#METABOLOMICS WORKBENCH youngh_20231101_183536 DATATRACK_ID:4435 STUDY_ID:ST002960 ANALYSIS_ID:AN004861 PROJECT_ID:PR001842 VERSION 1 CREATED_ON November 3, 2023, 5:49 am #PROJECT PR:PROJECT_TITLE Mid-Old Cells are Potential Target for Anti-aging Interventions in the Elderly PR:PROJECT_SUMMARY The biological process of aging is thought to result in part from accumulation PR:PROJECT_SUMMARY of senescent cells in organs. However, the present study identified a subset of PR:PROJECT_SUMMARY fibroblasts and smooth muscle cells which are the major constituents of organ PR:PROJECT_SUMMARY stroma neither proliferative nor senescent in tissues of the elderly, which we PR:PROJECT_SUMMARY termed “mid-old status” cells. Upregulation of pro-inflammatory genes (IL1B PR:PROJECT_SUMMARY and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were PR:PROJECT_SUMMARY detected in mid-old cells. In the stroma, SAA1 promotes development of the PR:PROJECT_SUMMARY inflammatory microenvironment via upregulation of MMP9, which decreases the PR:PROJECT_SUMMARY stability of epithelial cells present on the basement membrane, decreasing PR:PROJECT_SUMMARY epithelial cell function. Remarkably, the microenvironmental change and the PR:PROJECT_SUMMARY functional decline of mid-old cells could be reversed by a young cell-originated PR:PROJECT_SUMMARY protein, SLIT2. Our data identify functional reversion of mid-old cells as a PR:PROJECT_SUMMARY potential method to prevent or ameliorate aspects of aging-related tissue PR:PROJECT_SUMMARY dysfunction. PR:INSTITUTE Ajou University Medical Center PR:LAST_NAME Kim PR:FIRST_NAME Young Hwa PR:ADDRESS 206, World cup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea PR:EMAIL skyblue32@nate.com PR:PHONE +82-10-5153-3636 #STUDY ST:STUDY_TITLE Analysis of Lipids Secreted from Fibroblast Young Cells ST:STUDY_SUMMARY In this experimental study, we aimed to understand the potential factors within ST:STUDY_SUMMARY the secretions of young cells that could trigger the reverse aging of Mid-old ST:STUDY_SUMMARY cells. To investigate this phenomenon, we co-cultured young cells with Mid-old ST:STUDY_SUMMARY cells and observed a fascinating outcome: the Mid-old cells exhibited reverse ST:STUDY_SUMMARY aging and transformed into a more youthful state. To uncover the specific ST:STUDY_SUMMARY factors responsible for this reverse aging effect, we conducted a detailed ST:STUDY_SUMMARY analysis of the secreted factors from the young cells. Our analysis focused on a ST:STUDY_SUMMARY range of biomolecules, including lipids. However, despite our efforts, we did ST:STUDY_SUMMARY not identify any distinct factors that could be directly attributed to this ST:STUDY_SUMMARY remarkable reverse aging process. ST:INSTITUTE Ajou University Medical Center ST:LAST_NAME Kim ST:FIRST_NAME Young Hwa ST:ADDRESS 206, World cup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea ST:EMAIL skyblue32@nate.com ST:PHONE +82-10-5153-3636 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - 20210625_AJU_Y1_lipidomics *Factor:Test sample RAW_FILE_NAME=20210625_AJU_Y1_lipidomics.mzML SUBJECT_SAMPLE_FACTORS - 20210625_AJU_Y2_lipidomics *Factor:Test sample RAW_FILE_NAME=20210625_AJU_Y2_lipidomics.mzML SUBJECT_SAMPLE_FACTORS - 20210625_AJU_Y3_lipidomics *Factor:Test sample RAW_FILE_NAME=20210625_AJU_Y3_lipidomics.mzML SUBJECT_SAMPLE_FACTORS - 20210625_AJU_M1_lipidomics *Factor:Control sample RAW_FILE_NAME=20210625_AJU_M1_lipidomics.mzML SUBJECT_SAMPLE_FACTORS - 20210625_AJU_M2_lipidomics *Factor:Control sample RAW_FILE_NAME=20210625_AJU_M2_lipidomics.mzML SUBJECT_SAMPLE_FACTORS - 20210625_AJU_M3_lipidomics *Factor:Control sample RAW_FILE_NAME=20210625_AJU_M3_lipidomics.mzML #COLLECTION CO:COLLECTION_SUMMARY The experiment was initiated by seeding the cells in a 100mm cell culture dish, CO:COLLECTION_SUMMARY followed by a 2-day incubation period, during which the culture medium was CO:COLLECTION_SUMMARY carefully harvested from the cell culture dish. Use a sterile technique to CO:COLLECTION_SUMMARY minimize contamination. Transfer the harvested culture medium to a suitable CO:COLLECTION_SUMMARY container. CO:SAMPLE_TYPE Cultured fibroblasts #TREATMENT TR:TREATMENT_SUMMARY no treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For lipid extraction from samples, a two-step method involving neutral and SP:SAMPLEPREP_SUMMARY acidic extraction were used. At first, in neutral extraction, lipids from the SP:SAMPLEPREP_SUMMARY samples were extracted according to the Folch method using a mixture of SP:SAMPLEPREP_SUMMARY chloroform and methanol (2:1, v/v). The samples were vortexed and incubated on SP:SAMPLEPREP_SUMMARY ice for 10 min. The samples were centrifuged at 13,800×g for 2 min at 4°C, SP:SAMPLEPREP_SUMMARY supernatant was transferred to a new 1.5 mL tube. Next, in Acidic extraction, SP:SAMPLEPREP_SUMMARY 750 μL of chloroform:methanol:HCl (1N, 37%) (40:80:1, v/v/v) was added to the SP:SAMPLEPREP_SUMMARY remaining samples. After incubating for 15 min at room temperature, 250 μL of SP:SAMPLEPREP_SUMMARY cold chloroform and 450 μL of cold 0.1 M HCl were added to the sample. The SP:SAMPLEPREP_SUMMARY mixture was vortexed for 1 min and centrifuged at 6,500×g for 2 min at 4°C. SP:SAMPLEPREP_SUMMARY The lower organic phase was collected and combined with the prior extract. The SP:SAMPLEPREP_SUMMARY sample was then dried using HyperVAC-MAX VC2200 centrifugal vacuum concentrator SP:SAMPLEPREP_SUMMARY (Hanil Scientific Inc., Korea). Dried metabolite contents were reconstituted in SP:SAMPLEPREP_SUMMARY 50 µL of mobile phase solvent A:solvent B (2 :1, v/v) and then subjected to SP:SAMPLEPREP_SUMMARY LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Q Exactive™ Hybrid Quadrupole-Orbitrap MS coupled to a 1290 Infinity UHPLC CH:COLUMN_NAME e Hypersil GOLD™ C18 HPLC column(2.1 × 100 mm; 1.9 μm particle size; Thermo CH:COLUMN_NAME Fischer Scientific) CH:SOLVENT_A Acetonitrile:Methanol:Water (19:19:2, v/v/v) + 20 mmol/L ammonium formate + 0.1% CH:SOLVENT_A formic acid (v/v) CH:SOLVENT_B 2-propanol + 20 mmol/L ammonium formate + 0.1% formic acid (v/v) CH:FLOW_GRADIENT 0–5 min, 5% B; 5–15 min, 5–30% B; 15–22 min, 30–90% B; 22–25 min, CH:FLOW_GRADIENT 90% B; 25–26 min, 90–5% B; 26–30 min, 5% B. Parameters were set as CH:FLOW_GRADIENT follows: positive mode, spray voltage; 3.8 kV CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 320℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Q Exactive™ Hybrid Quadrupole-Orbitrap MS MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Obtained UHPLC-Orbitrap-MS/MS RAW files were processed using Lipid Search 4.2TM MS:MS_COMMENTS (Thermo Fisher Scientific, Waltham, MA, USA). Lipid profiling under the MS:MS_COMMENTS following conditions: parent search; 0.2 Da, product search; 5.0 ppm, precursor MS:MS_COMMENTS ion mass tolerance; 8.0 ppm, M-score threshold; 2.0. MS:MS_RESULTS_FILE ST002960_AN004861_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END