Liu et al. BMC Molecular and Cell Biology(2023) 24:30
https://doi.org/10.1186/s12860-023-00491-4

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Comprehensive brain tissue metabolomics
and biological network technology
to decipher the mechanism of hydrogenrich water on Radiation-induced cognitive
impairment in rats
Xiaoming Liu1†, Mengya Liu1†, Huan Liu1, Hui Yuan1, Yong Wang2, Xiaoman Chen2, Jianguo Li1 and Xiujun Qin1*
Abstract
Background Hydrogen-rich water (HRW) has been shown to prevent cognitive impairment caused by ionizing
radiation. This study aimed to investigate the pharmacological effects and mechanisms of HRW on ionizing radiationby coupling the brain metabolomics and biological target network methods.
Methods and results HRW significantly improves the cognitive impairment in rats exposed to ionizing radiation.
Based on metabolomics and biological network results, we identified 54 differential metabolites and 93 target genes.
The KEGG pathway indicates that glutathione metabolism, ascorbic acid and aldehyde acid metabolism, pentose
and glucuronic acid interconversion, and glycerophospholipid metabolism play important roles in ionizing radiation
therapy.
Conclusion Our study has systematically elucidated the molecular mechanism of HRW against ionizing radiation,
which can be mediated by modulating targets, pathways and metabolite levels. This provides a new perspective for
identifying the underlying pharmacological mechanism of HRW.
Keywords Hydrogen-rich water, Ionizing radiation, Brain tissue metabolomics, Biological network†

Xiaoming Liu and Mengya Liu contributed equally to this work.

*Correspondence:
Xiujun Qin
xiujunqin@163.com
1
Department of Radiology and Environmental Medicine, China
Institute for Radiation Protection, CAEA Center of Excellence on
Nuclear Technology Applications for Non-Clinical Evaluation forRadiopharmaceutical, Shanxi Key Laboratory for Pharmaceutical
Toxicology & Radiation Injury Pharmaceuticals, CNNC Key Laboratory forRadiotoxicology and Preclinical Assessment of Radiopharmaceuticals,
Taiyuan 030006, P. R. China
2
School of forensic medicine, Shanxi Medical University, Taiyuan
030001, P. R. China

Introduction
Ionizing radiation can produce a variety of free radicals,
damage nucleic acids, proteins, carbohydrates and lipid
compounds, thereby destroying the structure and function of cells [1, 2]. Approximately 60–70% of the damage is caused by hydroxyl radicals [3]. The brain contains
high levels of polyunsaturated fatty acids (PUFAs) andrelatively low levels of antioxidant enzymes, making it
highly susceptible to oxidative damage [2]. Radiation canaffect cognitive functions such as learning and memory
because it can damage neurogenesis, promote brain cell
apoptosis, and cause neurodegenerative changes [3].

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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Liu et al. BMC Molecular and Cell Biology(2023) 24:30

Radiation may cause long-term cognitive impairment,
especially in children whose nervous system is still developing [3, 4].
Hydrogen has great potential in the clinical treatmentof various neurological diseases [5]. Previous studies haveshown that hydrogen treatment can effectively prevent
and alleviate neonatal brain injury and cognitive dysfunction, especially cerebral ischemia-reperfusion injury[6–8]. Hydrogen can cross the blood-brain barrier andquickly cross cell membranes to interact with toxic ROS.
Therefore, hydrogen has great potential in the clinicaltreatment of various diseases of the nervous system.
Metabolomics, as a new omics technique, study thecellular processes involving metabolites, intermediates
and products of cell metabolism, and has find applications in drug metabolism, biomarkers, and environmental toxicants. Metabolomics elucidates the end pointsof biological processes, reflecting the physiological andpathological state through metabolic levels [9]. Therefore,
metabolomics is a useful tool to investigate pharmacodynamics and mechanism of action of radioprotectants
which reflect the end state of diseases and treatments[10–14]. However, the changes in endogenous metabolites are unknown, and using metabolomics alone as atool may not reveal the effectiveness of radiation protection agents in the treatment of radiation-induced
diseases.
Computer virtual computing based on high throughput omics data analysis and network database search
may reveal complex network signal relationships among
drugs, targets and diseases [15]. For radioprotective
agents, their pharmacological effects are characterized bymulti-targets and multi-levels. The biological target network has the advantage to analyze and explain the action
mechanisms from a holistic perspective, promoting thein-depth investigation [16]. Combining the two key technologies of computer simulation and metabionomics, themolecular mechanism of agents’ action on diseases canbe explained from the metabolic level and genetic levels. For example, Liu et al. linked the biological targets
network with metabolomics and found that amino acidmetabolism is a potential target for Xiaoyaosan antidepressant [17].
To the best of our knowledge, no studies have used
metabolomics combined with biological network
approaches to study the effect of hydrogen on radiationinduced cognitive impairment. This study used metabolomics combined with a biological target network toinvestigate the molecular biological mechanism of HRW
on radiation-induced cognitive impairment in rats.

Page 2 of 11

Materials and methods
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 andEthics Committee of the China Institute for Radiation
Protection. All laboratory operations were performed inaccordance 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 wateror 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 forradiotherapy 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 theabdominal aorta, the brains of the animals were collected.
Morris Water Maze Test

On the 30th day after irradiation, the rats in each groupwere subjected to a cognitive test in the form of a position navigation test and a spatial search test. In the siteLiu et al. BMC Molecular and Cell Biology(2023) 24:30

navigation task, rats acclimated themselves with thetest environment by swimming for 2 min in the circularpool 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 poolwall, the swimming route of the rats were tracked, theescape 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 escapelatency was recorded as 120 s if the rat did not find theplatform 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 waswithdrawn and the rats were placed at any entry point in
the pool on day six. The swimming distance of rats wererecorded 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 numberof 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 ℃ to28 ℃) 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 ofmethanol and water (1/4, vol/vol) were added to eachsample, 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 collectedfrom each tube using a crystal syringe and was filteredthrough 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 analysis
UHPLC-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, bothPage 3 of 11

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 theflow rate was 0.35 mL/min. The injection volume was 2µL. All samples were kept at 4℃ during analysis.
Data acquisition was performed in full scan mode (m/zranges from 100 to 1000) combined with MSE mode,consisting of alternating acquisition of 2 independent
scans with different collision energies (CE) during therun. The mass spectrometry parameters were as follows:a low-energy scan (CE 4 eV) and a high-energy scan (CEramp 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 (negativemode) and 3.8 kV (positive mode); capillary and heatertemperatures 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 theanalysis to provide a data set from which repeatability
can be assessed.
Data processing

The raw data were exported using Xcalibur workstationand 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 datacontaining 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 screenedusing Metlin, HMDB, Pubchem, KEGG, m/zcloud, andothers 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 withionizing radiation. Metabolites were introduced into the
Cytoscape 3.9.2 plug-in MetScape for metabolic enzyme

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

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 signaltransduction, DAVID (https://david.ncifcrf.gov/) wasused to evaluate the KEGG and GO enrichment profilesof 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.

Page 4 of 11

Results
Effect of HRW on learning and memory in rats with ionizing
radiation

Morris water maze test was used to assess the functional
consequences of radiation exposure on the brain andwhether HRW produced any neurocognitive benefits.
During the training period of the first five days, the radiation group had significantly longer escape latency thanthe control group, the HRW group and the radiation-HRW treatment group (P < 0.05, Fig. 1A.). The spatial
search test was performed from day six. The radiationHRW group had significantly longer retention time in theoriginal platform quadrant, greater platform crossings,
and longer swimming distance than the radiation group(P < 0.05 Fig. 1B, C, D). These results indicated that brain
radiation damaged the spatial memory and spatial searchability of rats, and HRW had a protective effect on radiation-induced cognitive impairment.
HRW regulated metabolic disorders in ionizing radiation
rats

PLS-DA was used for metabolic profiling to reduce thedimensionality of complex data obtained from brain tissue samples from the control and radiation groups. The
model was clearly separated from the control group,
indicating that this radiation had a significant effect on
the endogenous metabolic spectrum of rat brain tissue(Fig. 2A). The PLS-DA model was then validated withan arrangement test (Fig. 2B). All the permutation values of R2 and Q2 were smaller than the original values,

Fig. 1 Effects of hydrogen-rich water (HRW) on cognitive function by Morris water maze test. (A) The escape latencies of different groups during
the five-day training period. *P < 0.05 vs. radiation-HRW, one-way ANOVA. (B) The retention time in the original platform quadrant. (C) The swimming
distance in the original platform quadrant. (D) The number of platform crossings. *P < 0.05

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

Page 5 of 11

Fig. 2 Multivariate statistical analysis of LC-MS data. (A) PLS-DA score chart of control and radiation group. (B) PLS-DA model verification diagram.
(C) OPLS-DA score chart of control rats and model rats. (D) S-plot of control rats and model rats. (E) PLS-DA scores of brain tissue from different groups

indicating that the established model was stable and reliable. We further analyzed the differences between groups
using PLS-DA (Fig. 2E). The control group, the radiationgroup, and radiation-HRW group could be clearly distinguished, indicating that HRW was effective for radiationgroup rats. The QC samples were collected together in
PLS-DA to demonstrate the robustness and applicabilityof the system and method.
Brain tissue sample data from control and modelgroups were analyzed with OPLS- DA to identify radiation-related differential metabolites. The combinationof VIP > 1 (in S-plot) and P < 0.05 (in T-test) revealed asignificant contribution of metabolites to homeostasis(Fig. 2 C and 2D). Compared with the control group, a
total of 80 metabolites including LysoPE(18:1(11Z)/0:0),
pantothenic acid, gamma-glutamylleucine, 1-phosphateribose, stearoylethanolamide, etc., changed significantlyin the model group. After the intervention of HRW, suchchanges were significantly adjusted (Table 1), indicatingthat HRW dramatically reversed the metabolic disordersof ionizing radiation rats.

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

Page 6 of 11

Table 1 Statistics of differential metabolites recovered by HRW
No

Metabolites

m/zRetention Ion
time (min) mode

Formula

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

Bisdiphosphoinositol tetrakisphosphate
PC(20:1(11Z)/24:0)
Cholinealpha-methyl butyric Acid
Ribothymidine
2-N,6-N-Bis(2,3-dihydroxybenzoyl)-L-lysine
Ribose 1-phosphate
Norophthalmic acid
Caproic acid
Drazoxolon
S-Cysteinosuccinic acid
Succinylproline
Glutathionate(1-)
gamma-Glutamylglutamic acid
Maleic hydrazide(4 S,5 S)-4,5-dihydroxy-2,6-dioxohexanoic acid
Itaconic acid
Aristolodione
Isobutyric acid
D-Lysine
Propaphos
ADP-ribose
a-L-threo-4-Hex-4-enopyranuronosyl-D-galacturonic acid
D-Glucuronic acid
Glutathione
Letrozole
3,4,5-trihydroxy-6-(2-oxo-1,3-diphenylpropoxy)oxane2-carboxylic acid
Pseudouridine(R)-3-hydroxybutyrylcarnitine[2-hydroxy-5-(2-hydroxy-3-methoxy-3-oxopropyl)phenyl]oxidanesulfonic acid2-(alpha-D-Galactosyl)-sn-glycerol 3-phosphate
Cysteine-Homocysteine disulfide
Isoguanosine
Guanosine
L-Ascorbic acid-2-glucoside
1,4-beta-D-Glucan
Allopurinol-1-ribonucleoside
Arabinosylhypoxanthine
Allopurinol
5-fluoroindole-2-carboxylic Acid
Elephantorrhizol
Saphenamycin
Pyrocatechol glucuronide
Isosorbide Dinitrate
Carotamine
Glutamyltyrosine
Pantothenic Acid
S-(4,5-Dihydro-2-methyl-3-furanyl) ethanethioate
5β-cholanic Acid-3α, 12α-diol N-(2-sulphoethyl)-amide

704.8074441
880.7169796
104.1073217
120.1020555
276.1187823
399.1192105
229.0114577
274.1046339
134.1174357
238.0377343
236.0230962
233.1129797
308.090742
275.0886761
113.0348169
351.0571659
175.0237955
306.0765161
175.098179
191.1036811
343.0535487
604.0705895
351.0571152
175.0238612
306.0763654
308.0905898
385.1282845

0.74
0.74
0.78
0.78
0.8
0.81
0.82
0.82
0.84
0.86
0.87
0.88
0.88
0.89
0.89
0.89
0.89
0.89
0.91
0.94
0.95
1.05
1.17
1.17
1.23
1.24
1.24

Neg
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Pos
Neg
Pos
Pos
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Pos
Pos

C6H20O24P8
C52H102NO8P
C5H13NO
C5H10O2
C10H14N2O6
C20H22N2O8
C5H11O8P
C10H17N3O6
C6H12O2
C10H8ClN3O2
C7H11NO6S
C9H13NO5
C10H17N3O6S
C10H16N2O7
C4H4N2O2
C6H8O6C5H6O4
C18H13NO4
C4H8O2
C6H14N2O2
C13H21O4PS
C15H23N5O14P2
C12H16O12
C6H10O7
C10H17N3O6S
C17H11N5
C21H22O8

Radiation Radivs. Control ationHRW vs.
Radiation
↓
↑
↓
↑
↓
↑
↓
↑
↑
↓
↑
↓
↑
↓
↑
↓
↓
↑
↑
↓
↑
↓
↓
↑
↑
↓
↑
↓
↓
↑
↑
↓
↑
↓
↑
↓
↑
↓
↓
↑
↓
↑
↑
↓
↑
↓
↑
↓
↑
↓
↑
↓
↑
↓

245.0766217
248.1490203
310.0586886

1.27
1.28
1.38

Pos
Pos
Pos

C9H12N2O6
C11H21NO5
C10H12O8S

↑
↓
↑

↓
↑
↓

143.0702116
299.037998
284.0986875
282.0844741
383.0846362
537.1685822
269.0878499
267.0736398
137.0457685
357.0689298
303.0502085
803.2361443
571.1311958
281.0272222
310.0588562
311.1235281
220.1178617
176.0739269
538.2619276

1.39
1.39
1.42
1.44
1.44
1.45
1.45
1.45
1.45
1.45
1.45
1.45
1.46
1.49
1.8
2.82
3.09
4.07
4.20

Pos
Neg
Pos
Neg
Neg
Pos
Pos
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos

C7H12O4
C7H14N2O4S2
C10H13N5O5
C10H13N5O5
C12H18O11
C18H32O18
C10H12N4O5
C10H12N4O5
C5H4N4O
C9H6FNO2
C15H14O8
C23H18N2O5
C12H14O8
C6H8N2O8
C14H13N3O3
C14H18N2O6
C9H17NO5
C7H10O2S
C26H45NO6S

↓
↑
↓
↓
↑
↑
↑
↑
↑
↑

↑
↓

28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49

↑
↑
↑
↑
↓
↓
↑
↑

↑
↓
↓
↓
↓
↓
↓
↓
↓
↓
↓
↓
↑
↑
↓
↓

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

Page 7 of 11

Table 1 (continued)
No

Metabolites

m/zRetention Ion
time (min) mode

Formula

50
51
52
53
54

gamma-Glutamylleucine
LysoPE(0:0/18:1(11Z))
LysoPE(18:1(11Z)/0:0)
Stearoylethanolamide4-(beta-Acetylaminoethyl)imidazole

261.1444168
480.3084895
480.3083533
328.3207028
154.0973617

4.55
11.46
11.61
14.15
15.53

C11H20N2O5
C23H46NO7P
C23H46NO7P
C20H41NO2
C7H11N3O

Pos
Pos
Pos
Pos
Pos

Radiation Radivs. Control ationHRW vs.
Radiation
↓
↑
↓
↑
↓
↑
↓
↑
↑
↓

Fig. 3 Metabolic pathway analysis of MetPA. (A) Analysis of ionizing radiation-related metabolic pathways. (B) Analysis of HRW-regulated metabolic
pathways

Regulation of key metabolite-related metabolic pathways
by HRWTo explore the metabolic pathways of ionizing radiation rats, differential metabolites were introduced into
MetaboAnalyst for pathway enrichment analysis. Pathway effect > 0.1 was considered to be a key metabolic
pathway (the results are shown in Fig. 3), and five metabolic pathways related to ionizing radiation were identified, including: glutathione metabolism, ascorbic acidand aldonic acid metabolism, pentose and glucuronic
acid interconversion, glycerophospholipid metabolism, alanine, aspartic acid and glutamic acid metabolism. Four metabolic pathways (glutathione metabolism,ascorbic acid and aldonic acid metabolism, pentose andglucuronic acid interconversion, and glycerophospholipid metabolism) are specifically regulated by HRW. The
above results suggest that HRW may improve ionizing
radiation by regulating multiple metabolic pathways. It

reflects the multi-objective and multi-pathway mechanism of HRW.
Integrative analysis of metabolomics and network
pharmacology

An interaction network based on metabolomics andnetwork pharmacology was constructed to fully understand the mechanism of HRW against ionizing radiation(Fig. 4). Differential metabolites were introduced into the
Cytoscape’s MetScape plug-in to collect a compoundresponse-enzyme-gene network. By matching potential
targets identified in network pharmacology to genes from
the MetScape analysis, we identified one key target. The
key relevant metabolites are phosphatidylcholine, choline
and glucuronide salts. The pathways affected are glutathione metabolism, ascorbic acid and aldehyde acid metabolism, pentose and glucuronic acid interconversion,and glycerophospholipid metabolism. They may play an

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

Page 8 of 11

Fig. 4 The “active ingredient-target/gene-metabolite” biological network constructed by Cytoscape. (Blue nodes: metabolite-related target
genes, red nodes: differential metabolites.)

important role in the therapeutic effects of FWR on ionizing radiation.
Pathway analysis

The KEGG enrichment analysis was performed on thetargets and genes associated with recovery of differential metabolites, and the functions of these 93 key target
genes were predicted and analyzed. The KEGG enrichment results showed that key signaling pathways sensitive to ionizing radiation, such as glutathione metabolism(C2), arachidonic acid metabolism(C1), glycerophospholipid metabolism(C1), and ether lipid metabolism(C1)were significantly enriched [18]. There were interactions
between these signal pathways that can synergisticallyaffect pathways that in turn, synergistically affected the
HRW treatment with ionizing radiation (Fig. 5).
Therapeutic performance evaluation of biomarkers

Brain tissue biomarkers are the cornerstone of ionizing
radiation diagnosis, and their function was assessed by
ROC analysis. A total of three metabolites were selected
as potential biomarkers including phosphatidylcholine,
choline, and D-glucuronate. Figure 6 A shows that the
AUC for phosphatidylcholine, choline, and D- glucuronide were 0.8147, 0.6843, and 0.6503, respectively. In
addition, using a biomarker combination that provided
greater predictive power than a single biomarker, thecombined result for phosphatidylcholine, choline, andD-glucuronide was 0.8771 by logistic regression analysis,
as shown in Fig. 6B. These metabolites may be potential
biomarkers of HRW in ionizing radiation therapy.

Discussion
Radiation remains a health threat and safety hazards,
and. radiation protection agents are attracting increasing
attention. WR-2721 is the only FDA-approved radioprotective agent that can prevent radiation-induced damage, especially hydroxyl radical damage. WR-2721 cancause dizziness, vomiting and other adverse reactions.
Therefore, there is an urgent need to develop an effective and safe radioprotectants. Hydrogen is a safe, convenient, non-toxic substance with little or no side effects
and selective antioxidant function. Hydrogen scavenging prevents oxidative stress-induced damage [19, 20].
Hydrogen has a protective effect on various tissues fromradiation damage. For example, intake of hydrogenrich water after radiation can alleviate damages to theimmune system, skin, hematopoietic system, small intestine, and heart by reducing reactive oxygen species (ROS)[21–25]. Their selective neutralization of harmful oxygen
radicals has attracted increasing attention in the field ofradiation protection. Hydrogen can cross the blood-brain
barrier and quickly cross cell membranes to interact with
toxic ROS. However, the material basis and potential

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

Page 9 of 11

Fig. 5 The KEGG enrichment analysis of HRW regulation-related targets

Fig. 6 ROC curves of the combined markers used to evaluate the effect of the HRW treatment. (A) Phosphatidylcholine; (B) Choline; (C) D-Glucuronate. The combination of phosphatidylcholine, choline, and D-Glucuronate, and had the highest AUC = 0.8771

mechanisms of hydrogen utilization in many neurological diseases remain unclear.
In our previous study, a single partial whole-brain
irradiation with a 6-MeV electron beam of 30 Gy could
successfully induce cognitive dysfunction in rats [26].
Compared with control rats, the number of new neurons
decreased by 67% on the 7th day after 30 Gy whole brain
irradiation [27]; and on the 30th day after irradiation,

there were no surviving new neurons, indicating that
irradiation with 30 Gy successfully induced acute cognitive dysfunction. In this study, a single 30 Gy electron
beam whole brain irradiation was used to establish amodel of cognitive dysfunction in rats. Then, we investigated the effects of HRW on cognitive function and its
possible mechanism using Metabolomics combined withbiological networks. The results showed that after HRW

Liu et al. BMC Molecular and Cell Biology(2023) 24:30

treatment, the cognitive dysfunction of rats was alleviated, and the protective effect was related to the regulation of phosphatidylcholine, choline, and D-Glucuronate.
The results of brain metabolomics showed that HRW cansignificantly regulate the levels of 54 different metabolites, including glutathione metabolism, ascorbate andaldarate metabolism, pentose and glucuronate interconversions, and glycerophospholipid metabolism. Thesepathways were discussed below.
Pyroglutamic acid is a cyclized derivative of L- glutamic
acid produced by non-enzymatic reaction of glutamic
acid, glutamine and γ-glutamine peptide. It can also be
generated by the reaction of γ-glutamyl cyclotransferasewith amino acids. Acquired pyroglutamic acid deficiency(penicillins) and glutathione deficiency (e.g., malnutritionor sepsis) often result in moderate to severe encephalopathy clinically [28]. In our study, decreased levels of relevant metabolites were observed to decrease in ionizing
radiation rats, and these metabolites could be restoredby HRW, indicating that glutathione metallurgy may be
the mechanism by which HRW treats ionizing radiationinduced brain injury.
Cognitive impairment seriously affects people’s physical and mental health and also causes a substantial economic burden [29]. This study showed that HRW canregulate the glycerophospholipid metabolism, which is
the most abundant phospholipid in the body, and canactivate the phospholipase A2 pathway to release arachidonic acid, thereby regulating the metabolism ofglycerophospholipids and improve depression [30]. In
addition, ascorbate and aldarate metabolism, pentose
and gluconate interconversions are closely related to cognitive impairment [31, 32]. Our study found that HRW
can improve ionizing radiation-induced brain cognitive
impairment by modulating these pathways.
Although this study systematically explained the mechanism by which HRW improves cognitive impairment,
there are limitations that require further study: This
study only used non-targeted brain Metabolomics andbiological target network at the level of metabolites andmetabolic pathways. This model could be easily exploitedin future works for the elucidation of pharmacologicaleffects and mechanisms using a multi-omics approachbased on integrated transcriptomics, proteomics andmetabolomics studies.Further studies warrant adding
corresponding pathway inhibitors at the cellular and animal levels for verification. In depth analysis though might
be worth being conducted in future studies.

Conclusions
In summary, the mechanisms of action of HRW on ionizing radiation were predicted through integrating biological networks and metabolomics. HRW may exert its
potential effects by regulating 54 metabolites. In addition,

Page 10 of 11

glutathione metabolism, ascorbate and aldonic acidmetabolism, pentose and glucuronate interconversions,and glycerophospholipid metabolism as well as the levelsof phosphatidylcholine, choline, and D-Glucuronate are
key metabolic pathways/metabolites for HRW function.
These findings will help reveal the mechanism of HRW
on ionizing radiation and provide a rational way to clarify
this mechanism.
Acknowledgements
We thank the support of the Department of Radiology and Environmental
Medicine, China Institute for Radiation Protection,Shanxi,China.
Authors’ contributions
LXM conceived and performed the experiment. LMY wrote the manuscript.
LH and WY analyzed the data, YH and CXM assisted in research. QXJ and LJG
designed the study and protocol. All authors have understood, agreed andapproved the final edition of this manuscript.
Funding
The authors of this study thank their supporters and financial support. This
work was supported by the Natural Science Foundation of Shanxi Province(grant no. 202203021211005 and 201801D1213).
Data Availability
All data generated or analyzed during this study are included in this publishedarticle.

Declarations
Ethics approval and consent to participate
All procedures performed in studies were approved by the Animal Protection
and Ethics Committee of the China Institute for Radiation Protection, and wasreported in accordance with ARRIVE guidelines. All methods are performed inaccordance with the Regulations of China Institute for Radiation Protection on
the administration of laboratory animals.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 17 January 2023 / Accepted: 12 September 2023

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