Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2020, Article ID 6978784, 16 pages
https://doi.org/10.1155/2020/6978784

Research Article
Hydrogen Gas Attenuates Hypoxic-Ischemic Brain Injury via
Regulation of the MAPK/HO-1/PGC-1a Pathway in Neonatal Rats
Peipei Wang ,1,2 Mingyi Zhao ,1 Zhiheng Chen,1 Guojiao Wu,1 Masayuki Fujino,2,3Chen Zhang,1 Wenjuan Zhou,1 Mengwen Zhao,1 Shin-ichi Hirano,4 Xiao-Kang Li ,1,2and Lingling Zhao 1
1

Department of Pediatrics, the Third Xiangya Hospital, Central South University, Changsha, China
Division of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan
3
AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan4
MiZ Co., Ltd., Kanagawa, Japan
2

Correspondence should be addressed to Xiao-Kang Li; ri-k@ncchd.go.jp and Lingling Zhao; llzhao2011@qq.com
Received 11 October 2019; Revised 9 December 2019; Accepted 31 December 2019; Published 13 February 2020
Academic Editor: Paolo Tucci
Copyright © 2020 Peipei Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of death in neonates with no eﬀective treatments. Recent
advancements in hydrogen (H2) gas oﬀer a promising therapeutic approach for ischemia reperfusion injury; however, theimpact of this approach for HIE remains a subject of debate. We assessed the therapeutic eﬀects of H2 gas on HIE and theunderlying molecular mechanisms in a rat model of neonatal hypoxic-ischemic brain injury (HIBI). H2 inhalation signiﬁcantly
attenuated neuronal injury and eﬀectively improved early neurological outcomes in neonatal HIBI rats as well as learning andmemory in adults. This protective eﬀect was associated with initiation time and duration of sustained H2 inhalation.
Furthermore, H2 inhalation reduced the expression of Bcl-2-associated X protein (BAX) and caspase-3 while promoting theexpression of Bcl-2, nuclear factor erythroid-2-related factor 2, and heme oxygenase-1 (HO-1). H2 activated extracellular signalregulated kinase and c-Jun N-terminal protein kinase and dephosphorylated p38 mitogen-activated protein kinase (MAPK) inoxygen-glucose deprivation/reperfusion (OGD/R) nerve growth factor-diﬀerentiated PC12 cells. Inhibitors of MAPKs blocked
H2-induced HO-1 expression. HO-1 small interfering RNA decreased the expression of peroxisome proliferator-activatedreceptor gamma coactivator 1-alpha (PGC-1α) and sirtuin 1 (SIRT1) and reversed the protectivity of H2 against OGD/Rinduced cell death. These ﬁndings suggest that H2 augments cellular antioxidant defense capacity through activation of MAPK
signaling pathways, leading to HO-1 expression and subsequent upregulation of PGC-1α and SIRT-1 expression. Thus,
upregulation protects NGF-diﬀerentiated PC12 cells from OGD/R-induced oxidative cytotoxicity. In conclusion, H2 inhalation
exerted protective eﬀects on neonatal rats with HIBI. Early initiation and prolonged H2 inhalation had better protective eﬀects
on HIBI. These eﬀects of H2 may be related to antioxidant, antiapoptotic, and anti-inﬂammatory responses. HO-1 plays animportant role in H2-mediated protection through the MAPK/HO-1/PGC-1α pathway. Our results support further assessmentof H2 as a potential therapeutic for neurological conditions in which oxidative stress and apoptosis are implicated.

1. Background
Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe
disease with high neonatal morbidity and mortality; 25% of
HIE survivors have permanent neurological defects [1].
Before irreversible brain injury occurs, the speciﬁc pathological process of hypoxia-ischemia (HI) occurs through a
combination of multiple mechanisms [2, 3]. The severity

and duration of these mechanisms determine the extent ofbrain injury in HIE [4].
Molecular hydrogen (H2) easily penetrates the bloodbrain barrier and is a novel antioxidant [5]. Previous studies
have shown that H2 mitigates ischemia/reperfusion- (I/R-)
induced injury to diﬀerent organs [6–8]. However, the neuroprotective eﬀects of H2 treatment on hypoxic-ischemic
brain injury (HIBI) are controversial. For example, a recent

2Oxidative Medicine and Cellular Longevity
3% H2 inhalation
HIBI

Postnatal day

7

ReflexesIF
TTC Western blot

8

10

Morris water maze

40

44

Morris water maze

70

74

Figure 1: Experimental diagram in vivo.

study showed that H2-enriched water exerts neuroprotective
eﬀects on brain tissue after induction of HIBI [9]. However,another study found no protective eﬀects [10].
Oxidative stress is a well-recognized consequence of HIE
and is considered an important contributor to early brain
injury after HI [11] working in combination with inﬂammation [12]. Growing evidence suggests that oxidative stress andneuroinﬂammation underpin a diverse range of central nervous system (CNS) diseases including stroke, traumatic brain
injury, multiple sclerosis, Alzheimer’s, Parkinson’s, and otherneurodegenerative diseases [13–15]. Heme oxygenase-1(HO-1) is an important component of the cellular defense
enzyme that is induced by and acts against oxidant-induced
I/R injury [16]. HO-1 overexpression may also aﬀect the regulation of apoptotic pathway genes such as B-cell lymphoma
2 (Bcl-2), Bcl-2-associated X protein (BAS), and caspases[17]. HO-1 has antineuroinﬂammatory and neuroprotective
properties in the CNS [18, 19]. Thus, therapies targeting
HO-1 may be potential treatments for protection against
inﬂammation, oxidative stress, and apoptosis after HI.
Multiple signaling kinases related to cell survival andproliferation reportedly regulate the nuclear translocationof HO-1. Mitogen-activated protein kinases (MAPKs) aresome of the most common signaling pathways, which
serve to coordinate the cellular response to a variety of
extracellular stimuli [20]. These are well characterized in
mammals and include c-Jun N-terminal kinase (JNK),
p38 MAPK, and extracellular signal-regulated kinase(ERK) [21, 22]. Activation of MAPKs modulates HO-1
expression [23].
In this study, we evaluated the neuroprotective eﬀects of
H2 on neonatal HIBI rats through behavioral tests, immunoﬂuorescence, and western blot analysis to determine theappropriate therapeutic window for of H2. In vivo, we also
tested the expression of high-mobility group protein B1(HMGB1) and toll-like receptor 4 (TLR-4), the apoptosisrelated factors Bcl-2/BAX-caspase3, and the oxidative stresssignaling molecules p38 MAPK/Nrf2/HO-1. Then, we investigated the potential MAPK/HO-1/peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC-1α) signaling pathway in nerve growth factor- (NGF-) diﬀerentiated
PC12 cells after oxygen-glucose deprivation/reperfusion(OGD/R).

2. Methods
2.1. Experimental Design In Vivo. A total of 320 neonatal rats
were randomly divided into the following eight groups(n = 40/group): HIBI, HIBI+H2-30 min, HIBI+H2-60 min,
HIBI+H2-90 min, HIBI+H2-90 min (12 h), HIBI+H290 min (24 h), sham surgery, and control. Animals in
the HIBI and all HIBI+H2 treatment groups were rats
with induced HIBI. After establishing the HIBI model,
neonatal rats in the HIBI+H2-30 min, HIBI+H2-60 min,and HIBI+H2-90 min groups were immediately placed ina box for 30, 60, or 90 min of H2 inhalation, respectively.
This was followed by twice daily H2 inhalation (30, 60, or
90 min each time) over the next 3 days (7 : 00–9 : 00 am
and 7 : 00–9 : 00 pm). Animals in the HIBI+H2-90 min(12 h) and HIBI+H2-90 min (24 h) groups underwent 3%
H2 inhalation for 90 min at 12 and 24 h, respectively,
after induction of HI. This was followed by twice daily
H2 inhalation for 90 min for 3 days. Animals in the shamsurgery group underwent left carotid artery isolation
without ligation and hypoxia induction. Animals in thecontrol group were healthy neonatal rats without induction of HIBI. Detection of early neurological reﬂexes
was performed ~24 h after HIBI induction, and thensome neonatal rats were subjected to triphenyltetrazolium
chloride (TTC) staining (n = 8/group). At 96 h after
inducing HIBI, brain samples from neonatal rats werecollected for western blotting, paraﬃn-embedded, andsectioned for terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) and immunoﬂuorescencestaining. In addition, some of the neonatal rats
(n = 8/group) in adulthood (postnatal days 70–74) underwent testing in a Morris water maze (Figure 1). The
study was performed in a blinded and randomized matterwith respect to treatment administration and histologicaland functional assessments.
2.2. Experimental Animals. Neonatal Sprague-Dawley rats
(males and females, 7 days old, 12–18 g), were provided
by the Department of Laboratory Animals, Central South
University (Hunan Province, China). All pups werehoused with their mothers before and after the experiments in a barrier environment (all measurement indicators were in conformity with the national standard“Experimental Animal Environment and Facilities
GB14925, 2010”). All of the experiments were conductedin accordance with the Guide for the Care and Use ofLaboratory Animals (National Institute of Health Publication No. 80-23, revised 1996), and approved by the Center for Medical Ethics of the Third Xiangya Hospital(Hunan, China). Sincere eﬀorts were made to minimize
animal suﬀering and to reduce the number of animals
used.

Oxidative Medicine and Cellular Longevity
2.3. HIBI Modeling and H2 Inhalation. Neonatal rats at 7 daysof age were anesthetized with isoﬂurane inhalation. Thiswas followed by making a 0.7 cm incision to the left ofthe cervical midline to separate the left common carotid
artery, which was severed after bilateral ligation. Surgicalsuture 6-0 was used to close the incision. The entire surgical procedure was completed within 8 min. Neonatal rats
recovered in the same cage as their mother for 2 h after
surgery. Next, they were placed in an anoxic tank (Gaoke
Gas Technology Co. Ltd., Changsha, China) containing 8%
oxygen and 92% nitrogen at 37°C to induce hypoxia for
100 min. After hypoxic exposure, neonatal rats wereallowed to recover or underwent H2 inhalation. The
inhaled gas for H2 intervention was a mixture of H2 andair. H2 was generated by electrolysis of water in the
MHG-2000 Hydrogen Generator (MiZ Co. Ltd., Kanagawa, Japan) at a concentration of 3%.
2.4. TTC Staining. The infarct volume was determined by
TTC staining. Using this method, the brain sections
were prepared as follows. First, the brains were removed
and frozen at -20°C for 10 min. Next, consecutive 2 mm
coronal sections were obtained by slicing the brains witha Brain Matrix (ASI Instruments, Warren, MI, USA).
The subsequent incubation of the sections was performed in a dark environment with a 30 min immersion
in 2% TTC solution at 37°C. TTC stained normal areasof brain deep red but did not stain the infarcted tissue.
Infarction volumes were measured and analyzed with
ImageJ software (version 1.61; NIH Image, Bethesda,
MD, USA).
2.5. Immunoﬂuorescence. After the last H2 treatment, rats
were perfused with cold 0.1 M phosphate-buﬀered saline(PBS, pH 7.4) through the left ventricle. The brains were ﬁxed
in 4% paraformaldehyde overnight. Fixed brain tissues were
further processed by dehydration in graded ethanol prior to
paraﬃn embedding. Finally, the blocks were serially cut into
5 μm thick sections and stored at 4°C. After deparaﬃnization,
the sections were pretreated within antigen unmasking
solution (Vector Laboratories, Burlingame, CA, USA) for
15 min in the microwave oven (medium ﬁre for 8 min, ceased
ﬁre for 8 min, and low ﬁre for 7 min). After washing with
PBS, the sections were quenched with spontaneous ﬂuorescence quenching agent (Servicebio, Wuhan, China) for
5 min at room temperature. Nonspeciﬁc binding sites were
blocked by incubating the sections in 5% bovine serum albumin for 30 min. This was followed by an overnight incubation at 4°C with the following primary antibodies: anti-glialﬁbrillary acidic protein (GFAP) (1 : 1000; Sigma, St. Louis,
MO, USA), anti-ionized calcium-binding adaptor molecule
1 (1 : 1000; Wako, Osaka, Japan), anti-HMGB1 (1 : 500;
Abcam, Cambridge, MA, USA), anti-TLR4 (1 : 100; Servicebio), anti-Nrf2 (1 : 100; Abcam), anti-HO-1 (1 : 100; Abcam),anti-BAX (1 : 100; Abcam), anti-Bcl2 (1 : 100; Abcam), anticaspase3 (1 : 100; Servicebio), and anti-p-p38 MAPK
(1 : 100, Abcam). Then the sections were immersed in PBS
and incubated with the secondary antibody (Servicebio) for
50 min at room temperature. Finally, the sections were incu-

3
bated for 10 min with DAPI. After washing with PBS, the sections were sealed with antiﬂuorescence quenching sealantand scanned using Pannoramic MIDI (3DHISTECH Ltd.,
Budapest, Hungary). CaseViewer software was used to merge
the stained images and integrate the ﬂuorescence emission
intensity.
2.6. Detection of Early Neurological Reﬂexes. Twenty-fourhours (24 h) after establishing the HIBI model, early neurological reﬂexes [24] including righting, cliﬀ aversion,and geotaxis reﬂexes were evaluated in neonatal rats asfollows. (1) The righting reﬂex was assessed by placing
each neonatal rat in a supine position and recording thetime (in seconds) to roll over to a normal prone position.
For each neonatal rat, the test was performed three times
to obtain a mean value. (2) The cliﬀ aversion reﬂex wasassessed by dangling the upper limbs of each neonatal
rat from a board edge and recording the time to turn
90° away from the cliﬀ edge. The maximum duration ofobservation was 20 s. Neonatal rats that did not turn 90°
within the observation period were recorded at a time of
20 s. (3) The geotaxis reﬂex was measured by placing thehead of each neonatal rat downward on a 40° inclined
plate and recording the time required for turning around(with the head facing upward and >90° rotation). The
maximum duration of observation was 20 s. Neonatal ratsthat did not turn within the observation period wererecorded at a time of 20 s.
2.7. Morris Water Maze Test. The Morris water maze wasused to study the eﬀects on spatial learning and memory inrats [25, 26]. The test was performed at week 10 after establishing the HIBI model (postnatal days 70–74, representing
rats in adulthood [27]). Rats were tested in a pool of 160 cm
in diameter. The pool was ﬁlled with water at a depth of
40 cm. The rescue landing platform (8 cm in diameter)
was submerged by 1 cm under the water surface in oneof four quadrants. Water temperature was maintained at
23°C–24°C. Testing consisted of four consecutive training
days during which four attempts (starts from four quadrants) were given to each rat to ﬁnd the platform within
60 s. Each rat was given 20 min to rest between swimmingtrials. The training session occurred at the same time of
day throughout the training and testing periods. A rat
was placed in the water in the quadrant opposite to thelanding, and the latency time (in seconds) spent by eachanimal to ﬁnd the platform was recorded. If the rat was
not able to locate and climb on the landing platform
within the allotted time, the experimenter gently placed
the animal on the platform and let the animal become
accustomed to it for 20 s. On day 5 of probe training testing, the rescue platform was removed and the time spentin the quadrant where the landing used to be wasrecorded (allotted time 60 s). Only one attempt was given
to each rat during the probe trial on day 5 of testing.
2.8. Western Blot Analysis. Rats were sacriﬁced by decapitation after the last H2 treatment, and the brain was microdissected and stored at -80°C. NGF-diﬀerentiated PC12 cells

4were washed with PBS and lysed in RIPA buﬀer (Proteintech, Rosemont, PA, USA) for 30 min on ice. The protein
concentration in the extracts was determined using the
BCA assay (Wellbio, Changsha, China). Equal amounts ofprotein (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred topolyvinylidene ﬂuoride membranes (Millipore, Billerica,
MA). Membranes were blocked in 5% skim milk powder
dissolved in phosphate-buﬀerred saline with 0.5% Tween
20 in PBS (PBST) at 4°C overnight and then incubated withthe following antibodies: anti-GFAP (1 : 1000; Abcam),anti-HMGB1 (1 : 1000; Abcam), anti-TLR4 (1 : 500; Proteintech, Rosemont, IL, USA), anti-phosphorylated nuclear factor kappa B (p-NF-κB) p65 (1 : 1000; Abcam), anti-caspase3(1 : 1000; Abcam), anti-BAX (1 : 1000; Abcam), anti-Bcl-2(1 : 500; Proteintech), anti-p-p38 MAPK (1 : 1000; Abcam),anti-p-ERK1/2 (1 : 1000; Abcam), anti-p-JNK antibody(1 : 1000; Abcam), anti-Nrf2 (1 : 1000, Abcam), anti-HO-1(1 : 1000; Abcam), anti-PGC-1α (1 : 1000; Abcam), andanti-SIRT1 (1 : 1000; Abcam). β-Actin (Proteintech) wasused as the gel loading control. Following overnight incubation, blots were washed three times with PBST and thenincubated with the secondary antibody (IRDye 800CW
Goat Anti-Rabbit IgG, 1 : 10000; Invitrogen, Carlsbad, CA,
USA). Proteins were detected using the Odyssey Infrared
Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
Band intensity was quantiﬁed using ImageJ software.
2.9. Cell Culture and Diﬀerentiation. The PC12 cell line wasobtained from American Type Culture Collection (Manassas,
VA, USA) and maintained at 37°C in a humidiﬁed
atmosphere containing 5% CO2 in high-glucose Dulbecco’s
Modiﬁed Eagle’s Medium supplemented with 10% heatinactivated fetal calf serum, 10% heat-inactivated horse
serum, 100 kU/L penicillin, and 100 mg/L streptomycin. For
diﬀerentiation, PC12 cells were plated at a low density on
collagen-coated plastic in DMEM plus 1% horse serum andNGF (50 ng/mL) for 7 days.
2.10. PC12 Model of OGD. The cell culture was incubated at
37°C in a low oxygen incubator (1% O2, 94% N2, and 5%
CO2) for 6 h (OGD). Then, glucose-free DMEM was replacedwith normal medium, followed by incubation for 3/6/24 h(OGD/R) in standard culture gas (N2 as base gas, O221%, and CO2 5%). For the OGD/R+H2 group, cells wereincubated for 3/6/24 h in standard culture gas with H2 gas(N2 as base gas, O2 21%, CO2 5%, and H2 2–3%). Immediately after inducing OGD/R or OGD/R+H2, cells werecollected for western blotting, quantitative PCR (qPCR),
ﬂuorescence-activated cell sorting (FACS), and immunoﬂuorescence staining.
2.11. RNA Extraction and qPCR Analysis. All RNA wasextracted from cells using a DNA/RNA Mini Kit (QIAGEN,
Germantown, MD USA). Reverse transcription was doneusing the High Capacity cDNA Reverse Transcription Kit(Thermo Fisher Scientiﬁc, Waltham, MA USA). Quantitative
measurements of target gene expression relative to 18 s RNA
were performed in triplicate using the TaqMan Real-Time

Oxidative Medicine and Cellular Longevity
PCR assay following the manufacturer’s recommendations
in a Real-Time PCR system.
2.12. Annexin V and Propidium Iodide Staining Assay. Cells
were detached and collected into a 15 mL centrifuge tube,
washed twice with ice-cold PBS, and centrifuged at1000 rpm for 5 min to remove the supernatant. A volume of
0.1 mL binding buﬀer containing 0.14 M NaCl, 2.5 mM
CaCl2, and 0.01 M HEPES/NaOH pH 7.4 was added to resuspend the cells, followed by adding 5 μL Annexin V-FITC and5 μL of 50 μg/mL propidium iodide (PI) staining reagents.
After mixing homogeneously and reacting at 25°C for
15 min in the dark, the apoptotic cell population was analyzed by a ﬂow cytometer.
2.13. Statistical Analysis. Mean ± standard deviation was calculated for all parameters determined in this study.
Repeated measures of two-way analysis of variance(ANOVA) were used for each parameter of the water
maze task, followed by multiple t-test analyses. One-way
ANOVA followed by Tukey’s multiple-comparison andwestern blotting was used for histopathological parameters(volume and cell density). P < 0:05 was considered statistically signiﬁcant.

3. Results
3.1. H2 Signiﬁcantly Reduces Areas of Cerebral Infarction,
Neuronal Loss, and Glial Activation in HIBI Rats. As shownin Figures 2(a) and 2(c), TTC staining showed no obvious
white infarcts in either the left or right cerebral hemispherein the normal and sham surgery groups but showed obvious white infarcts in the left cerebral hemisphere of the
HIBI group, with ligation of the left common carotid
artery. The area of cerebral infarction was signiﬁcantly
reduced with an increase in duration of H2 inhalation(∗ p < 0:05; ∗∗ p < 0:01). As shown in Figure 2(b), antibodies
for two diﬀerent biomarkers, neuronal nuclei (NeuN) and
GFAP, were used in immunoﬂuorescence staining toobserve changes in neurons and astrocytes in the hippocampus. Compared with the normal and sham surgery groups,
the immunoreactivity of NeuN was signiﬁcantly reduced in
the cortex and hippocampal CA3 regions of the HIBI group(∗ p < 0:05; ∗∗ p < 0:01; Figure 2(d)), which suggested that
HIBI caused neuronal loss. In addition, the immunoreactivity of GFAP was signiﬁcantly increased in the HIBI group(∗ p < 0:05; ∗∗ p < 0:01; Figure 2(e)), which suggested thatastrocytes were activated. However, compared with the
HIBI group, the immunoreactivity of NeuN was signiﬁcantly increased in the cortex and hippocampal CA3
regions of the H2 inhalation groups, especially in the H290 min group, and the immunoreactivity of GFAP was signiﬁcantly decreased in the H2 inhalation groups. Thus, H2inhalation might protect neurons from injury and reduce
activation of astrocytes. We also found that the decreaseof neuronal loss was signiﬁcantly associated with the duration and initiation time of H2 inhalation. H2 inhalation for
90 min immediately after HIBI had the most signiﬁcantprotective eﬀect (∗ p < 0:05; ∗∗ p < 0:01), while 90 min H2

Oxidative Medicine and Cellular Longevity
NormalSham surgery

5

HIBI

H2-30 minH2-60 minH2-90 minH2-90 min (12 h) H2-90 min (24 h)(a)1000 𝜇m1000 𝜇m1000 𝜇m1000 𝜇m1000 𝜇m1000 𝜇m1000 𝜇m1000 𝜇m

200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m

(c)

(d)

⁎⁎

250

⁎
⁎⁎

200
150
100
50
H2-90 minH2-60 minH2-30 minHIBI

Sham surgery

0
NormalNumber of GFAP+ cells(/mm2)

0H2-90 minH2-90 minH2-60 minH2-30 minHIBI

Sham surgery

025
H2-60 min550

H2-30 min

10

⁎

75

HIBI

15

⁎

⁎⁎

100

Normal20

125

Sham surgery

⁎

⁎⁎
⁎⁎

NeuN+ cells(each/control group) (%)

25

NormalInfarct volume(% of ipsilateral hemisphere)

(b)

(e)

Figure 2: H2 reduced the area of infarction, neuronal loss, and glial activation in HIBI rats. (a) Representative results of TTC staining of eachgroup. (b) Double-immunoﬂuorescence staining (NeuN, green, expressed by neurons; GFAP, red, expressed by activated astrocytes) of eachgroup (scale bar: 1000 μm and 200 μm). (c) Percentage of cerebral infarct volume in the ipsilateral hemisphere of diﬀerent groups. Percentage
of cerebral infarct volume was calculated as follows: percentage of cerebral infarct volume = cerebral infarct volume/normal cerebral
hemisphere volume × 100%. Compared with the HIBI group, the percentage of cerebral infarct volume was signiﬁcantly reduced in the H2intervention groups (n = 8/group; ∗ p < 0:05; ∗∗ p < 0:01). (d, e) Bar graphs comparing relative NeuN immunoreactivity (NeuN+ cells) ofeach group/sham surgery group and the number of GFAP-immunoreactive cells (/mm2), respectively, in diﬀerent groups (n = 8/group;∗p < 0:05; ∗∗ p < 0:01).

inhalation 24 h after HIBI had no signiﬁcant protective
eﬀect on neuronal injury but did reduce activation ofastrocytes, which suggests that astrocytes may be involvedin the processes of HIBI.
3.2. H2 Improves Early Behavioral Reﬂexes in HIBI Rats. To
evaluate the neuroprotective eﬀects of H2 on HI brain injury,
we evaluated the righting, geotaxis, and cliﬀ aversion reﬂexesof rats 24 h after HIBI. Neonatal rats in the HIBI group had asigniﬁcantly slower righting reﬂex, and H2 inhalation
improved the righting reﬂex in this group. Times for therighting reﬂex in the neonatal HIBI rats were signiﬁcantly
shortened with earlier initiation and extended duration of
H2 inhalation (∗ p < 0:05, ∗∗ p < 0:01 vs. the sham surgery
group ∗∗∗ p < 0:01; Figure 3(a)). In assessment of the geotaxis
reﬂex, neonatal rats in the HIBI group took signiﬁcantly longer to turn from facing downward on the inclined plate, withthe time to turn being three times greater than that of thesham surgery group. H2 intervention improved the geo-

taxis reﬂex (∗ p < 0:05, ∗∗ p < 0:01 vs. the sham surgery
group ∗∗∗ p < 0:01; Figure 3(b)). However, no signiﬁcantdiﬀerence was found between the H2-60 min and H290 min groups (p > 0:05). Improvement in time for turning
was less pronounced with later H2 intervention in neonatal
HIBI rats (Figure 3(b)). Assessment of the cliﬀ aversion
reﬂex showed results similar to those of the righting andgeotaxis reﬂexes. Neonatal rats in the HIBI group had longer cliﬀ avoidance times, and H2 inhalation decreased cliﬀ
avoidance time in this group. However, signiﬁcant
improvement in cliﬀ avoidance time was only found in
the H2-60 min and H2-90 min groups (∗∗ p < 0:01 vs. thesham surgery group ∗∗∗ p < 0:01; Figure 3(c)).
3.3. H2 Improved Learning and Memory of HIBI Rats during
Adulthood. The spatial memory performance was evaluatedusing the Morris water maze. Rats were subjected to repeatedMorris water maze tests 10 weeks after HI exposure or shamoperation. The results showed that neonatal HIBI induced

6

Oxidative Medicine and Cellular Longevity

⁎⁎⁎

25

10
5
0

15
10
5

(a)

(b)

H2-90 min (24 h)

H2-90 min (12 h)

H2-90 minH2-60 minH2-90 min (24 h)

H2-90 min (12 h)

H2-90 minH2-60 minH2-30 minHIBI

Sham surgery

0
Normal

H2-90 min (24 h)

H2-90 min (12 h)

H2-90 minH2-60 minHIBI

H2-30 minSham surgery

Normal

0

20

HIBI

2

15

⁎⁎⁎
⁎⁎⁎
⁎⁎

H2-30 min4

20

Sham surgery

6⁎⁎⁎ ⁎⁎

25

Normal8Time (seconds)

Time (seconds)

⁎

⁎⁎⁎
⁎⁎⁎
⁎⁎
⁎
Time (seconds)

⁎⁎⁎

10

⁎⁎⁎
⁎⁎⁎
⁎
⁎⁎
⁎⁎

(c)

Figure 3: H2 improved the righting, geotaxis, and cliﬀ aversion reﬂexes in HIBI rats. (a) Righting, (b) geotaxis, and (c) cliﬀ aversion reﬂex
performance in neonatal 8-day-old rats (n = 8/group; ∗ p < 0:05; ∗∗ p < 0:01).

signiﬁcant long-lasting cognitive deﬁcits throughout brain
maturation and adulthood. As shown in Figures 4(a) and
4(b), the mean latency to ﬁnd the platform declined progressively during the period of training days in all animals
except the HIBI and H2-90 min (24 h) groups. The latent
period of the HIBI group was signiﬁcantly longer thanthat of the control and sham surgery groups. H2 inhalation signiﬁcantly shortened the latent period. This eﬀect
was correlated with the duration of H2 inhalation, andthe H2-90 min group had the most signiﬁcant improvement among the H2 intervention groups. Delays in initiation of H2 intervention reduced the neuroprotective eﬀects
of H2 against brain injury. The navigational memory of
the rats was assessed by removing the escape platform
on the ﬁfth day of the Morris water maze test. Neonatal
HIBI rats with H2 intervention in diﬀerent groups demonstrated better navigational memory than the HIBI group,and the H2-60 min, H2-90 min, and H2-90 min (12 h)groups showed signiﬁcant diﬀerences (∗ p < 0:05; ∗∗ p <0:01; Figures 4(c) and 4(d)).
3.4. H2 Inhibits Activation of HMGB1 and TLR-4 Pathways in
HIBI Rats. To explore the protective mechanism of H2 on
HIBI, we assessed the expression of HMGB1, TLR-4, and
NF-κB-p65 in the hippocampus of neonatal rats. As
shown in Figure 5, we found signiﬁcantly enhanced
expression of TLR-4 and GFAP in the HIBI group, suggesting that astrocytes were associated with enhanced
TLR-4 signaling. In the HIBI+H2 groups, TLR-4 and
GFAP expression was lower compared with the HIBI
group and the longer the H2 inhalation, the lower the
TLR-4 and GFAP expression. We also found that the
HMGB1 expression was mainly localized in the nuclei.
In the HIBI group, HMGB1 expression was signiﬁcantly
enhanced and detected outside the nuclei and GFAP
expression was also enhanced. In the HIBI+H2 groups,
GFAP expression was reduced compared with the HIBI

group and HMGB1 expression was conﬁned to the nuclei,
suggesting that the secretion of HMGB1 was reduced after
H2 inhalation (∗∗ p < 0:01; Figure 5).
3.5. H2 Improves Hippocampal Bcl-2/BAX/Caspase-3 Protein
Expression in HIBI Rats. As shown in Figure 6(a), TUNEL
staining showed no detectable neuronal apoptosis in thesham surgery group but showed elevated apoptosis neurons in the cortex and hippocampal CA3 region of the
HIBI group. The number of apoptotic neurons was significantly reduced in the H2-90 min group. The AnnexinV/PI double staining assay further conﬁrmed that H2
attenuated apoptotic events in NGF-diﬀerentiated PC12
cells following OGD/R. Representative assay results areshown in Figure 6(b), and statistical analyses are summarized in Figure 6(c). Both early (Annexin V+/PI-) and late
(Annexin V+/PI+) stages of apoptotic PC12 cells increasedin the OGD/R group, while H2 reduced the number of
apoptotic PC12 cells (∗ p < 0:05; ∗∗ p < 0:01). Bcl-2 andBAX genes have been recognized as the two most important regulatory genes that are functionally antagonistic toeach other in the regulatory processes of apoptosis. Bcl-2
inhibits apoptosis, while BAX promotes apoptosis.
Caspase-3 is the most critical apoptosis executioner protease. In this study, the HIBI group showed downregulated
Bcl-2 protein expression and upregulated BAX andcaspase-3 protein expression (Figure 6(d)). Bcl-2 protein
expression in the H2 intervention groups was higher thanthat in the HIBI and OGD/R groups, while BAX andcaspase-3 protein expression in the H2 intervention groups
was lower than that in the HIBI and OGD/R groups(∗∗ p < 0:01; Figures 6(e)–6(g)).
3.6. H2 Regulates the Expression of MAPK/Nrf2/HO-1. We
examined the expression of p-p38 MAPK, HO-1, andNrf2 in the cortex of HIBI rats. As shown in
Figure 7(a), H2 signiﬁcantly enhanced the p-p38 MAPK,Oxidative Medicine and Cellular Longevity
Sham surgery

HIBI

H2-30 minH2-60 minH2-90 minH2-90 min (12 h) H2-90 min (24 h)Day 4Normal

7

Day 5

(a)

(b)

⁎
⁎
⁎
⁎⁎ ⁎

⁎⁎
⁎
⁎
⁎
⁎⁎ ⁎

60
⁎⁎
Time spent in the“platform” quadrant (%)

Latency (seconds)

60

40

20

0
Day 2

Day 3

Day 4

Training day
Normal

H2-60 minSham surgery

H2-90 minHIBI
H2-30 minH2-90 min (12 h)

⁎

⁎⁎

⁎

40

20

0
Day 1

⁎

⁎⁎

Day 5
Probe training day

H2-90 min (24 h)
(c)

(d)

Figure 4: H2 improved learning and memory of HIBI rats during adulthood. (a) Diagrams indicating movement of rats in the normal (A),sham surgery (B), HIBI (C), H2-30 min (D), H2-60 min (E), H2-90 min (F), H2-90 min (12 h) (G), and H2-90 min (24 h) (H) groups to locate
the escape platform during the day 4 training trial. (b) Diagrams indicating 60 s movement of rats in the normal (A), sham surgery (B), HIBI(C), H2-30 min (D), H2-60 min (E), H2-90 min (F), H2-90 min (12 h) (G), and H2-90 min (24 h) (H) groups to locate the escape platform
during the day 5 probe trial. (c) Bar graph of latency to locate the escape platform during training trials on days 1 through 4 in diﬀerent
groups of rats (n = 8/group; ∗ p < 0:05; ∗∗ p < 0:01). (d) Spatial preference (percentage of time spent) in the escape platform quadrant
during the day 5 probe trial (n = 8/group; ∗ p < 0:05; ∗∗ p < 0:01).

HO-1, and Nrf2 expression in the HIBD rat cortex. As
shown in Figures 7(b) and 7(c), H2 signiﬁcantly enhanced
the p-p38 MAPK, p-ERK1/2, p-JNK, HO-1, and Nrf2
expression in NGF-diﬀerentiated PC12 cells following
OGD/R (∗∗ p < 0:01).
3.7. Role of p38 MAPK, ERK, and JNK in HO-1 Expression
Induced by H2. The eﬀects of p38 MAPK, ERK, and JNK inhibition on the expression of HO-1 are shown in Figure 8. The
p38 MAPK pathway inhibitor SB203580, ERK inhibitor
U0126, and JNK inhibitor SP600125 signiﬁcantly reduced

OGD/R-induced injury, and H2 plus OGD/R injury induced
HO-1 expression (∗∗ p < 0:01).
3.8. Role of HO-1 in PCG1-α and SIRT1 Expression Inducedby H2. The eﬀects of HO-1 silencing on apoptosis and expression of PGC-1α and SIRT1 are shown in Figure 9. NGFdiﬀerentiated PC12 cells treated with HO-1 small interfering
RNA (siRNA) showed increased apoptosis events following
OGD/R injury, and H2 did not attenuate apoptosis(Figures 9(a) and 9(b)). As shown in Figures 9(c)–9(i), HO1 siRNA markedly decreased the expression of HO-1, PGC-

8

Oxidative Medicine and Cellular Longevity
Sham surgery

HIBD

Sham surgery

H2-90 min

HIBD

H2-90 min

HMGB1

TLR4
20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

GFAP

GFAP

DAPI

DAPI
20 𝜇m

20 𝜇m

20 𝜇m

Merge

Merge
20 𝜇m

20 𝜇m

20 𝜇m

(a)
HIBD

H2-90 min
0.8

𝛽-Actin

0.6

TLR4/𝛽-actin

TLR4

HMGB1

⁎⁎

0.4
0.2

𝛽-Actin

⁎⁎

⁎⁎

0.8
0.6
0.4
0.2
0.0

0.0
Sham surgery
(c)

1.0

⁎⁎
HMGB1/𝛽-actin

Shamsurgery

(b)

HIBD
(d)

H2-90 minSham surgery

HIBD

H2-90 min(e)

Figure 5: H2 inhibited HMGB1/TLR-4 expression in the hippocampal CA3 region of neonatal HIBI rats. (a) The representative images of
TLR-4 (red) and glial ﬁbrillary acidic protein (GFAP, green) and 4 ′ ,6-diamidino-2-phenylindole (DAPI, blue) immunoﬂuorescencestaining as well as merged immunoﬂuorescent signals of all markers in the hippocampal CA3 region of sham surgery, HIBI, and H290 min groups (scar bar: 50 μm). (b) Representative images of HMGB1 (red) and glial ﬁbrillary acidic protein (GFAP, green) and 4 ′ ,6diamidino-2-phenylindole (DAPI, blue) immunoﬂuorescence staining as well as merged immunoﬂuorescent signals of all markers in the
hippocampal CA3 region of sham surgery, HIBI, and H2-90 min groups (scale bar: 50 μm). (c) Western blot analysis of TLR-4, HMGB1,
and β-actin proteins in the hippocampus of each group. (d, e) Bar graphs of the relative expression of TLR-4, HMGB1 in the
hippocampus of each group (n = 3/group; ∗∗ p < 0:01).

1α, and SIRT1 induced by H2 in NGF-diﬀerentiated PC12
cells following OGD/R (∗ p < 0:05; ∗∗ p < 0:01).

4. Discussion
Neonatal HIE is the most common cause of neonatal deathand disability, often causing motor, sensory, and cognitive
impairments [28]. Mild hypothermia has been recognized
as an eﬀective treatment to reduce HIE mortality. However,the disability and mortality rates of HIE are still very high[29]. Therefore, studying new eﬀective and safe treatments
for HIE is an important task in medical research. The neuroprotective eﬀects of H2 treatment against HIBI are controversial. Cai et al. [9] found that H2 therapy signiﬁcantly reduced
the number of TUNEL-positive cells and attenuated caspase
activity, suggesting that H2 gas exerts neuroprotective eﬀects

by inhibiting cellular apoptosis after HI. However, in anotherstudy of HI, hydrogen treatment failed to show neuroprotective eﬀects, possibly as a result of the severity of injury [10].
In both studies, HIBI rats received H2 inhalation only once,
without systematically studying possible pathways. Therefore, we considered whether H2 gas treatment of HIE isrelated to the degree of HI and the duration of inhalation.
In this study, we reported for the ﬁrst time that theprotective eﬀects of H2 inhalation are related to the start
time and duration. First, anatomical, cerebral, and hippocampal infarction, neuronal loss, and astrocyte activation
were found in neonatal rats during the early HIBI stage;in H2 inhalation groups, these injuries were alleviated,with the most signiﬁcant results occurring in the H290 min group (Figure 2). We also conducted short-term
behavioral analyses of rats and showed that HIBIOxidative Medicine and Cellular Longevity

9

Hippocampal CA3 Cerebral cortex

Sham surgery

HIBD

H2-90 min

100 𝜇m

100 𝜇m

100 𝜇m

100 𝜇m

100 𝜇m

100 𝜇m(a)Normal

OGD6h-R24 h

OGD6h-R24 h+H2

200 𝜇m200 𝜇m

3

3

102

101

100

100

101

102

10
FL1 INT LOG: Fl1 INT LOG

10
FL1 INT LOG: Fl1 INT LOG

FL1 INT LOG: Fl1 INT LOG

10PI200 𝜇m

3

102

101

100

103

100

FL1 INT LOG: Fl1 INT LOG

101

102

102

101

100

103

100

FL1 INT LOG: Fl1 INT LOG

101

102

103

FL1 INT LOG: Fl1 INT LOG

Annexin V(b)

Percentage of cells (%)

100
80
60

Sham surgery

⁎⁎
⁎⁎ ⁎⁎
⁎⁎
⁎⁎

40

HIBD

H2-90 min

Bcl-2

⁎⁎ ⁎

200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m200 𝜇m20
0Annexin V(+)
/ PI(−)

Annexin V(−)
/ PI(+)

Bax

Annexin V(−)
/ PI(−)

Normal
OGD6h-R24h
OGD6h-R24h+H2

Caspase-3(c)(d)
Shamsurgery

HIBI

H2-90 minControl

Bcl-2
Bax
Caspase-3
𝛽-Actin(e)

Figure 6: Continued.

OGD6h
R24h

OGD6h
R24h+H210

Oxidative Medicine and Cellular Longevity
⁎⁎
⁎⁎ ⁎⁎

⁎⁎
⁎⁎ ⁎⁎

1.0
Target protein/𝛽-actin

Target protein/𝛽-actin

2.5
2.0

⁎⁎ ⁎⁎

1.5

⁎⁎ ⁎⁎

1.0
0.5

⁎⁎
⁎⁎ ⁎⁎

0.8
0.6

⁎⁎ ⁎⁎

0.4
0.2
0

0

Bcl-2

Bax

Bcl-2

Caspase-3

Bax

Caspase-3

Sham-surgery
HIBD
H2-90minControl
OGD6h-R24h
OGD6h-R24h+H2

(f)

(g)

Figure 6: H2 inhibited apoptosis in HIBI rat brain neurons and NGF-diﬀerentiated PC12 cells. (a) Apoptotic cells were detected by TUNEL
staining (dark brown, indicated by arrows) in the brains of neonatal rats 3 days after HIBI. (b) Images of NGF-diﬀerentiated PC12 cells weretaken under an inverted microscope immediately after OGD/R. Then, apoptosis analysis was done by Annexin-V/PI double staining. (c) Dataof viable (Annexin V-negative/PI-negative), early apoptotic (Annexin V-positive/PI-negative), and late apoptotic (Annexin V-positive/PIpositive) cells from three independent experiments (∗ p < 0:05; ∗∗ p < 0:01). (d) Representative immunoﬂuorescent signals (green) of Bcl-2,
BAX, and caspase-3 with DAPI nuclear counterstain (blue) (scale bar: 20 μm). (e) Western blot analysis of β-actin, Bcl-2, BAX, andcaspase-3 proteins in each group. (f, g) Bar graph showing the relative expression of Bcl-2, BAX, and caspase-3 in each group (n = 3/group;∗∗p < 0:01).
DAPI

Merge

HO-1

DAPI

Merge

Nrf2

DAPI

Merge

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

20 𝜇m

H2-90 min

HIBD

Sham surgery

P38-MAPK

OGD6h-R6h+H2

OGD6h-R6h

OGD6h-R3h+H2

Control

OGD6h-R6h+H2

OGD6h-R6h

OGD6h-R3h+H2

OGD6h-R3h

Control

OGD6h-R6h+H2

OGD6h-R3h+H2

OGD6h-R6h+H2

OGD6h-R3h+H2

OGD6h-R6h+H2

OGD6h-R6h

𝛽-Actin

OGD6h-R3h

𝛽-ActinControl𝛽-Actin
OGD6h-R6h

𝛽-Actin
OGD6h-R3h

ERKControl

Nrf2

OGD6h-R3h+H2

HO-1

OGD6h-R6h

p-JNK

OGD6h-R3h

p-P38
MAPK

Control

p-ERK

OGD6h-R3h

(a)

(b)

0.5
OGD6h-R6h+H2

OGD6h-R3h+H2

0
OGD6h-R6h

0

OGD6h-R3h

0.5
0

1.0

Control

0.5

1.5

OGD6h-R6h+H2

1.0

OGD6h-R3h+H2

1.0

OGD6h-R6h

2.0
1.5

OGD6h-R3h

OGD6h-R6h+H2

OGD6h-R3h+H2

OGD6h-R6h

OGD6h-R3h

OGD6h-R6h+H2

OGD6h-R3h+H2

OGD6h-R6h

Control

Control

0

0

⁎⁎

2.5
2.0

Control

0.5

Nrf2/𝛽-actin

⁎⁎

1.5

OGD6h-R6h+H2

1.0

HO-1/𝛽-actin⁎⁎

OGD6h-R3h+H2

1.5

1

⁎⁎

2.5

OGD6h-R6h

⁎

2.0

2

OGD6h-R3h

p-JNK/𝛽-actin

p-P38 MAPK/𝛽-actin⁎⁎

Control⁎⁎

OGD6h-R3h

p-ERK/ERK
3(c)

Figure 7: Eﬀects of H2 gas on MAPK/Nrf2/HO-1 protein expression. (a) Representative images of p-p38 MAPK (green), Nrf2 (red), HO-1
(red), and DAPI (blue) immunoﬂuorescence staining of the rat cortex in the sham surgery, HIBI, and H2-90 min groups (scar bar: 20 μm). (b)p-ERK, p-P38 MAPK, p-JNK, Nrf2, and HO-1 protein expressions were analyzed by western blot analysis in each group of β-NGFdiﬀerentiated PC12 cells. (c) Bar graph showing the relative expression of p-ERK, p-p38 MAPK, p-JNK, Nrf2, and HO-1 in each group(n = 3/group; ∗ p < 0:05; ∗∗ p < 0:01).

Oxidative Medicine and Cellular Longevity

11

HO-1
𝛽-Actin
−
−
−
−
−

OGD/R
H2
U0126
SB203580
SP600125

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(a)

HO-1 RNA

HO-1/𝛽-actin
10

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OGD/R −
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SB203580 −SP600125 −

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(c)

Figure 8: Involvement of MAPK in the expression of HO-1 induced by H2 gas in diﬀerentiated PC12 cells. β-NGF-diﬀerentiated PC12 cells
were pretreated with U0126 (10 mM), SB203580 (20 mM), or SP600125 (10 mM) for 1 h followed by oxygen-glucose deprivation (6 h) andthen reperfusion with or without H2 gas. The expression of HO-1 protein and mRNA was measured by western blotting and qPCR,
respectively. (a) Western blot analysis of β-actin and HO-1 proteins in each group. (b) Protein expression of HO-1 is shown as the HO1/β-actin ratio for each sample. (c) The mRNA expression of HO-1 was normalized with 18S to determine the relative expression ratio(log2 fold) for each group (n = 3/group; ∗∗ p < 0:01).

decreased these neurological reﬂexes, which suggested
impaired neuronal function. However, H2 alleviated this
impairment in neonatal rats (Figure 3). The Morris water
maze test, which was conducted once the neonatal rats
reached adulthood (Figure 4), evaluated navigational abilities in learning and memory. The results indicated thatrats in the HIBI group had signiﬁcant impairments inlearning and memory, while H2 intervention improved
performance. Shortening the duration of H2 inhalationand delaying H2 intervention reduced the protective eﬀects
of H2. Encouraged by these protective eﬀects, we further
investigated the related mechanisms and found that themechanisms underlying the protective eﬀects included
anti-inﬂammation, antiapoptosis, and antioxidative stress.
The main mechanisms of neonatal HIBI include oxidative stress, excitability, inﬂammation, and apoptosis [2].
Many researchers believe that similar to nitric oxide, hydrogen sulﬁde, and carbon monoxide, H2 may be an importantbioactive gas [30, 31]. There are no reported side eﬀects with
H2, which may confer a unique advantage in disease prevention and treatment [31]. In addition, human safety has been
demonstrated and standards have been established for inhalation of high H2 concentrations; high-pressure H2 gas isused in deep diving gas mixes to prevent decompression sickness and arterial gas thrombi [32].
Many neurological studies have associated the activation of astrocytes with CNS injury [33]. Activation of glial
cells may induce subsequent inﬂammatory responses,
attributed to neuronal injury and repair mechanisms laterin life and resulting in neurodevelopmental disorder [34].
In this study, we found that HI activated astrocytes in
the hippocampus of HIBD rats and H2 inhalation reduced
the activation of astrocytes (Figure 5). These results suggest that H2 may inhibit subsequent inﬂammatory
responses by reducing glial cell activation. We also foundthat H2 suppressed the expression of HMGB1 and TLR-4(Figure 5). The HMGB1/TLR-4/NF-κB signaling pathway
is well known to be a central regulator of several typesof inﬂammatory responses including astrocyte-mediatedneuroinﬂammation [35].
Apoptosis plays a more prominent role in the progressof ischemic brain injury in neonatal rodents and humans
than in adult brain ischemia [36]. HI can promote a seriesof pathological changes in neurons, which result in ectopic
expression of the death promoter BAX, leading to the formation of the apoptosome. Then, the apoptosome activates12

Oxidative Medicine and Cellular Longevity
OGD6h-R24h
HO-1 siRNA (+)

102
101
100

103

FL1 INT LOG: Fl3 INT LOG

103

102
101
100

103
100
101
102
FL1 INT LOG: Fl1 INT LOG

PI

200 𝜇m200 𝜇m
FL1 INT LOG: Fl3 INT LOG

FL1 INT LOG: Fl3 INT LOG

200 𝜇m

OGD6h-R24h+H2
HO-1 siRNA (+)

103
102
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100

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100
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FL1 INT LOG: Fl1 INT LOG

103
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FL1 INT LOG: Fl1 INT LOG

Annexin V
(a)
60

HO-1

40

PGC1𝛼
20

SirT1
𝛽-Actin0
Annexin V(+)
/ PI(−)

Annexin V(−)
/ PI(+)

Annexin V(−)
/ PI(−)

Control

OGD/R
H2

−
−

+

+

−

−

+
+

+
+

HO-1 siRNA

−

−

+

−

+

OGD6h-R24h HO-1 siRNA (+)
OGD6h-R24h+H2 HO-1 siRNA (+)

(b)

(c)
SirT1/𝛽-actin

PGC1𝛼/𝛽-actin
2.0

2.0

⁎

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1.5

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H2
HO-1
siRNA

⁎⁎

⁎⁎

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+
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+

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+
−

+
+
+

OGD/R
H2
HO-1
siRNA

−
−
−

(d)

+
−
−
(e)

Figure 9: Continued.

+
−
+

+
+
−

+
+
+

HO-1 siRNA (+)

Control
HO-1 siRNA (+)

Oxidative Medicine and Cellular Longevity

13
HO-1

HO-1/𝛽-actin

1.5

6

⁎⁎

⁎
1.0

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H2
HO-1
siRNA

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OGD/R

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HO-1
siRNA

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+

(f)

(g)

PGC1𝛼
20

SirT 1

⁎⁎

25

6

⁎

15

4

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5
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H2
HO-1
siRNA

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OGD/R
H2
HO-1
siRNA

(h)

−
−
−

+
−
−

+
−
+

(i)

Figure 9: Knockdown of HO-1 expression in the HO-diﬀerentiated PC12 cells was followed by OGD and reperfusion with or without H2 gas.
β-NGF-diﬀerentiated PC12 cells were pretreated with HO-1 siRNA for 24 h followed by OGD (6 h) and then reperfusion with or without H2gas. (a) Percentage of apoptotic cells (provided for each condition) was determined by combined Annexin V/PI staining and FACS analysis.
(b) Data of early apoptotic (Annexin V-positive/PI-negative), late apoptotic (Annexin V-positive/PI- positive), and dead (Annexin Vnegative/PI-negative) cells are summarized for three independent experiments. (c) Western blot analysis of β-actin, HO-1, PGC-1α, and
SIRT1 proteins in each group. (d–f) The protein expression of HO-1, PGC-1α, and SIRT1 is shown as the ratio of the protein to β-actin
for each sample. (g–i) The mRNA expression of each gene was normalized with 18S to determine the relative expression ratio (log2 fold)for each group (n = 3/group; ∗ p < 0:05; ∗∗ p < 0:01).

P13K

AKT
Oxidative stress

ATP
O2

ERK1/2

H2

p38 MAPK

Nrf2

P
Nrf2

Keap1
JNK

HO-1
Keap1
SirT1

PKC

PGC1𝛼

Gene
expression

Figure 10: The proposed mechanism by which H2 gas regulate the MAPK/Nrf2/HO-1 pathway. Step 1: H2 gas upregulates the expression of
MAPKs (ERK1/2, P38 MAPK, and JNK). Step 2: HO-1 can be expressed by stimuli mainly via MAPK-dependent Nrf2 activation. Step 3: HO1 inhibits oxidative stress and increases SIRT1 expression. Step 4: SIRT1 directly deacetylates PGC-1α. In summary, the functional outcome isdecreased apoptosis and oxidative stress.

14
procaspase-9, which is followed by the activation ofprocaspase-3 and ultimately cell death [37, 38]. As membersof the Bcl-2 family of proteins, BAX and Bcl-2 serve as a classof apoptosis regulators at its early stages. Bcl-2 is an antiapoptotic protein that counteracts the proapoptotic eﬀects of BAX.
Above all, an appropriate ratio of BAX/Bcl-2 can maintain ahomeostatic state in cells and ensure cell survival [39]. In
our study, we found that H2 could signiﬁcantly suppress theexpression of BAX and caspase-3 and concurrently promote
the expression of antiapoptotic protein Bcl-2 in neurons,
which strongly supports the notion that H2 has an anticell
death activity (Figure 6).
Recent studies have proposed that oxidative injury tovital cellular structures contributes to the pathogenesis of
HIE [40]. To explore the potential oxidative stress signaling
pathways underlying the oxidative stress response after HIBI,
we investigated the expression of antioxidant enzyme HO-1.
Recent studies have demonstrated that HO-1 activation contributes to the inhibition of glial hyperactivation and protection against gliosis-induced neuronal damage in the brain[41, 42]. HO-1 activation not only confers antioxidativeeﬀects but also modulates redox homoeostasis and regulates
neuroinﬂammatory conditions in activated glial cells [43].
PGC-1α has been demonstrated to be essential for the regulation of mitochondrial oxidative stress and biogenesis [44].
To explore the potential oxidative stress signaling pathways
underlying the oxidative stress response after HIBI, we investigated MAPKs, HO-1, and Nrf2 in neurons (Figure 7) andsuggested that the HO-1 system is one of the major protective
pathways of H2 in HI-induced injury. Another novel ﬁndingof this study is the importance of MAPK/HO-1/PGC-1αsignaling in H2’s neuroprotective actions. H2 activated
MAPKs (ERK1/2, p38 MAPK, and JNK) and increased HO1 expression, and MAPK inhibitors blocked the increase of
HO-1 induced by H2 (Figure 8). HO-1 siRNA inhibited HO1/PCG1-/SIRT1 expression and enhanced PC12 cell apoptosis
induced by OGD/R (Figure 9). We believe that H2 exerts
cytoprotection against HI by reducing neuron apoptosis andregulating the MAPK/HO-1/PCG1-α pathway (Figure 10).

5. Conclusions
Our results demonstrate that H2 inhalation is a potential
treatment for neonatal HIE. We propose early (<12 h after
HIE or HIBI) and prolonged (>60 min) H2 inhalation to protect against HIBI. Furthermore, our study suggests possible
underlying mechanisms mediated by the suppression of
HMGB1, TLR-4, BAX, and saspase-3 and activation of the
MAPK/HO-1/PGC-1α pathway. We believe that the protective eﬀects of H2 may be related to anti-inﬂammation, antiapoptosis, and antioxidative stress.

Data Availability
The datasets used and/or analyzed during the current study
are available from the corresponding authors on reasonable
request.

Oxidative Medicine and Cellular Longevity

Ethical Approval
All experiments were conducted in accordance with the
Guide for the Care and Use of Laboratory Animals (National
Institute of Health Publication No. 80-23, revised 1996) andwere approved by the Center for Medical Ethics of the Third
Xiangya Hospital.

Conflicts of Interest
The authors have no competing interests to declare.

Authors’ Contributions
PW, LX-K, and LZ designed this research. PW performed theexperiments. PW, MZ, ZC, GW, CZ, WZ, and MZ analyzed
the data. PW, MF, HS, L-XK, and LZ wrote the manuscript.
All authors read and approved the ﬁnal manuscript.

Acknowledgments
We thank LetPub (https://www.letpub.com) for their linguistic assistance during the preparation of this manuscript. This
work was supported by grants from the project of Xiangya
famous doctor of Central South University (2014, 68), theproject of new Xiangya talent of the Third Xiangya Hospitalof Central South University (2014, 97), the Natural Science
Foundation of Hunan Province of China (2018 JJ 2620), theNational Center for Child Health and Development, andthe Ministry of Education, Culture, Sports, Science and Technology of Japan.

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