FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of vascular dementia

Accepted Manuscript Title: FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of vascular dementia Authors: Xin Jiang, Xiaoli Niu, Qingjun Guo, Yanhong Dong, Jing Xu, Nan Yin, Qi Qianqian, Yanqiu Jia, Liwei Gao, Qihui He, Peiyuan Lv PII: DOI: Reference:

S0166-4328(18)30010-X https://doi.org/10.1016/j.bbr.2018.05.023 BBR 11444

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

8-1-2018 21-4-2018 24-5-2018

Please cite this article as: Jiang X, Niu X, Guo Q, Dong Y, Xu J, Yin N, Qi Q, Jia Y, Gao L, He Q, Lv P, FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of vascular dementia, Behavioural Brain Research (2018), https://doi.org/10.1016/j.bbr.2018.05.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Apr. 19, 2018, the Research Paper for Behavioural Brain Research

FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of vascular dementia Running title: FoxO1-mediated autophagy plays an important role in the

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neuroprotective effects of hydrogen in VD

a

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Xin Jianga,b, Xiaoli Niub, Qingjun Guoc, Yanhong Dongb, Jing Xub, Nan Yinb, Qianqian Qib, Yanqiu Jiab, Liwei Gaoa, Qihui Hed, Peiyuan Lva,b

Department of Neurology, Hebei Medical University, No. 361 Zhongshan East Road,

Changan District, Shijiazhuang 050017, Hebei Province, People’s Republic of China b

Department of Neurology, Hebei General Hospital, No. 348 Heping West Road,

Department of Surgery, Hebei Medical University, No. 361 Zhongshan East Road,

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c

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Xinhua District, Shijiazhuang 050051, Hebei Province, People’s Republic of China

d

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Changan District, Shijiazhuang 050017, Hebei Province, People’s Republic of China Department of mangement, North China University of Science and Technology, No.

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21 Bohai avenue, Caofeidan District, Tangshan 063210, Hebei Province, People’s

231 words

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Abstract:

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Republic of China

793 words

Discussion:

1472 words

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Introduction:

7874 words

Text pages:

34

Figures:

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Total text:

Corresponding author: Peiyuan Lv, Ph. D. Department of Neurology, Hebei General Hospital,

348 Heping West Road, Xinhua District, Shijiazhuang, Hebei, P.R. China 050051 E-mail: [email protected]

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Tel: +86-311-85988906

Highlights

HRW attenuates learning and memory impairments in a rat model of VD.

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HRW attenuates the neuronal apoptosis and autophagy levels in VD rats.

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FoxO1-mediated autophagy is partially involved in the protective effects of HRW

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

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Excessive autophagy and apoptosis are both activated 4 weeks after bilateral

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

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in VD rats.

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occlusion of the common carotid artery surgery in rats.

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Abstract

Vascular dementia (VD) is a heterogeneous group of brain disorders in which

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cognitive impairment is attributed to cerebrovascular pathologies. Autophagy, a self-cannibalization mechanism, has been demonstrated to be involved in VD progression.

Molecular

hydrogen is known for

its powerful anti-oxidative,

anti-apoptotic, and anti-inflammatory activities, and it is also involved in autophagy. However, the effects of hydrogen on VD remain unclear. The current study found that

hydrogen-rich water (HRW) significantly alleviated spatial learning and memory impairments. Similar to donepezil treatment, HRW also inhibited neuron loss and shrinkage in the hippocampal CA1 region. In addition, we found that HRW significantly increased the Bcl-2/Bax expression ratio and decreased cleaved

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caspase-3 expression levels in the hippocampus of VD rats. Moreover, electron microscopy revealed that HRW decreased the number of autophagosomes. We also

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observed that HRW reduced the increased ratio of LC3-II/I and Beclin 1 expression and saliently upregulated p62 expression. Furthermore, FoxO1 (a major mediator of

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autophagy regulation) and Atg7 levels were apparently decreased in the

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hippocampus of HRW-treated bilateral common carotid artery occlusion (2VO) rats.

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Taken together, these data show that molecular hydrogen exerts beneficial effects on

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cognitive impairment induced by chronic cerebral hypoperfusion. FoxO1-mediated

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autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of VD. Furthermore, the present findings highlight that HRW should be further

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investigated as a new therapeutic strategy for VD treatment in the future.

Abbreviations:

Atg7, autophagy associated gene 7; Bax, Bcl-2 associated X protein; Bak, Bcl-2 homologous antagonist/killer; Bcl-2, B-cell lymphoma-2; Beclin 1, Bcl-2 interacting

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protein 1; CCH, chronic cerebral hypoperfusion; Cleaved caspase-3, Cleaved cysteinyl aspartate specific proteinase-3; DHW, degassed hydrogen-rich water; FoxO1,

forkhead

box

proteins

1;

HRW,

hydrogen-rich

water;

LC3,

microtubule-associated protein 1 light chain 3; MWM, Morris water maze; Sequestosome-1/p62, SQSTM1/p62, p62; VD, vascular dementia; 2VO, 2-vessel occlusion, permanent bilateral occlusion of the common carotid artery

Keywords: vascular dementia; hydrogen; autophagy; apoptosis; FoxO1

1. Introduction

Vascular dementia (VD) is a heterogeneous group of brain disorders in which

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cognitive impairment is attributed to cerebrovascular pathologies[1]. VD is the second

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most common cause of dementia[2] and causes approximately 15-20 % of all dementia cases[2-3]. However, the treatment strategies for VD focus on controlling the underlying cardiovascular and cerebrovascular risk factors and treating the

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associated symptoms[3]. There is currently no effective treatment for VD. Therefore,

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finding a highly potent drug has become a key medical issue.

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Numerous studies have demonstrated that chronic cerebral hypoperfusion (CCH) is associated with the initiation and progression of VD[4]. The deprivation of oxygen

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and glucose from brain tissue after CCH could result in serious homeostasis

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alterations, including oxidative stress[5], inflammation[6], apoptosis[7], mitochondrial and neurotransmitter dysfunction[4], lipid metabolism disturbance[8], and growth

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factor alterations[9]. In recent years, autophagy has become an emerging therapeutic target for diseases, particularly vascular diseases[10-12]. Autophagy has become a

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relevant target for aging and diseases affecting the central nervous system[13].

Autophagy is a self-cannibalization mechanism that involves the engulfment of

cytoplasmic material and intracellular organelles within a double-membrane autophagosome; this autophagosome then fuses with a lysosome to form an

autolysosome where the captured material is subsequently degraded by specific acidic hydrolases[14]. Autophagosomes, which can be observed by electron microscopy, are hallmark structures of autophagy. Autophagy has been implicated in diverse physiological and pathological processes, including starvation, oxidative

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stress, inflammatory reactions, immune responses and a wide range of diseases[15]. Autophagy is executed by autophagy-associated genes (Atgs). B-cell lymphoma-2

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interacting protein 1 (Beclin 1), a mammalian homologue of Atg6 that can bind to

multiple proteins, such as B-cell lymphoma-2 (Bcl-2), is a key regulator of autophagy.

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The autophagy-inducible Beclin 1 complex contributes to the activation of

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downstream autophagy components[16].Microtubule-associated protein 1 light chain

and

adaptor

proteins,

such

as

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cargo

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3 (LC3-II) contributes autophagosome closure and enables the docking of specific sequestosome-1/p62

(SQSTM1/p62,

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SQSTM1/p62, hereafter referred to as p62). Concurrent increases in both Beclin 1 and LC3-II and decreases in p62 indicate the activation of autophagy[17]. Forkhead

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box protein 1 (FoxO1), which is highly expressed in the brain, is considered a major

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mediator of autophagy regulation. Of the multiple stages of autophagy, FoxO1 is involved in initiation, vesicle nucleation, and vesicle elongation. A very recent report shows that FoxO1 is located in the cytoplasm of cells and interacts with Atg7 (a key

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regulator of autophagosome formation), which induces autophagy[18].

Increasing evidence indicates that autophagy and apoptosis may share common molecular inducers and regulatory mechanisms. Beclin 1 may be involved in the cysteinyl aspartate-specific proteinase-3 (caspase-3) activation process because the

increased expression levels of Beclin 1 colocalize with activated caspase-3 after adult focal cerebral ischemia and hypoxia-ischemia[19]. Bcl-2 negatively regulates autophagy by inhibiting Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak)[20] and blocking Beclin 1[21]. A recent report showed that CCH

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caused rat hippocampal neuronal apoptosis and enhanced and redistributed autophagy[22]. Moreover, consistent with this report, our previous studies also found

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that autophagy[11-12] and apoptosis[8] were induced in VD animal models. Thus, the

induction of autophagy, which is correlated with apoptosis, plays a pathogenic role in

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aggravating the effects of VD development. The inhibition of robust autophagy in the

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central nervous system of VD rats may be a neuroprotective effect.

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Molecular hydrogen, which is small, electrically neutral, and nonpolar, can rapidly diffuse across all cell membranes; as such, it can easily penetrate the blood-brain

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barrier and the placental and testis barriers. In 2007, Ohsawa reported that gas hydrogen (H2) acts as a therapeutic and preventive anti-oxidant by selectively

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reducing the levels of strong oxidants, such as the hydroxyl radical (•OH) and

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peroxynitrite (ONOO–), in cells and that H2 exhibits cytoprotective effects against oxidative stress[23].Since this astonishing therapeutic effect of H2 on a rat model of

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cerebral infarction was reported, multiple lines of evidence have shown that many disease models and human diseases can benefit from hydrogen[24]

An increasing

number of studies have revealed the protective effects of hydrogen in various models of brain injury[25]. Molecular hydrogen has been intensively studied in the past ten years, and it has attracted great interest due to its anti-oxidative, anti-apoptotic, and

anti-inflammatory activities[26].Recently, there have been an increasing number of studies on the relationship between hydrogen and autophagy. However, the role of hydrogen in autophagy remains controversial. Some studies have shown that hydrogen can downregulate autophagy[27-28], and other studies have demonstrated

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that hydrogen induces autophagy[25, 29]. However, no literature has highlighted the protective effects of hydrogen on VD. Therefore, the role of hydrogen in CCH and its

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underlying mechanism still need to be further investigated.

The current study aims to investigate whether hydrogen-rich water (HRW) would

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autophagy would be involved in this process.”

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ameliorate the cognitive decline caused by 2VO, and whether apoptosis and

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2. Materials and methods

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2.1 Animals

Forty-eight male Sprague-Dawley (SD) rats weighing 200-250 g were purchased

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from the Laboratory Animal Center of Hebei Medical University. All rats were housed

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in a climate-controlled environment (temperature-controlled at 23 °C ± 2 °C, humidity-controlled at 60 % ± 5 %) under a 12-hour light/dark cycle with free access to

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food and water. A one-week acclimation period was allowed before the start of the experiments. All procedures were performed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Animal Use Committee of Hebei Medical University.

2.2 Drugs and hydrogen

HRW (H2 concentration > 1.6 ppm) was provided by Beijing Hydrovita Beverage Co., Ltd. (Beijing, China) and stored under atmospheric pressure at 23 ± 2 °C in an aluminum pot with no dead volume. Degassed hydrogen-rich water (DHW) was

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generated by exposing HRW to air and gently stirring for 24 h. Donepezil, a positive

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control drug, was purchased from Eisai China Inc. Cleaved caspase-3, Bax, LC3B,

FoxO1, and Atg7 antibodies were all purchased from Cell Signaling Technology (Boston, MA, USA). Bcl-2, Beclin 1, and p62 antibodies were all purchased from

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Abcam (Cambridge, MA, USA). Beta-actin (β-actin) was purchased from Bioworld

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Technology, Inc. Dylight 800 goat anti-rabbit IgG (H + L) was purchased from KPL

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(Milford, MA, USA).

2.3 Surgery

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Rats were subjected to CCH using a bilateral common carotid artery occlusion

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(2-vessel occlusion, 2VO) model. Briefly, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (i.p., 2 %, 50 mg/kg), and their limbs

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were fixed on a board. A midline cervical incision was performed, and the bilateral carotid arteries were exposed and gently separated from the carotid sheath and vagus nerve using glass dissecting tools. The bilateral carotid arteries were doubly and permanently ligated with 4-0 silk sutures, and the area between the ligations was cut in CCH rats. These arteries were not ligated or cut in the sham-operated control

animals. The temperature was monitored and maintained at 37 ± 0.5 °C throughout the surgical procedure. Following the surgical operation, the rats were returned to

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their cages with food and water ad libitum.

2.4 Groups and drug administration

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Rats were divided randomly into four groups (n = 12 per group): (1) Sham group: sham operation, intragastric (i.g.) administration of DHW at 3 ml/d; (2) Model group:

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2VO operation, i.g. DHW at 3 ml/d; (3) HRW group: 2VO operation, i.g. HRW at 3 ml/d;

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and (4) Don group: 2VO operation, i.g. donepezil at 3 ml/d (dissolved in DHW; the

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drug concentrations were adjusted to an appropriate volume for 1 mg/kg body weight).

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Water with fresh HRW or DHW was administered every day. Each group was

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administered treatment via gavage for 28 consecutive days (between 10:00 and 12:00

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a.m.) postoperatively.

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2.5 Morris water maze

All rats were trained and tested in a Morris water maze (MWM) after 28 days of drug

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administration to assess spatial learning and memory. The MWM and the training and testing procedures were the same as those in our previously described procedures[8, 11]. Briefly, the MWM was a black circular pool (160 cm diameter and 45 cm height) filled with 23 ± 1 °C water. The pool was divided equally into 4 parts, with an escape platform (10 cm × 10 cm) submerged 2 cm below the surface at the center of a fixed

quadrant. First, the rats were trained with four trials per day for five consecutive days. For every training trial, the rats were placed gently in the water facing the pool wall at one of four starting positions and allowed to swim freely to the escape platform. The procedure was repeated for all four starting positions and in a different order each day.

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For each trial, the escape latency (time to reach the submerged platform), the swimming route to reach the platform, and the swimming speed were recorded by a

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computer-operated video tracking system. If the rat reached the platform within 120 s, it was allowed to remain on the platform for 10 s for orientation purposes. Rats that

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failed were gently guided to the platform by the experimenter and permitted to stay

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there for 10 s. The rats were then removed and placed in their heated cage to rest

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until the next trial. On day 6, memory retention tests (spatial probe) were performed

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without the platform in the tank. Each rat was placed in the quadrant opposite the

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target quadrant (where the platform had been located) and allowed to swim freely for

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120 s. The time spent in each quadrant was recorded.

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2.6 Histopathology and immunohistochemistry

Three rats, chosen randomly from each group, were sacrificed for Nissl staining

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shortly after the MWM tests. The animals were anesthetized with pentobarbital sodium (i.p., 2 %, 50 mg/kg) and transcardially perfused with saline through the left cardiac ventricle and ascending aorta until the liver and lungs were cleared of blood. Then, the animals were perfused with 4 % paraformaldehyde. After perfusion, the brains were removed and immersed in 4 % paraformaldehyde for 48 h. Serial sections

(5 μm) were cut coronally from paraffin blocks of cerebrum containing the hippocampus and stained with Nissl staining. Mounted slides selected from the same site in each rat were examined and photographed under an Olympus light microscope to study the morphological changes to pyramidal neurons in the cornu ammonis 1

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(CA1) region of the hippocampus. In addition, the other mounted slides from the same site were used for immunohistochemical staining for cleaved caspase-3 (1:500),

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LC3B (1:300), and p62 (1:200). This process was conducted as previously described[30]. The intensity of positively stained neurons in the pyramidal cell layers

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of the bilateral CA1 region was quantified for five visual fields from each animal at

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2.7 Transmission Electron Microscopy

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software (Media Cybernetics, USA).

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400× magnification with integrated optical density (IOD) using Image-Pro Plus

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The rats were sacrificed one day after the MWM tests. After they were anesthetized,

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the rats were transcardially perfused with saline, followed by a mixed solution of 4 % paraformaldehyde and 2.5 % glutaraldehyde. The tissue samples collected from the

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CA1 region of the hippocampus were dissected into 1-mm3 pieces, fixed in 4 % glutaraldehyde overnight and then processed as described in previous studies[31].

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Finally, ultra-thin sections of at least three blocks per sample were cut and observed using a Hitachi TEM.

2.8 Western blot assay

Following behavioral testing, the rats were decapitated under anesthesia. The hippocampi were rapidly removed, placed into liquid nitrogen and stored at -80 °C until use. The segments for Western blotting were ground in liquid nitrogen and homogenized on ice in RIPA buffer containing protease inhibitors. Total protein from

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the hippocampi was extracted according to the manufacturer’s protocols (Beijing Solarbio Science & Technology Co., Ltd, Beijing, China). Protein concentrations were

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determined with a BCA kit (Beijing ComWin Biotech Co., Ltd, Beijing, China). The

protein samples were mixed with loading buffer and subjected to sodium dodecyl gel

electrophoresis

(SDS-PAGE).

As

previously

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sulfate-polyacrylamide

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described[11-12], the transferred membranes were blocked and incubated overnight

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at 4 °C with the following antibodies: Bax (1:1000), Bcl-2 (1:1000), cleaved caspase-3

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(1:1000), LC3B (1:1000), Beclin 1 (1:1000), p62 (1:500), FoxO1 (1:1000), and Atg7

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(1:1000). β-actin was blotted on the same membranes and served as a control. On the second day, the membranes were washed three times with TBST and incubated

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with fluorescent-labeled secondary antibodies (DyLight 800-conjugated goat

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anti-rabbit, 1:10000 dilution, KPL) for 1 h at room temperature. The bands on the membranes were subsequently detected using the Odyssey two-color infrared laser imaging system (LI-COR, Lincoln, NE, USA).The relative intensity of each band was

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normalized to β-actin.

2.9 Data analysis

All quantitative data in this study are presented as the mean ± SEMs. The escape latencies for the MWM test were analyzed by two-way analysis of variance (ANOVA) (treatment × day) with repeated measures, followed by the least-significant difference (LSD) test for multiple comparisons among various groups. Differences in the other

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data were analyzed using one-way ANOVA, followed by the LSD test for intergroup comparisons. All data were analyzed using SPSS v20.0 software (SPSS Inc.,

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Chicago, IL, USA). A P-value of p < 0.05 was considered statistically significant.

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3. Results

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3.1 HRW attenuated learning and memory impairments induced by CCH

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All rats were trained in a MWM for 5 days to assess whether treatment with HRW

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might improve spatial learning. Fig. 1A shows the changes in the daily mean escape latencies to find the hidden platform 4 weeks after 2VO surgery. There was a

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significant overall group difference for the four groups (main effect of treatment: F =

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23.158, p < 0.01; training day effect: F = 171.208, p < 0.01; interaction of group x test: F = 1.912; p < 0.05). As the MWM training days progressed, the escape latencies of the four groups of rats gradually shortened. Further analysis by LSD tests revealed

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that compared with those in the Sham group, rats in the Model group showed significantly longer mean escape latencies from day 1 to day 5 (days 1-5: p < 0.01). Compared with those in the Model group, rats in the HRW and Don groups took less time to find the platform, particularly from day 3 day to day 5 (day 1: p < 0.05 and p >

0.05, respectively; day 2: p < 0.05 and p > 0.05, respectively; days 3-5: p < 0.01 and p < 0.01, respectively). Moreover, during the five days, the mean escape latencies were not significantly different between rats treated with HRW and donepezil (days 1-5: p > 0.05). In the probe trial, there was a significant difference among all groups for the

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percentage of time that the rats spent in the target quadrant (F = 22.52, p < 0.01). LSD tests demonstrated that rats in the 2VO group stayed in the target quadrant for

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significantly less time than those in the Sham group (Model group: p < 0.01; HRW

group: p < 0.01; Don group: p < 0.01). Compared with those in the Model group, rats

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in the HRW group spent an increased proportion of time in the target quadrant (p <

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0.01). Rats in the Don group exhibited a similar trend (p < 0.01); however, no

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significant difference was found between the HRW group and the Don group (p > 0.05)

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(Fig. 1B). These results indicate that HRW attenuates not only the spatial learning

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impairment but also the memory impairment that is induced in 2VO rats.

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……Figure 1 about here……

3.2 HRW reversed the pathologic changes in the hippocampal CA1 region in CCH

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rats

Representative Nissl staining is shown in Fig. 2. Our results revealed that in the

Sham group, the pyramidal neurons in the CA1 region of the hippocampus were tightly ranked in order, and the neurons were clearly stained and moderate in size with normal microstructure. However, four weeks after CCH, obvious pathological changes

were seen in the hippocampal CA1 area. These changes included hippocampal atrophy, significant neuronal shrinkage, neuronal loss, and loosely arranged neurons in the Model group. The administration of HRW, like treatment with donepezil, markedly reversed these morphologic changes. These results indicate that HRW

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reversed the pathologic impairment induced by CCH in the CA1 region.

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……Figure 2 about here……

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3.3 HRW attenuates neuronal apoptosis in the hippocampal CA1 areas of rats

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subjected to CCH

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To explore the effects of HRW on the apoptosis of hippocampal neurons in 2VO

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rats, we used immunohistochemistry to detect the expression of cleaved caspase-3 in

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the hippocampal CA1 area. We also assessed the protein expression levels of Bcl-2 and Bax in the hippocampus by Western blot. As shown in Fig. 3A, positive staining

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for cleaved caspase-3 was observed in the nuclei of neurons in the hippocampus. Our

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results showed that the IOD/area value of cleaved caspase-3 in the CA1 region was significantly different among all groups (F = 47.356, p < 0.01). Cleaved caspase-3

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levels were dramatically higher in the Model group than in the Sham group (p < 0.01). In addition, the IOD/area values of cleaved caspase-3 were significantly lower in the HRW (p < 0.01) and Don (p < 0.01) groups than in the Model group. However, the reduction measured in the HRW group was less than that measured in the Don group (p < 0.01) (Fig. 3C). Furthermore, we detected changes in Bcl-2 and Bax protein

levels in the hippocampus after 2VO and determined whether HRW had any effect on their expression. The results showed significant differences in the expression levels of Bcl-2 and Bax as well as the Bcl-2/Bax ratio among the four groups (F = 8.195, p < 0.01; F = 12.276, p < 0.01; F = 33.305, p < 0.01, respectively). Compared with the

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levels in the Sham group, Bcl-2 expression levels were significantly decreased in the Model group (p < 0.05), and Bax expression levels were significantly increased (p <

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0.01). Thus, the ratio of Bcl-2/Bax was markedly reduced in the rats of the Model

group (p < 0.01). However, rats receiving HRW orally had increased Bcl-2 expression

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levels (p < 0.01) and decreased Bax expression levels (p < 0.01), which led to a

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significant increase in the Bcl-2/Bax ratio (p < 0.01). The relative expression of Bcl-2

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in the Don group was similar to that in the HRW group (p > 0.05), but the reduction in

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Bax expression levels was less than that in the HRW group (p < 0.05); thus, the ratio

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of Bcl-2/Bax was obviously lower than that in the HRW group (p < 0.05) (Fig. 3D-E). These results suggest that the daily oral administration of HRW attenuates neuronal

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apoptosis induced by 2VO surgery.

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……Figure 3 about here……

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3.4 HRW inhibited ultrastructure damage in the hippocampal CA1 region in CCH rats

The electron microscopy results revealed that the sham-operated rats had linear, rough endoplasmic reticulums, clear mitochondria crests, rich ribosome and glycogen particles, clear nuclear membranes, visible nucleoli, and a small number of

autophagosomes. There were more autophagosomes, swollen mitochondria and loose endoplasmic reticulums and worse nuclear borders in the Model group. We also observed that compared with the Model group, the HRW and Don groups had varying decreases in the numbers of autophagosomes and ameliorated nucleus and

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organelle microstructure damage. However, mitochondrial swelling was not significantly improved in the HRW group, unlike in the Don group (Fig. 4). These

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results indicate that HRW inhibited the ultrastructural damage to neurons and decreased the number of autophagosomes in the hippocampal CA1 region.

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……Figure 4 about here……

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3.5 HRW suppressed autophagy in the hippocampus under CCH.

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To investigate whether HRW can inhibit autophagy induced by 2VO, we examined the expression of several proteins related to autophagy. LC3-II expression was

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examined by immunohistochemistry (Fig. 5A). We found that the IOD/area value of

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LC3-II among all groups was significantly different (F = 7.830, p < 0.01). CCH increased the IOD/area value of LC3 in the CA1 region relative to that in the Sham

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group (p < 0.01). After four weeks of HRW administration, the IOD/area value of LC3 was significantly lower than that in the Model group and was similar to the value after donepezil administration (p < 0.01) (Fig. 5C). According to these results, we next investigated the expression of Beclin 1, LC3-II, and p62 in the hippocampi by Western blotting (Fig. 5D-E). As expected, there were significant differences in the expression

levels of Beclin 1 and p62 among the four groups (F = 21.643, p < 0.01; F = 18.618, p < 0.01, respectively). An obvious increase in the relative expression of Beclin 1 was found in the Model group compared to that in the Sham group (p < 0.01), and Beclin 1 expression levels were significantly ameliorated in the HRW and Don groups (p < 0.01;

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p < 0.01, respectively). In addition, our results showed an obvious decrease in LC3-I levels after four weeks of 2VO surgery when compared with those in the Sham group

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(p < 0.01); in contrast, the LC3-II expression levels increased slightly (p > 0.05), and the LC3-II/I ratio increased significantly (p < 0.01). HRW and donepezil significantly

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reduced the increase in the LC3-II/I ratio (p < 0.01; p < 0.01, respectively), but there

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was no significant difference between the two groups (p > 0.05). Additionally, the

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relative expression of p62 was obviously lower in the Model group than in the Sham

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group (p < 0.01) and higher in the HRW group than in the Model group (p < 0.01).

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Furthermore, the p62 level was significantly higher in the Don group than in the Model group (p < 0.01); however, there was no significant difference between the HRW

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group and the Don group (p > 0.05). These results indicate that HRW suppressed the

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activation of autophagy in the hippocampus 4 weeks after the animals were subjected to 2VO surgery.

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……Figure 5 about here……

3.6 HRW decreased FoxO1 and Atg7 activation

To further explore the autophagy signaling pathway in CCH and whether this

signaling could be affected by HRW, we used Western blots to measure FoxO1 and Atg7 proteins in the hippocampus. The FoxO1 and Atg7 protein expression levels presented in Fig. 6 were significantly different among the four groups (F = 29.083, p < 0.01; F = 135.497, p < 0.01, respectively). Our results showed that FoxO1 and Atg7

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expression levels in the hippocampus were significantly higher in the Model group than in the Sham group (p < 0.01; p < 0.01, respectively). However, these increases in

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the 2VO groups were significantly decreased in the HRW group (p < 0.01 for FoxO1; p < 0.01 for Atg7). FoxO1 and Atg7 levels were considerably lower in the HRW group

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than in the Don group. However, there were no differences between the Don group

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and the Model group (p > 0.05 for FoxO1; p > 0.05 for Atg7) (Fig. 6B).

4. Discussion

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……Figure 6 about here……

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In the present study, we demonstrated the following for the first time: (1) HRW

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treatment could attenuate cognitive impairment caused by 2VO-induced VD; (2) HRW treatment could prevent neuronal death and diminish hippocampal atrophy and

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neuronal apoptosis in the hippocampal CA1 area; and (3) HRW treatment could at least partially increase the FoxO1-mediated autophagy induced by CCH in rats.

Studies

have

reported

that

hydrogen

has

neuroprotective

effects

via

anti-apoptosis mechanisms in neonatal hypoxia-ischemia rat models[32] and in early brain injury following subarachnoid hemorrhage[33]. Moreover, drinking hydrogen

water can ameliorate cognitive impairment in senescence-accelerated mice[34]. Based on these studies, we hypothesized that HRW might have a neuroprotective effect on preventing VD development. Rats that were subjected to 2VO surgery, a classic and useful CCH animal model[11], were used in our experiments. Because of

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its known improvements to cognition and behavior, donepezil was chosen as a positive control drug. Our study revealed that the rats in the four groups all learned

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and memorized the peripheral environment during the 4 weeks of post-operative cognitive testing; however, their abilities were different. Compared with that of the

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Sham group, the performance of the 2VO group decreased significantly from the

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preoperative levels, which was consistent with previous studies[8, 11, 35]. However,

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the rats drinking HRW every day could successfully find the platform much faster than

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those in the Model group. Moreover, the time spent in the target quadrant was shorter

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for the HRW group than for the Model group. This implies that drinking HRW might improve the impaired learning and memory abilities induced by CCH. Even more

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surprising, the cognitive improvement induced by chronic HRW administration was

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the same as that induced by donepezil.

The hippocampus is an important area of the brain that is associated with

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learning and memory. It is also a site predisposed to brain damage caused by factors such as ischemia/hypoxia. The structure of the hippocampus can be divided into three sub-regions, namely, CA1, CA3 and dentate gyros (DG). These three sub-regions and their associated neural networks play an important role in the formation of learning and memory[36]. Some studies have shown that neurons in the CA1 region

are very sensitive to local hypoxia/ischemia factors. The initial damage caused by ischemia-induced neurodegeneration occurs in the CA1 region. Therefore, in the CCH-induced cognitive impairment model, the CA1 region is often chosen to observe the damage to neurons in the hippocampus caused by ischemia and hypoxia[37].

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Therefore, we verified the protective effects of HRW on neurons in the hippocampal CA1 region of 2VO rats by Nissl staining. As expected, drinking HRW significantly

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reduced neuronal loss and improved cell morphology in the hippocampal CA1 region.

These results indicated that HRW had a neuroprotective effect via enhancing cell

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survival. Amazingly, HRW treatment achieved the same neuroprotective effect as

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donepezil treatment. These findings provoked our interest in investigating the

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underlying mechanism(s) of the neuroprotective effect of HRW in more detail.

Mounting literature has demonstrated that apoptosis is widely appreciated as a

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major mechanism of regulated death; apoptosis is employed not only upon cell

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damage or stress but also during normal development and morphogenesis[14], and neuronal apoptosis is closely related to the cognitive deficits induced by CCH[8].

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However, hydrogen can suppress apoptosis in several ischemic diseases[32-33, 38-39]. Therefore, we investigated the expression of important apoptosis-related

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proteins in the hippocampus of rats after 2VO with or without HRW treatment. The ratio of Bcl-2 (an anti-apoptotic protein) to Bax (a proapoptotic protein) is closely related to cell survival, and cleaved caspase-3 is an important factor contributing to the process of apoptosis and thus cell destruction. Our results revealed that the ratio of Bcl-2/Bax decreased and the expression of cleaved caspase-3 increased in the

hippocampus of rats in the Model group. However, HRW, as well as donepezil, could markedly increase the ratio of Bcl-2/Bax. Moreover, we also found that HRW treatment significantly decreased the expression of cleaved caspase-3. These data, which are consistent with those of previous studies, indicate that HRW could

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effectively inhibit apoptosis in the hippocampus during CCH.

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It has been reported that apoptosis and autophagy might share common molecular

inducers and regulatory mechanisms[14]. There is crosstalk between apoptosis and autophagy activation. However, the role of autophagy in cell survival and death is

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complex. It has been associated with not only increased susceptibility to apoptosis but

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also resistance to anti-apoptosis therapie[40]. Previous studies demonstrated that

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A

excessive autophagy induction in response to ischemia injury may contribute to cell death, which is characterized by the presence of a large number of autophagosomes

ED

in the cytoplasm, and the inhibition of over-activated autophagy can attenuate

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cerebral ischemia-associated neuronal damage[41]. Lines of research have shown that autophagy is involved in cognitive impairment in CCH, and inhibiting autophagy

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has neuroprotective effects on brain ischemia[8, 11, 35]. Interestingly, a study demonstrated that hydrogen obviously enhanced neuronal autophagy and exerted

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neuroprotective effects against hypoxic-ischemic brain damage[25]. In contrast, other results

have

also

demonstrated

that

hydrogen

attenuates

myocardial

ischemia-reperfusion injury by attenuating autophagy[27]. What role hydrogen plays in CCH-induced autophagy and what the underlying mechanism is remain incompletely understood. Therefore, in the present study, we measured the

expression of autophagy-related proteins to determine whether autophagy was induced or inhibited by HRW. Consistent with the results of previous studies, our results showed that LC3 was expressed at higher levels in the hippocampus of the Model group, whereas HRW treatment for 4 weeks significantly decreased the

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number of LC3-positive cells. As autophagy progresses, LC3-I in the cytoplasm is conjugated to phosphatidylethanolamine to form LC3-II. An increase in LC3-II and a

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concomitant decrease in LC3-I (increased LC3-II/I ratio) are commonly used as a marker of autophagy[20]. Our Western blot results showed that LC3-I was

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significantly decreased in the hippocampus of 2VO rats, whereas LC3-II changed

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slightly. However, the ratio of LC3-II/I was greatly increased in 2VO rats but was

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significantly attenuated after 4 weeks of HRW administration. We also demonstrated

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that Beclin 1 expression was significantly increased, and p62 expression was

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decreased in the Model group. Fortunately, HRW administration inhibited Beclin 1 accumulation and increased p62 accumulation in the hippocampus after 2VO. These

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results implied that autophagy was highly induced in the Model group. High levels of

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autophagy might lead to decreased neuronal survival, which can impair cognitive function. Given our results, we considered that HRW might confer neuroprotective effects via suppressing autophagy activated by CCH. We further verified our

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hypothesis by transmission electron microscopy. Our results revealed that the numbers of autophagosomes and autolysosomes in the neurons of the hippocampal CA1 area in 2VO rats were significantly increased. These findings were consistent with our previous conclusions. HRW treatment could partly reverse organelle damage

and decrease the numbers of autophagosomes and autolysosomes. The above evidence suggested that the neuroprotective effects of HRW might be closely related to its regulation of autophagy to enhance cell survival and reduce apoptosis. Our results further confirmed that HRW could attenuate autophagy and thus improve

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CCH-induced cognitive impairment.

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Then, we investigated how HRW modulates autophagic cell death. Recently, accumulating evidence suggests that FoxO1 has a uniquely specific function in

eliciting autophagy in various diseases[18, 40, 42]. Endogenous FoxO1 is a key

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molecule required for autophagy in response to oxidative stress and apoptosis[43]. In

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addition, it is a key regulator of autophagy in neurons due to its transcriptional

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A

regulation of autophagy-related genes[44]. FoxO1 interacts specifically with Atg7 to induce the autophagic process[43], and FoxO1 knockdown inhibits autophagy[45].

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The translocation of FoxO1 from the cytoplasm to the nucleus in neurons triggers cell

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death. However, there have been no reports about whether hydrogen benefits neuronal survival in FoxO1-mediated autophagy. In this study, our data clearly

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demonstrated FoxO1 upregulation, accompanied by Atg7 upregulation, after 2VO. These results showed that 2VO surgery might induce high levels of autophagy, which

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is consistent with our previous results. According to our findings, both HRW and donepezil remarkably attenuated CCH-induced autophagy induced. FoxO1 and Atg7 expression levels were significantly reduced by HRW, compared with those in the Model group; however, donepezil seemed to have little effect on FoxO1 and Atg7. A possible explanation for these results is that FoxO1 may be a major target that

mediates autophagy to accelerate the adverse effects of CCH, and HRW may downregulate FoxO1-dependent autophagy associated with Atg7 in 2VO rats. Thus, FoxO1-mediated autophagy plays an important role in the neuroprotective effects of hydrogen in a rat model of VD.

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In summary, we provided evidence that HRW has neuroprotective effects on CCH.

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HRW not only plays a critical role in regulating apoptosis but also has been implicated in autophagy. We conclude that HRW decreases the levels of cytoprotective autophagy in CCH, possibly through decreasing FoxO1 expression. Taken together,

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our findings suggest a novel role for HRW in suppressing the harmful autophagic

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process and directly promoting cell survival. Thus, HRW may be a potential

Acknowledgments:

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multi-target therapeutic strategy for treating VD in the future.

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This work was supported by the National Natural Science Foundation of China

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(No.81241037) and the Youth Foundation of Hebei province Health and Family Planning Commission (No. 20160459).

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Figure Legends

Fig. 1. Effect of HRW on 2VO-induced spatial learning and memory deficits in rats, as

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measured with the MWM. Rats in the HRW and Don groups were postoperatively

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intragastric administered of HRW at 3 ml/d or donepezil at 3 ml/d (dissolved in DHW; the drug concentrations were adjusted to an appropriate volume for 1 mg/kg body weight), respectively, daily for 28 days. Rats in the sham and model groups were

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postoperatively intragastric administered an equal volume of DHW, given daily for 28 days. (A) Changes in the daily escape latencies to find the hidden platform 4 weeks after 2VO (n = 10 in each group). (B) Percentage of time spent in the target quadrant during the probe trial without the platform 4 weeks after 2VO (n = 10 in each group).

All data are expressed as means±SEMs. *p<0.05 vs. sham group; **p<0.01 vs. sham

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group; #p<0.05 vs. model group; ##p<0.01 vs. model group.

Fig. 2. Nissl staining revealed the effects of HRW on morphological changes in the hippocampal CA1 area. Rats in the HRW and Don groups were postoperatively intragastric administered of HRW at 3 ml/d or donepezil at 3 ml/d (dissolved in DHW; the drug concentrations were adjusted to an appropriate volume for 1 mg/kg body

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weight), respectively, daily for 28 days. Rats in the sham and model groups were postoperatively intragastric administered an equal volume of DHW, given daily for 28

(n = 3 in each group). (A) Representative images of pyramidal neuron damage

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days.

in the rats 4 weeks after 2VO. Scale bar = 100 μm. Magnification, ×200. (B)

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Representative images of pyramidal neuron damage 4 weeks post-operation in the

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four groups. Scale bar = 50 μm. Magnification × 400.

Fig. 3. HRW attenuates neuronal apoptosis in the hippocampus of rats subjected to 2VO. Rats in the HRW and Don groups were postoperatively intragastric administered of HRW at 3 ml/d or donepezil at 3 ml/d (dissolved in DHW; the drug concentrations were adjusted to an appropriate volume for 1 mg/kg body weight), respectively, daily

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for 28 days. Rats in the sham and model groups were postoperatively intragastric administered an equal volume of DHW, given daily for 28 days.(A) Representative

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photomicrographs of cleaved caspase-3-positive cells in the hippocampal CA1 area 4 weeks after 2VO. Scale bar = 20 μm. Magnification, ×400.(n = 3 in each group).

(B)

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Representative Western blot bands for Bcl-2 and Bax in the hippocampus of rats 4

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weeks after 2VO. β-actin was used as an internal control.(n = 6 in each group). (C)

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IOD/area value of cleaved caspase-3 in the CA1 region of each group. (n = 3 in each

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group). (D) Quantitative analysis of Bcl-2 and Bax relative expression (fold of the

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Sham group). (E) Quantitative analysis of the Bcl-2/Bax ratio (fold of the Sham group). (mean ± SEM, n = 6 in each group). *p<0.05 vs. sham group; **p<0.01 vs. sham

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group; #p<0.05 vs. model group; ##p<0.01 vs. model group; &p<0.05 vs. Don group;

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&&p<0.01 vs. Don group.

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Fig. 4. Electron microscopy results revealed the effects of HRW on ultrastructural damage in the hippocampal CA1 region in 2VO rats. Representative images of pyramidal neuron damage in the rats 4 weeks after 2VO. The arrows indicate autophagosomes. (A) Sham group. (B) Model group. (C) HRW group. (D) Don group.

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Scale bar = 500 nm. Magnification × 20000.

Fig. 5. Effects of HRW on suppressing autophagy in the hippocampal neurons of 2VO rats. (A) Representative photomicrographs of LC3-positive cells in the hippocampal CA1 area 4 weeks after 2VO. Scale bar = 20 μm. Magnification, ×400. (B) Representative Western blot bands for Atg7 and FoxO1 4 weeks after 2VO. (C)

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IOD/area value of LC3 in the CA1 region in each group (mean ± SEM, n = 3 in each group). (D) Quantitative analysis of Beclin 1 and p62 relative expression (fold of the

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Sham group) (mean ± SEM, n = 6 in each group). (E) Quantitative analysis of LC3-I, LC3-II, and the ratio of LC3-II/I (fold of the Sham group) (mean ± SEM, n = 6 in each

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group).(mean ± SEM, n = 6 in each group). *p<0.05 vs. sham group; **p<0.01 vs.

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sham group; ##p<0.01 vs. model group.

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Fig. 6. Effects of HRW on Atg7 and FoxO1 protein expression levels induced by CCH in the rat hippocampal CA1 area. (A) Representative Western blot bands for Atg7 and FoxO1 4 weeks after 2VO. (B) Quantitative analysis of Atg7 and FoxO1 expression (mean ± SEM, n = 6 in each group). **p < 0.01 vs. sham group; ##p < 0.01 vs. model

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group; &&p < 0.01 vs. Don group.