Tsp d4

Doxorubicin hydrochloride (DOX; Product Number: D1515; CAS Number: 25316-40-9; formula: C₂₇H₂₉NO₁₁.HCL) was obtained from Sigma–Aldrich Chemicals (St. Louis, MO, USA). DOX has a molecular weight of 579.98 g/mol and is in the form of powder. Methanol (CH₃OH) and chloroform (CHCl₃) were purchased from Sigma–Aldrich. Dulbecco’s Modified Eagle’s medium (DMEM; Lot No: 09221712, GenDEPOT, St. Louis, MI, USA) and 10% (v/v) fetal bovine serum (FBS; Lot No: WB0009) were purchased from GE Healthcare Life Science (Queensland, Australia). HCT116 colon cancer cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). TSP-d₄ is a 3-(trimethylsilyl) propionic acid that was purchased from Cambridge Isotope Laboratories in the United States.

0.30 × 25 mm stainless steel acupuncture needle (Suzhou Acupuncture Supplies Co., Ltd., Jiangsu, China); IITC-336G foot thermal pain tester (Shanghai Yuyan Scientific Instrument Co., Ltd.); LH202 Han’s acupoint neurostimulator (produced by Peking University Neuroscience Research Institute, produced by Beijing Huawei Industrial Development Co., Ltd.); Bruker 600 MHz Advanced III NMR spectrometer (Brook, Germany). Germany LEICA, Intelligent Biomicroscope (Olympus); BX53.Leica paraffin embedding station (EG 1,160, Leica Biosystems Nussloch GmbH, Germany); Leica rotary microtome (RM 2,135, Leica Biosystems Nussloch GmbH, Germany); TSPd4 (Cambridge Isotope Laboratories, Inc., USA); K2HPO4, NaH2PO4⋅2H2O (Xilong Scientific Co., Ltd., Guangdong, China); D2O (633,178, Sigma, USA); anhydrous ethanol, 0.9% sodium chloride injection, 4% polyoxymethylene (Changsha GugeBio Technology Co., Ltd., Changsha, China); NMR spectrometer (Bruker Biospin, Rheinstetten, Germany); NMR data preprocessing (MestReNova v9.0.1 software, Mestrelab Research, Santiago de Compostela, Spain); multivariate statistical analysis (SIMCA-P14.1, Umetrics, Sweden); R510 small animal anaesthesia machine (Rived R510IP).

For targeted NMR-based metabolomics analysis, brain samples were prepared and measured using methods previously described after slight modification [48 (link)]. Frozen brain samples (100 mg) were homogenized (Ultraturax T25, IKA, Staufen, Germany) in ice-cold methanol/chloroform (2:1, v/v, 3 mL) for 1 min and then sonicated in an ice-cold water bath for 30 min. After addition of 1 mL of ice-cold water and 1 mL of ice-cold chloroform, samples were centrifuged (1800g, 4 °C) for 35 min to achieve phase separation. The aqueous supernatant was collected, dried using an evacuated centrifuge (Savant, SVC 100H, Techtum Instrument AB, Umeaå, Sweden), and re-dissolved in 520 μL of sodium phosphate buffer (0.135 mol/L, pH 7.0). The residual proteins were then removed using Nanosep centrifugal filters (3 kDa, Pall Life Science, Port Washington, USA). The filtrate (390 μL) was mixed with extra phosphate buffer (130 μL, 0.135 mol/L, pH 7.0), D2O (50 μL), and sodium-3- (trimethylsilyl)-2,2,3,3-tetradeuteriopropionate solution (TSP-d4, 30 μL, 0.3 mmol/L, Cambridge Isotope Laboratories, Andover, USA). For NMR analysis 580 μL of mixture was added to 5 mm NMR tubes.