#METABOLOMICS WORKBENCH MingxinZhang_20250213_225847 DATATRACK_ID:5623 STUDY_ID:ST003731 ANALYSIS_ID:AN006122 PROJECT_ID:PR002317 VERSION 1 CREATED_ON February 15, 2025, 4:57 pm #PROJECT PR:PROJECT_TITLE Gut neurotransmitter targeting metabolome in sleep-deprived mice PR:PROJECT_SUMMARY This project investigates the changes in neurotransmitter levels in the jejunum PR:PROJECT_SUMMARY tissue of mice before and after 2 days of sleep deprivation, exploring the PR:PROJECT_SUMMARY alterations in intestinal neurotransmitter release caused by sleep deprivation. PR:PROJECT_SUMMARY Metabolomic results indicate that in mice subjected to 2 days of sleep PR:PROJECT_SUMMARY deprivation, intestinal neurotransmitters such as KynA, Gln, and Trp were PR:PROJECT_SUMMARY significantly reduced, while TrpA, 5-HT, and 5-HTP were significantly increased PR:INSTITUTE China Agricultural University PR:LAST_NAME Zhang PR:FIRST_NAME Mingxin PR:ADDRESS 2 West Yuanmingyuan Road, Beijing, 100193, China PR:EMAIL zmx15310686122@163.com PR:PHONE 18800152903 #STUDY ST:STUDY_TITLE Gut neurotransmitter targeting metabolome in sleep-deprived mice ST:STUDY_SUMMARY Sleep disturbances associate with pathogenesis of numerous chronic disorders, ST:STUDY_SUMMARY including chronic gastrointestinal diseases. However, the mechanism that ST:STUDY_SUMMARY transmits sleep disturbance-induced aberrant neural signaling from the brain to ST:STUDY_SUMMARY the gut remains elusive. We show that acute sleep deprivation (SD) impairs ST:STUDY_SUMMARY intestinal stem cell function, leading to shortening of crypt-villus ST:STUDY_SUMMARY architecture and Paneth cell loss. We identified dorsal motor nucleus of vagus ST:STUDY_SUMMARY (DMV) as the SD-sensitive central nervous system center that transmits sleep ST:STUDY_SUMMARY effects to the gut. SD aberrantly activates DMV neurons, driving excessive ST:STUDY_SUMMARY acetylcholine release from the vagus nerve into the gut. Acetylcholine triggers ST:STUDY_SUMMARY 5-hydroxytryptamine (5-HT) release by enterochromaffin cells and suppresses its ST:STUDY_SUMMARY reuptake via muscarinic receptors, thereby causing a spike in 5-HT levels. ST:STUDY_SUMMARY Elevated 5-HT induces excessive oxidative stress in intestinal stem cells ST:STUDY_SUMMARY through its receptor HTR4, promoting gut pathologies. Overall, we reveal ST:STUDY_SUMMARY SD-responsive neural circuit that controls intestinal stem cells and identify ST:STUDY_SUMMARY therapeutic strategies for mitigating SD-related gut diseases. ST:INSTITUTE China Agricultural University ST:LAST_NAME Zhang ST:FIRST_NAME Mingxin ST:ADDRESS 2 West Yuanmingyuan Road, Beijing, 100193, China ST:EMAIL zmx15310686122@163.com ST:PHONE 18800152903 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Not applicable #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 1 SD0d_1_B Sample source:jejunum | Treatment:Ctrl RAW_FILE_NAME(Raw name)=FZTM220066984-1A SUBJECT_SAMPLE_FACTORS 2 SD0d_2_B Sample source:jejunum | Treatment:Ctrl RAW_FILE_NAME(Raw name)=FZTM220066985-1A SUBJECT_SAMPLE_FACTORS 3 SD0d_3_B Sample source:jejunum | Treatment:Ctrl RAW_FILE_NAME(Raw name)=FZTM220066986-1A SUBJECT_SAMPLE_FACTORS 4 SD0d_4_B Sample source:jejunum | Treatment:Ctrl RAW_FILE_NAME(Raw name)=FZTM220066987-1A SUBJECT_SAMPLE_FACTORS 5 SD0d_5_B Sample source:jejunum | Treatment:Ctrl RAW_FILE_NAME(Raw name)=FZTM220066988-1A SUBJECT_SAMPLE_FACTORS 6 SD2d_1_B Sample source:jejunum | Treatment:SD2d RAW_FILE_NAME(Raw name)=FZTM220066989-1A SUBJECT_SAMPLE_FACTORS 7 SD2d_2_B Sample source:jejunum | Treatment:SD2d RAW_FILE_NAME(Raw name)=FZTM220066990-1A SUBJECT_SAMPLE_FACTORS 8 SD2d_3_B Sample source:jejunum | Treatment:SD2d RAW_FILE_NAME(Raw name)=FZTM220066991-1A SUBJECT_SAMPLE_FACTORS 9 SD2d_4_B Sample source:jejunum | Treatment:SD2d RAW_FILE_NAME(Raw name)=FZTM220066992-1A SUBJECT_SAMPLE_FACTORS 10 SD2d_5_B Sample source:jejunum | Treatment:SD2d RAW_FILE_NAME(Raw name)=FZTM220066993-1A #COLLECTION CO:COLLECTION_SUMMARY Fresh jejunum tissue weighing 0.3g was obtained from mice through dissection CO:COLLECTION_SUMMARY before and after sleep deprivation and immediately stored at -80°C.The samples CO:COLLECTION_SUMMARY were taken respectively and homogenized with 400 μL of acetonitrile (80%) which CO:COLLECTION_SUMMARY contained mixed internal standards and centrifuged at 12,000 rpm for 10 min. CO:COLLECTION_SUMMARY Finally, the supernatant (2 μL) was injected into the LC-MS/MS system for CO:COLLECTION_SUMMARY analysis. The supernatant was diluted 100 times by acetonitrile (80%) which CO:COLLECTION_SUMMARY contained mixed internal standards and centrifuged at 12,000 rpm for 10 min. The CO:COLLECTION_SUMMARY supernatant was injected into the LC-MS/MS system for analysis CO:SAMPLE_TYPE Jejunum #TREATMENT TR:TREATMENT_SUMMARY "jejunum tissue samples were obtained immediately after mice were subjected to TR:TREATMENT_SUMMARY 48 hours of continuous sleep deprivation(SD)." #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The stock solution of individual neurotransmitter was mixed and prepared in SP:SAMPLEPREP_SUMMARY neurotransmitter -free matrix to obtain a series of neurotransmitter SP:SAMPLEPREP_SUMMARY calibrators. Certain concentrations of L-Tyrosine-d4 was mixed as Internal SP:SAMPLEPREP_SUMMARY Standard (IS). The stock solution of all of these and working solution were SP:SAMPLEPREP_SUMMARY stored in refrigerator of -20°C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME QTRAP 6500+ CH:COLUMN_NAME Waters XSelect HSS T3 XP (150 x 3mm,2.5um) CH:SOLVENT_A 100% Water; 0.1% formic acid; 5mM ammonium acetate CH:SOLVENT_B 100% Acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT initial 5% B, 2min; 5-40% B, 4min; 40-70% B, 7 min; 70-100% B, 7.5min; 100% B, CH:FLOW_GRADIENT 9min; 100-5% B, 9.1min; 5% B, 11min. CH:FLOW_RATE 0.30 mL/min CH:COLUMN_TEMPERATURE 45°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS LC-MS was used to detect the concentration series of standard solution. The MS:MS_COMMENTS ratio of concentration of standard to internal standard as abscissa, and the MS:MS_COMMENTS ratio of peak area of standard to internal standard as ordinate to investigate MS:MS_COMMENTS the linearity of standard solution. The correlation coefficient (r) > 0.99 of MS:MS_COMMENTS each metabolites were the necessary condition. The limit of quantification (LOQ) MS:MS_COMMENTS were determined by the method of signal-to-noise ratio (S/N), which is comparing MS:MS_COMMENTS the signal measured by the standard solution concentration with the blank MS:MS_COMMENTS matrix. Generally, when the S/N = 10:1, the corresponding concentration is the MS:MS_COMMENTS LOQ #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/g MS_METABOLITE_DATA_START Samples SD0d_1_B SD0d_2_B SD0d_3_B SD0d_4_B SD0d_5_B SD2d_1_B SD2d_2_B SD2d_3_B SD2d_4_B SD2d_5_B Factors Sample source:jejunum | Treatment:Ctrl Sample source:jejunum | Treatment:Ctrl Sample source:jejunum | Treatment:Ctrl Sample source:jejunum | Treatment:Ctrl Sample source:jejunum | Treatment:Ctrl Sample source:jejunum | Treatment:SD2d Sample source:jejunum | Treatment:SD2d Sample source:jejunum | Treatment:SD2d Sample source:jejunum | Treatment:SD2d Sample source:jejunum | Treatment:SD2d 4-Aminobutyric acid 14323.57319 7112.329737 8318.95055 16479.41842 10131.12339 17338.58188 19411.66 16971.51962 6724.368773 12187.63653 Histamine 5.0893298 4.679517362 4.405787491 1.230804488 3.077804321 2.120054469 1.469570369 1.755492632 3.217986748 3.256092242 Acetylcholine chloride 170.9414849 40.71405101 102.4081922 244.5363062 248.2842358 162.7769096 133.4661634 136.6793818 87.26285415 71.03540054 Hydroxytyramine hydrochloride 5205.89958 3250.31173 4823.447938 4882.503063 3412.352358 5234.274888 4997.339125 4280.644699 4005.33812 3455.838048 Tryptamine 0.500780402 0.268112916 0.15654261 1.155301218 0.908619871 1.743070962 1.678291498 0.920754228 0.35042958 0.235335457 Noradrenaline hydrochloride 93585.28215 127101.6569 108588.8402 175383.4062 159544.0944 144260.7215 154930.0412 131747.2255 120255.8278 129023.321 Serotonin hydrochloride 1087.541975 569.8444296 110.0880672 338.1689991 769.7563757 894.7506367 621.4436827 305.4370573 837.6991127 406.8167658 L-Tyrosine 711030.1475 512008.5494 599801.3298 219437.7095 265112.9867 279817.7533 212215.9407 225911.9251 390770.7278 456210.0529 Kynurenic acid 80.35030803 61.19950387 73.59764815 29.37035432 57.70820625 37.56997264 22.35582179 29.25436286 41.42753135 41.35591403 5-Hydroxyindole-3-acetic acid 2248.011946 917.454291 1909.440073 2804.701292 1165.283157 2337.373831 2169.038714 2172.585716 1516.797983 1193.583784 Levodopa 881.3552934 804.8344889 953.2809015 263.4236128 422.9687804 406.8725576 348.9272745 450.3805687 629.3080044 742.2463372 L-Tryptophan 79440.89003 52077.7252 64854.16078 18477.55863 29249.63813 27362.52169 22458.98833 21137.37093 35271.1154 49234.07306 Xanthurenic acid 436.8036288 244.8840688 475.9243965 148.8917509 235.8218409 185.6547873 183.584327 152.2217096 200.7994536 381.6248482 DL-Kynurenine 3169.95713 1425.382973 3729.752834 6444.685506 2497.367109 4522.619388 3710.411312 3933.267761 2740.327552 1476.03904 5-Hydroxytryptophan 1006.089644 513.4287357 278.554471 630.864858 1283.316341 1333.514596 1003.812923 630.1099655 1208.049235 376.8698157 L-Glutamic acid 2858610.563 1715202.032 2369843.068 1746833.831 1458515.917 1741102.382 1536921.367 1459445.389 1565681.589 1763070.536 L-Histidine 94989.54484 65841.42231 74801.11879 42189.09193 44468.88412 46638.89729 36783.09487 44745.31428 53432.63512 89542.32476 L-Glutamine 755627.6112 444361.4758 543566.1437 363178.0655 351809.5591 379239.6656 330558.7227 309913.672 368911.4054 546092.0124 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 4-Aminobutyric acid Histamine Acetylcholine chloride Hydroxytyramine hydrochloride Tryptamine Noradrenaline hydrochloride Serotonin hydrochloride L-Tyrosine Kynurenic acid 5-Hydroxyindole-3-acetic acid Levodopa L-Tryptophan Xanthurenic acid DL-Kynurenine 5-Hydroxytryptophan L-Glutamic acid L-Histidine L-Glutamine METABOLITES_END #END