. Addition of extraction solvent: Add an appropriate amount of extraction solvent. For example, a common choice is a mixture of methanol and water (e.g., 80:20 v/v ratio). The volume of the solvent added should be proportional to the weight of the fecal sample, typically 3 - 5 times the weight of the feces (in mL per mg). Homogenization process: Use a homogenizer (such as a bead - beating homogenizer or a sonicator) to break down the fecal matrix and release the metabolites into the solvent. If using a bead - beating homogenizer, add sterile beads (e.g., zirconia beads) to the tube, and set the homogenizer to an appropriate speed and time (e.g., 3000 - 6000 rpm for 2 - 5 minutes). If using a sonicator, place the tube in the sonic bath and apply ultrasonic waves for a suitable duration (e.g., 10 - 20 minutes with intermittent cycles). 3. Centrifugation After homogenization, transfer the homogenized sample to a centrifuge tube. Centrifuge the sample at a high speed (e.g., 10,000 - 15,000 g) for 10 - 15 minutes at 4°C. This step will separate the insoluble fecal particles from the supernatant containing the extracted metabolites. Carefully transfer the supernatant to a new sterile tube using a pipette, avoiding disturbing the pellet at the bottom of the centrifuge tube. 4. Sample Clean - up (Optional) Solid - phase extraction (SPE): If necessary, perform SPE to further purify the metabolite extract. Select an appropriate SPE cartridge based on the properties of the metabolites to be analyzed. Condition the cartridge according to the manufacturer's instructions, then load the supernatant onto the cartridge. Wash the cartridge with appropriate solvents to remove impurities, and finally, elute the metabolites with a suitable elution solvent. Filtering: Pass the supernatant or the eluted sample through a 0.22 - μm or 0.45 - μm filter to remove any remaining particulate matter. This step helps to prevent clogging of the analytical instruments during metabolite sequencing. 5. Concentration and Drying (Optional) If the concentration of metabolites in the sample is too low for the subsequent sequencing analysis, the sample can be concentrated. This can be achieved using methods such as rotary evaporation or nitrogen - blowdown. For rotary evaporation, transfer the sample to a round - bottom flask and place it in a rotary evaporator. Evaporate the solvent under reduced pressure at a suitable temperature (e.g., 30 - 40°C). For nitrogen - blowdown, place the sample in a glass vial and gently blow a stream of nitrogen gas over the surface of the sample until the solvent is evaporated. After concentration, the sample may be dried completely. If the sample is to be stored for a long time, it is advisable to dry it under a gentle stream of nitrogen gas or in a vacuum desiccator. 6. Re - dissolution and Storage Re - dissolve the dried or concentrated sample in an appropriate solvent, such as a mixture of water and organic solvent (e.g., methanol or acetonitrile) suitable for the metabolite sequencing instrument. The volume of the re - dissolution solvent should be adjusted according to the required concentration for analysis. Transfer the re - dissolved sample to a clean, labeled vial for storage. Store the sample at - 80°C until it is ready for metabolite sequencing. Avoid repeated freeze - thaw cycles to maintain the stability of the metabolites.

Sampleprep ID:

SP003957

Sampleprep Summary:

1. Thawing the Samples Take the frozen fecal samples stored at - 80°C out of the freezer. Place them on ice in a cold room or a cold - box to thaw slowly. Avoid rapid thawing at room temperature, as this may cause chemical changes in the metabolites due to temperature shock. Ensure that the samples are completely thawed but still kept cold throughout the process. 2. Homogenization Weighing: Transfer the thawed fecal sample into a pre - weighed sterile tube. Weigh the tube with the sample to determine the weight of the fecal material. Record this value accurately. Addition of extraction solvent: Add an appropriate amount of extraction solvent. For example, a common choice is a mixture of methanol and water (e.g., 80:20 v/v ratio). The volume of the solvent added should be proportional to the weight of the fecal sample, typically 3 - 5 times the weight of the feces (in mL per mg). Homogenization process: Use a homogenizer (such as a bead - beating homogenizer or a sonicator) to break down the fecal matrix and release the metabolites into the solvent. If using a bead - beating homogenizer, add sterile beads (e.g., zirconia beads) to the tube, and set the homogenizer to an appropriate speed and time (e.g., 3000 - 6000 rpm for 2 - 5 minutes). If using a sonicator, place the tube in the sonic bath and apply ultrasonic waves for a suitable duration (e.g., 10 - 20 minutes with intermittent cycles). 3. Centrifugation After homogenization, transfer the homogenized sample to a centrifuge tube. Centrifuge the sample at a high speed (e.g., 10,000 - 15,000 g) for 10 - 15 minutes at 4°C. This step will separate the insoluble fecal particles from the supernatant containing the extracted metabolites. Carefully transfer the supernatant to a new sterile tube using a pipette, avoiding disturbing the pellet at the bottom of the centrifuge tube. 4. Sample Clean - up (Optional) Solid - phase extraction (SPE): If necessary, perform SPE to further purify the metabolite extract. Select an appropriate SPE cartridge based on the properties of the metabolites to be analyzed. Condition the cartridge according to the manufacturer's instructions, then load the supernatant onto the cartridge. Wash the cartridge with appropriate solvents to remove impurities, and finally, elute the metabolites with a suitable elution solvent. Filtering: Pass the supernatant or the eluted sample through a 0.22 - μm or 0.45 - μm filter to remove any remaining particulate matter. This step helps to prevent clogging of the analytical instruments during metabolite sequencing. 5. Concentration and Drying (Optional) If the concentration of metabolites in the sample is too low for the subsequent sequencing analysis, the sample can be concentrated. This can be achieved using methods such as rotary evaporation or nitrogen - blowdown. For rotary evaporation, transfer the sample to a round - bottom flask and place it in a rotary evaporator. Evaporate the solvent under reduced pressure at a suitable temperature (e.g., 30 - 40°C). For nitrogen - blowdown, place the sample in a glass vial and gently blow a stream of nitrogen gas over the surface of the sample until the solvent is evaporated. After concentration, the sample may be dried completely. If the sample is to be stored for a long time, it is advisable to dry it under a gentle stream of nitrogen gas or in a vacuum desiccator. 6. Re - dissolution and Storage Re - dissolve the dried or concentrated sample in an appropriate solvent, such as a mixture of water and organic solvent (e.g., methanol or acetonitrile) suitable for the metabolite sequencing instrument. The volume of the re - dissolution solvent should be adjusted according to the required concentration for analysis. Transfer the re - dissolved sample to a clean, labeled vial for storage. Store the sample at - 80°C until it is ready for metabolite sequencing. Avoid repeated freeze - thaw cycles to maintain the stability of the metabolites.

Combined analysis:

Analysis ID	AN006275	AN006276
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Triple TOF	Triple TOF
MS instrument name	ABI Sciex 5600 TripleTOF	ABI Sciex 5600 TripleTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Relative abundance	Relative abundance

Chromatography:

Chromatography ID:	CH004760
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	40
Flow Gradient:	0 - 5 min: A 95%→70%, B 5%→30%, flow rate 0.2 mL/min → 0.5 mL/min; 5 - 15 min: A 70%→20%, B 30%→80%, flow rate 0.5 mL/min; 15 - 17 min: A 20%→40%, B 80%→60%, flow rate 0.5 mL/min → 0.2 mL/min
Flow Rate:	0.2 - 0.5 mL/min
Solvent A:	0.1% formic acid in water
Solvent B:	0.1% formic acid in acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005977
Analysis ID:	AN006275
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	Mass spectrometry For secondary mass spectrometry (MS/MS), a quadrupole-time-of-flight mass spectrometer (AB Sciex TripleTOF 5600) equipped with an electrospray ionization (ESI) source was used. The analysis was conducted in both positive and negative ionization modes with an ion source temperature of 550°C and a capillary voltage of 4500 V. The ion source gas pressures (Gas1: 50, Gas2: 50) were optimized to achieve stable ionization. The mass-to-charge ratio (m/z) range for scanning was set from 50 to 1000. Parent ions were selected, and fragmentation was induced via collision-induced dissociation (CID) to analyze product ions. The generated spectra were processed to identify metabolites, comparing their m/z values to known databases and standards for metabolite identification. Data transformation The raw data obtained from the LC-MS/MS analysis of fecal metabolites were first processed to remove background noise and correct any baseline shifts. Data files were converted into a compatible format mzML using ProteoWizard software. The resulting data were then analyzed using PeakView to identify peaks corresponding to metabolites based on their retention times and m/z values. Peak intensity values were normalized using internal standards to correct for potential variability in sample preparation and instrumental response.
Ion Mode:	POSITIVE

MS ID:	MS005978
Analysis ID:	AN006276
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	Mass spectrometry For secondary mass spectrometry (MS/MS), a quadrupole-time-of-flight mass spectrometer (AB Sciex TripleTOF 5600) equipped with an electrospray ionization (ESI) source was used. The analysis was conducted in both positive and negative ionization modes with an ion source temperature of 550°C and a capillary voltage of 4500 V. The ion source gas pressures (Gas1: 50, Gas2: 50) were optimized to achieve stable ionization. The mass-to-charge ratio (m/z) range for scanning was set from 50 to 1000. Parent ions were selected, and fragmentation was induced via collision-induced dissociation (CID) to analyze product ions. The generated spectra were processed to identify metabolites, comparing their m/z values to known databases and standards for metabolite identification. Data transformation The raw data obtained from the LC-MS/MS analysis of fecal metabolites were first processed to remove background noise and correct any baseline shifts. Data files were converted into a compatible format mzML using ProteoWizard software. The resulting data were then analyzed using PeakView to identify peaks corresponding to metabolites based on their retention times and m/z values. Peak intensity values were normalized using internal standards to correct for potential variability in sample preparation and instrumental response.
Ion Mode:	NEGATIVE