Materials and reagents

Portable HRW machine (China Institute For Radiation Protection, Shanxi Zhongfu Nuclear Instruments Co.,Ltd.), Linear accelerator (Elekta Synergyt, Crawley, UK), ultra-high-performance liquid chromatography (Dionex Ultimate 3000 UHPLC, Thermo Scientific, USA) coupled to high-resolution mass spectrometry (Q-Exactive Plus, Thermo Scientific, USA). Water, methanol, acetonitrile and formic acid were purchased from CNW Technology Co., Ltd. (Dü sseldorf, Germany). L-2-chlorophenylalanine was obtained from Shanghai Hengchuang Biotechnology Co., Ltd. (Shanghai, China). All chemicals and solvents are of analytical or HPLC grade.

Animals and ethic statement

Male Sprague-Dawley rats weighing 140–170 g and, aging 6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. Animals were caged in a humidity and temperature-controlled room, with a 12/12 light-dark cycle. Water and food were provided to animals with free access. The animal study protocols were approved by the Animal Protection and Ethics Committee of the China Institute for Radiation Protection. All laboratory operations were performed in accordance with the Regulations of China Institute for Radiation Protection on the administration of laboratory animals. The study was carried out in compliance with the ARRIVE guidelines.

Establishment of the ionizing radiation model

Twenty-four rats were randomized into four groups (6 in each group), as follows: HRW group received HRW(hydrogen concentration: 0.8–0.9 ppm) without radiation treatment; Radiotherapy group received 30 Gy whole brain irradiation of purified water; Radiation -HRW group received 30 Gy whole brain irradiation with HRW; Control group received purified water without radiation treatment. Rats were dosed with purified water or HRW gavage at 20ml/kg per day 10 min before irradiation and 30 days after irradiation. Immobilize the animal directly with clamps before preparing the animal for radiotherapy with deionized water using a portable HRW machine. Then, at room temperature, a 6-MeV electron beam was emitted at a rate of 3 Gy/min by a linear accelerator to irradiate the whole brain. On the 36th day after irradiation, rats were sacrificed by intraperitoneal injection of pentobarbital and blood was taken from the abdominal aorta, the brains of the animals were collected.

Morris Water Maze Test

On the 30th day after irradiation, the rats in each group were subjected to a cognitive test in the form of a position navigation test and a spatial search test. In the site navigation task, rats acclimated themselves with the test environment by swimming for 2 min in the circular pool without a platform the day before the formal test. Rats were then trained for five consecutive days. To start training, rats were placed in the pool facing the pool wall, the swimming route of the rats were tracked, the escape latency (the time from entering the water to finding platform–all limbs on the platform) was measured. Rats were allowed to rest on the platform for a few seconds before returning them to their cages. The escape latency was recorded as 120 s if the rat did not find the platform within 2 min and were guided to the platform. The arithmetic mean of the escape latencies was calculated four times per day. The position navigation test was followed by a spatial probe test in which the platform was withdrawn and the rats were placed at any entry point in the pool on day six. The swimming distance of rats were recorded and analyzed within 1 min. Record the passage time they cross the original platform, the time the rat retained in the original platform quadrant, their swimming distance in the platform quadrant, and the number of times the rat crossed the platform.

Brain tissue samples collection and preparation

After the brain tissue was collected, 30 mg brain tissue sample were accurately weighed and transferred to a 1.5 mL Eppendorf tube. Two small steel spheres and 20 µL of the internal standard (2-chloro-l-phenylalanine in methanol, 0.3 mg/mL) and 400 µL of methanol/water (4/1, v/v) extraction solvent were added to the sample tube. The samples were stored at -80 ℃ for 2 min, ground at 60 HZ for 2 min, sonicated at ambient temperature (25 ℃ to 28 ℃) for 10 min and stored at -20 ℃ for 30 min Centrifuge the extract at 13,000 rpm, 4 ℃ for 15 min. Dry 300 mL of the supernatant in a brown glass vial in a freeze concentration centrifugal dryer. Then 200 µL mixture of methanol and water (1/4, vol/vol) were added to each sample, the sample was vortexed for 30 s and, placed at 4 ℃ for 2 min. Samples were centrifuged at 13,000 rpm, 4 ℃ for 5 min. The supernatant (150 µL) was collected from each tube using a crystal syringe and was filtered through a 0.22 μm microfilter and transferred to an LC vial. Before LC -MS analysis, the vials were stored at -80 ℃ until. QC samples were prepared through mixing aliquots of all samples into a pooled sample.

Metabolomics analysisUHPLC-MS parameters

Samples were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry in both ESI positive and ESI negative ion modes. In both positive and negative ionization modes, an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) were employed. Water and acetonitrile, both containing 0.1% formic acid, were used as mobile phases A and B, respectively. The linear gradient was: 0 min, 5% B; 2 min, 5% B; 4 min, 25%B; 8 min, 50% B; 10 min, 80% B; 14 min, 100% B; 15 min, 100% B; 15.1 min, 5%B and 16 min, 5%B. The column temperature was 45 ℃ and the flow rate was 0.35 mL/min. The injection volume was 2 µL. All samples were kept at 4℃ during analysis.

Data acquisition was performed in full scan mode (m/z ranges from 100 to 1000) combined with MSE mode, consisting of alternating acquisition of 2 independent scans with different collision energies (CE) during the run. The mass spectrometry parameters were as follows: a low-energy scan (CE 4 eV) and a high-energy scan (CE ramp 20-45 eV) to fragment the ions. Argon (99.999%) was used as collision-induced dissociation gas; scan rate was 0.2 s/scan; capillary voltages were − 3 kV (negative mode) and 3.8 kV (positive mode); capillary and heater temperatures were 320℃ and 300℃. The auxiliary gas volume flow was 8 arb, sheath gas volume flow was 35 arb. The stepped normalized collision energy (NCE) was set to 10, 20, and 40 eV respectively. The QCs were injected periodically (every 10 samples) throughout the analysis to provide a data set from which repeatability can be assessed.

Data processing

The raw data were exported using Xcalibur workstation and imported into Composite Discoverer 2.0 software to obtain matched and aligned peak data. The parameters are as follows: the scanning range is 100–1000 m/z. Ass deviation is 5 ppm. The retention time is 0.05 min. The signal-to-noise ratio threshold is 1.5. The peak data containing retention time, molecular formula, accurate molecular weight, and peak area information were imported into Excel for peak area normalization. Finally, the peak area normalization data were imported into SIMCA-P 14.0 for partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA). VIP > 1 in the S-plot and P < 0.05 in the independent sample t-test were used to screen for the most contributing differential metabolites. Differential metabolites were matched and screened using Metlin, HMDB, Pubchem, KEGG, m/zcloud, and others online databases. Finally, the identified differential metabolites were directed to MetaboAnalyst 5.0 for pathway enrichment analysis.

Construction of biological network of HRW for ionizing radiation therapy

The GeneCards database (https://www.genecards.org/) and the DisGeNET database (http://www.disgenet.org/) were used to identify potential genes associated with ionizing radiation. Metabolites were introduced into the Cytoscape 3.9.2 plug-in MetScape for metabolic enzyme analysis. Finally, Cytoscape 3.9.2 was used to construct a “gene-metabolite” regulatory network for ionizing radiation therapy.

Biological function and pathway analysis

To elucidate the gene functions and their role in signal transduction, DAVID (https://david.ncifcrf.gov/) was used to evaluate the KEGG and GO enrichment profiles of HRW against ionizing radiation (P < 0.05).

ROC analysis

ROC curve analysis and calculation of AUC were performed using the Omicstudio tool (https://www.omicstudio.cn/home) to screen for potential therapeutic biomarkers and assesse the efficacy of central metabolites in the HRW treatment of ionizing radiation. These metabolites were combined by logistic regression analysis. P < 0.05 with a 95% confidence interval was considered statistically significant.

Statistical analysis

All data were presented as mean ± standard deviation. SPSS 22.0 and GraphPad Prism 7.0 were used for statistical analysis. The independent samples t test and one-way analysis of variance were used for comparison between two groups and multiple groups. P < 0.05 was considered statistically significant.