Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice

Qi He; Xiang Lan; Mengyuan Ding; Na Zhang

doi:10.1371/journal.pone.0317080

. 2025 Feb 14;20(2):e0317080. doi: 10.1371/journal.pone.0317080

Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice

Qi He ^1,^2,^#, Xiang Lan ^1,^2,^#, Mengyuan Ding ^1,^2,^#, Na Zhang ^3,^*

Editor: David Chau⁴

PMCID: PMC11828380 PMID: 39951412

Abstract

Background

Chronic stress has emerged as a prevalent facet of contemporary existence, significantly jeopardizing overall bodily health. The liver, a pivotal organ responsible for metabolic equilibrium, is particularly vulnerable to its adverse effects. This study delves into the hepatoprotective properties of extended consumption of HRW in mice subjected to chronic stress.

Methods

Mice subjected to chronic stress via CUMS and HRW administration for seven months underwent liver pathological examination. Key liver function indicators (AST, ALT), oxidative stress markers (SOD, CAT, GSH), and markers related to lipid peroxidation and ferroptosis (MDA, Fe) were measured using standard kits. ELISA determined corticosterone and 4-HNE levels. Immunofluorescence evaluated ROS, Nrf2, and apoptosis in liver tissues. Western blotting analyzed markers for ferroptosis (GPX4, SLC7A11, HO-1, Nrf2), apoptosis (Bax, Bcl-2, Cytc, Caspase-3, Caspase-8), mitochondrial biogenesis (Nrf1, PGC-1α, Tfam), and quality control (Drp1, Fis1, Mfn1, Mfn2, OPA1, PINK1, Parkin, LC3 I/II).

Results

The findings indicate a noteworthy improvement in liver health among mice exposed to HRW, as evidenced by histological analysis. Furthermore, the consumption of HRW exhibited hepatoprotection, as evidenced by the normalization of AST and ALT levels. Mechanistically, our results indicate that HRW elevates the levels of SOD, CAT, and GSH, while effectively clearing ROS within mitochondria. It was observed led to a regulation in the expression of mitochondrial quality control proteins, consequently improving mitochondrial biogenesis (Nrf1, PGC-1α, Tfam), and increasing ATP production. Furthermore, HRW decreased Cytc, Bax, Caspase-3, and Caspase-8 levels, and increasing the expression of Bcl-2. Additionally, HRW reduced MDA and 4-HNE levels, alleviating ferroptosis through the Nrf2/HO-1 pathway, and upregulating the expression of GPX4 and SLC7A11. By mitigating hepatocyte death through the aforementioned mechanisms, HRW fulfills its crucial role in safeguarding liver health.

Conclusions

This study reveals that long-term hydrogen-rich water (HRW) consumption provides significant hepatoprotection in mice under chronic stress. HRW normalizes liver enzyme levels, enhances antioxidant capacity, and reduces lipid peroxidation and ferroptosis. It improves mitochondrial biogenesis, function, and ATP production, and attenuates apoptosis by modulating related proteins. Behavioral tests show HRW alleviates stress-induced anxiety and enhances exploratory behavior. These findings suggest HRW is a promising non-invasive intervention for preventing and treating stress-related liver disorders by targeting oxidative stress and mitochondrial dysfunction.

Introduction

Stress, in its fundamental form, is the body’s innate response to perceived threats or challenges [1]. It initiates a series of physiological and psychological reactions intended to help the body deal with the situation. Stress can be categorized based on the duration of the stimulus, distinguishing between acute stress and chronic stress [2]. Acute stress is typically beneficial to the body [3], while chronic stress is detrimental both physiologically and psychologically. Chronic stress occurs when these reactions persist over an extended period, often without any direct threat. Various sources, including social, psychological, and physiological pressures, can induce chronic stress [4]. Chronic stress significantly impacts overall physical health, elevating the risk of numerous chronic diseases such as cardiovascular disorders, diabetes, and immune system dysregulation [5, 6]. Therefore, investigating effective methods to alleviate chronic stress has become a vital research focus in modern medicine.

In this context, hydrogen gas has gained significant attention as an emerging medical gas [7]. Hydrogen-rich water (HRW), which contains a high concentration of hydrogen gas (H₂), offers a unique advantage due to its small molecular size and exceptional solubility. These properties allow it to easily permeate cell membranes and enter cells, exerting its biological effects. Recent research has shown that hydrogen-rich water can alleviate various liver oxidative stress-induced injuries. For instance, Sun et al. [8] found that hydrogen-rich saline inhibited harmful ROS in the livers of mice with experimental liver injury and reduced the activity of pro-apoptotic factors such as JNK and caspase-3, thereby exerting a protective effect against liver injury. In the study by Iketani et al. [9], pre-drinking hydrogen-rich water was found to reduce liver damage in mice with LPS-induced sepsis. Li et al. [10] found that Hydrogen-rich saline protected liver tissues from ischemia-reperfusion injury by alleviating liver tissue ERS. Lin et al. [11] discovered that hydrogen-rich water mitigated ethanol-induced mouse fatty liver through its antioxidant and anti-inflammatory effects. Moreover, numerous studies have reported that drinking hydrogen-rich water can increase hydrogen concentration in the liver, allowing hydrogen molecules to exert their effects within the liver [9].

This study aims to investigate the protective effects of long-term consumption of HRW on the livers of mice subjected to chronic stress. A comprehensive range of experimental techniques, including behavioral observations, measurements of biochemical markers, histopathological analysis, Western blotting, and immunofluorescence, will be employed to systematically assess the impact of HRW on liver function and structure in mice experiencing chronic stress. The objective of these experiments is to uncover the mechanisms through which HRW influences mice under chronic stress, with a specific focus on the liver. This research endeavor aims to establish a scientific foundation for the clinical application of HRW. Through in-depth investigation, our goal is to provide theoretical support for the development of innovative stress resistance and liver protection strategies. Additionally, we aim to offer new insights into the clinical utilization of HRW, facilitate its broader application in the realm of chronic disease treatment, and contribute to the advancement of human and animal health.

Materials & methods

2.1 Animals and experimental scheme

2.1.1 Experimental animals grouping and experimental process

KM (Kunming) mice of both genders (n = 48, male = 24; female = 24) were weighed 30±5g (Liaoning Changsheng Biotechnology Co., Ltd) and were housed with unrestricted access to food and provided ample space for free movement. The mice, all eight weeks old, were regularly checked (mental status, fur quality, fecal consistency and so on) for good health and did not have any underlying diseases before the start of the experiment. They were kept in a controlled environment at 20±1°C with a relative humidity of 55%, following a 12-hour light-dark cycle. Water was made available to the mice during specific intervals: 8:00–10:00 am, 1:00–3:00 pm, and 6:00–8:00 pm. The mice were acclimated to the environment for 7 months before the commencement of the experiments. Ethical approval for all animal experiments conducted in this study was obtained from the Ethics Committee for Experimental Animals of Northeast Agricultural University (NEAUEC2022 03 21).

Mice were randomly divided into 3 groups using simple randomization., 16 mice per group (n = 16, male = 8; female = 8): the HRW treatment group (HRW group), the model group (Model group), and the control group (C group). Chronic stress modeling began after seven months of HRW feeding. The Model and HRW groups were subjected to Chronic Unpredictable Mild Stress (CUMS) experiments involving a series of stressors: heat stress, cold stress, crowding, slanted cages, starvation, vibration, and tail clipping. The HRW treatment group received daily consumption of freshly prepared HRW, while the model group and control group received distilled water. In the context of behavioral experiments, it is essential to measure and statistically analyze various parameters: total movement distance, frequency of grid crossings, number of times standing, and duration of resting in mice. After behavioral tests, mice were anesthetized using isoflurane inhalation. Subsequently, their eyes were enucleated for blood collection, after which they were euthanized using cervical dislocation and liver tissues were extracted for further analysis. The livers were partitioned into two sections: one part was fixed in formalin for liver morphology analysis, and the remaining samples were rapidly frozen in liquid nitrogen and stored at -80°C for frozen section preparation and various index measurements.

2.1.2 Preparation of HRW

Hydrogen gas produced by the hydrogen generator (QL-500, Saikesaisi Hydrogen Energy Co, Shandong, China) is introduced into 300 mL of distilled water through aeration. After 15 minutes, it yields HRW with a concentration of 0.5 mg/ml. The concentration of hydrogen in the water decreases at an average rate of 0.1 mg/mL per hour at room temperature, as measured by a HRW detector. The HRW is prepared and utilized as required.

2.1.3 Establishment of CUMS model

After seven months of feeding, both the HRW group and the Model group underwent a 21-day CUMS protocol. This protocol consisted of three cycles of stress, each lasting seven days. Behavioral testing was conducted after the completion of each stress cycle. (1) heat stress: Place the mice in a constant temperature oven at 45°C for 1 hour. (2) cold stress: Place the mice in a 4°C refrigerator for 1 hour. (3) crowding: Combine four cages of mice into one cage, and maintain crowding for 24 hours. (4) slanted cages: Tilt the mice cage at a 45-degree angle and maintain for 24 hours. (5) starvation: Withhold food from the mice for 24 hours, but allow free access to water. (6) vibration: Place the mice on a shaker for 30 minutes. (7) tail pinching: Use a clip to clip the base of the mice tail, and maintain for 3 minutes.

2.2 Behavioral testing

The open field test was employed to assess the spatial exploration behavior and anxiety levels of the mice in an unfamiliar environment. Mice were given unrestricted movement within a black, uncovered square box measuring 100×100×40 cm, devoid of any light source, for a duration of 3 minutes. A digital camera positioned above the box recorded the test data. Prior to the open field test, mice were placed in the testing environment for 30 minutes to acclimate to their surroundings. Following the 30-minute adaptation period, each mouse was placed individually in the center of the box and allowed to move freely within the dark, quiet enclosure for 3 minutes while the camera recorded their movement trajectories, total distance traveled, immobility time, number of standing instances, and grid crossings. The box was cleaned after each mouse’s activity to ensure the reliability of the results.

2.3 Corticosterone determination

Following the manufacturer’s guidelines, 10% liver tissue samples were homogenized and analyzed using ELISA kits specific for corticosterone (Cort) (Jingmei Biotechnology, Jiangsu, China). Standard wells were set up, with each receiving 50μl of standard solution at varying concentrations. Sample wells received 10μl of the sample to be tested, followed by 40μl of sample diluent. Subsequently, 100μl of horseradish peroxidase (HRP)-conjugated detection antibody was added to each well, both in the standard and sample wells. Reaction wells were sealed with a sealing membrane and then incubated at 37°C for 60 minutes in a constant temperature incubator. After removal of the liquid, the plate was blotted dry with absorbent paper, and each well was filled with wash buffer, left to stand for 1 minute, and then flicked off. This washing process was repeated five times. Subsequently, each well received 50μl of substrate A and B, and the plate was incubated at 27°C in the dark for 15 minutes. Following this, 50μl of stop solution was added to each well, the absorbance of the samples (n = 5 per group) was measured at 450 nm using an Epoch microplate reader (BioTek, Winooski, VT, USA). Expression levels of 4-HNE and Cort were determined utilizing standard curves.

2.4 Biomarkers of liver toxicity

A 10% liver tissue homogenate was prepared by homogenizing the liver tissue with a ratio of 9 parts normal saline to 1 part liver tissue. The homogenate was then centrifuged at 2500 rpm for 10 minutes, and the resulting supernatant was collected. Alanine transaminase (ALT) and, aspartate transaminase (AST) test kit used (C009-2-1, C010-2-1), 20μl of pre-warmed substrate solution was added to both the test wells and the control wells. Subsequently, 5μl of the sample to be tested was added to the test wells, while no sample was added to the control wells. Following a 30-minute incubation at 37°C, 20μl of 2,4-dinitrophenylhydrazine solution was added to both the test and control wells. Additionally, 20μl of the sample to be tested was added to the control wells, while no sample was added to the test wells. The plate was then incubated at 37°C for 20 minutes. Following this, 200μl of 0.4 mol/L sodium hydroxide solution was added to both the test and control wells. The plate was gently mixed and left at room temperature for 15 minutes. The optical density (OD) of each well was measured at 510nm using a microplate reader. The absolute OD value (OD of the test well minus OD of the control well) was used to construct a standard curve. The activity of ALT/AST was determined accordingly (n = 5 per group).

2.5 Liver enzymatic antioxidant activities

Superoxide dismutase test kit (SOD, A007-1-1, Jiancheng, Nanjing, China) is used according to the instructions. In the control and control blank wells, 20 μl of distilled water was added. Likewise, 20 μl of tissue homogenate was added to the test and test blank wells. Subsequently, 20 μl of enzyme working solution was added to the control and test wells, while 20 μl of enzyme dilution solution was added to the control blank and test blank wells. Following this, 200 μl of substrate application solution was added to the control, control blank, test, and test blank wells. After thorough mixing, the plates were incubated at 37°C for 20 minutes, and the OD values at 450 nm were determined using an enzyme marker. The SOD inhibition rate was calculated according to the formula: Cpr: Protein concentration to be tested (n = 5 per group).

S O D i n h i b i t i o n r a t e = \frac{[(O D_{c o n t r o l} ‐ O D_{c o n t r o l b l a n k}) ‐ (O D_{t e s t} ‐ O D_{t e s t b l a n k})]}{(O D_{c o n t r o l} ‐ O D_{c o n t r o l b l a n k})} \times 100 %

S O D a c t i v i t y = \frac{S O D i n h i b i t i o n r a t e \times 50 %}{C p r}

Catalase (CAT, A007-1-1, Jiancheng, Nanjing, China) test kit is used according to the instructions, 0.05mL of tissue homogenate was added to the test tube, while the control tube received no tissue homogenate. Subsequently, pre-warmed reagent one (1.0mL) and reagent two (0.1mL) were added to both the control and test tubes immediately after mixing, and the timer was started. After precisely 60 seconds at 37°C, reagent three (1.0mL) and reagent four (0.1mL) were added to both the control and test tubes. Additionally, 0.05mL of tissue homogenate was added to the control tube, while the test tube remained unchanged. After thorough mixing, the absorbance of each tube (n = 5 per group) was measured at a wavelength of 405nm. CAT activity was then calculated using the formula: T: reaction time, 60s.

C A T a c t i v i t y = (O D_{c o n t r o l} ‐ O D_{t e s t}) \times 271 \div V_{s a m p l e} \div T \div C p r

Glutathione (GSH, A006-2-1, Jiancheng, Nanjing, China) test kit is used according to the instructions., In the blank wells, 100μl of reagent one was added, while in the standard wells, 100μl of 20μmol/L GSH standard solution was added. In the test wells, 100μl of tissue homogenate was added. Subsequently, 25μl of reagent three and 100μl of reagent two were added to the blank, standard, and test wells. The plate was gently shaken and left to stand for 5 minutes. The absorbance of each well (n = 5 per group) was measured at 405nm. GSH content was calculated using the formula:

G S H c o n t e n t = \frac{(O D_{t e s t} ‐ O D_{b l a n k})}{(O D_{s t a n d a r d} ‐ O D_{b l a n k})} \times c_{s t a n d a r d} \times 5 \times 10

2.6 Liver lipid peroxidation detection

Malondialdehyde (MDA, A003-1, Jiancheng, Nanjing, China) test kit used according to the instructions, In the blank tubes, 100μl of anhydrous ethanol was added, while in the standard tubes, 100μl of 10nmol/ml standard solution was added. The test and control tubes received 100μl of tissue homogenate. Subsequently, 100μl of reagent one was added to the blank, standard, test, and control tubes, followed by thorough mixing. Then, 3ml of reagent two was added to the blank, standard, test, and control tubes, and 1ml of reagent three was added to the blank, standard, and test tubes, with 1ml of 50% ice-cold acetic acid added to the control tubes. Cover the centrifuge tubes with caps, puncture a small hole in the cap using a needle, vortex mix thoroughly, immerse in a 95°C water bath for 40 minutes, remove, and cool under running water, then centrifuge at 3500–4000 rpm for 10 minutes. Collect the supernatant and measure the absorbance values of each tube (n = 5 per group) at 532 nm. MDA content was calculated using the formula:

M D A c o n t e n t = \frac{(O D_{t e s t} - O D_{c o n t r o l})}{(O D_{s t a n d a r d} - O D_{b l a n k})} \times c_{s t a n d a r d} \div C p r

The ELISA detection method for 4-hydroxynonenal (4-HNE, Jingmei Biotechnology, Jiangsu, China) is the same as the corticosterone detection (Method 2.3).

2.7 ATP content detection

Following the instructions provided with the ATP content testing kit (A095-1-1, Jiancheng, Nanjing, China), 30 μL of 1 mmol/L standard solution was added to the blank tubes and standard tubes, while 30 μL of sample was added to the test tubes and control tubes. Subsequently, substrate solution one (100 μL) and substrate solution two (200 μL) were added to the blank tubes, standard tubes, test tubes, and control tubes. 30 μL of enhancer was added to the standard tubes and test tubes. After thorough mixing, the tubes were incubated in a 37°C water bath for 30 minutes. Then, 50 μL of precipitant was added to the blank tubes, standard tubes, test tubes, and control tubes. After thorough mixing, the tubes were centrifuged at 4000 rpm for 5 minutes, and 300 μl of supernatant was collected for measurement. Subsequently, 500 μL of color developing solution was added to all tubes, mixed thoroughly, and allowed to stand at room temperature for 2 minutes. Then, 500 μL of stop solution was added to all tubes, mixed thoroughly, and allowed to stand for 5 minutes. The absorbance of each tube was measured at 636 nm. ATP content was calculated using the formula: N: the dilution factor of the sample before measurement.

A T P c o n t e n t = \frac{(O D_{t e s t} - O D_{c o n t r o l})}{(O D_{s t a n d a r d} - O D_{b l a n k})} \times c_{s t a n d a r d} \times N \div C p r

2.8 Tissue iron content detection

According to the instructions of the tissue iron test kit, the iron content (Fe, A039-2-1, Jiancheng, Nanjing, China) in liver tissue was determined. 500μl of double-distilled water was added to the blank wells, 500μl of iron standard application solution with a concentration of 2mg/L was added to the standard wells, and 500μl of the test sample was added to the test wells. Subsequently, 1500μl of iron chromogenic reagent was added to all wells, mixed thoroughly, heated in a boiling water bath for 5 minutes, cooled under running water, centrifuged at 3500 rpm for ten minutes, and 1000μl of supernatant was collected. The absorbance OD values of each well were measured at a wavelength of 520nm.

i r o n c o n t e n t = \frac{(O D_{t e s t} - O D_{b l a n k})}{(O D_{s t a n d a r d} - O D_{b l a n k})} \times c_{s t a n d a r d} \div C p r

2.9 Histological examination

Liver tissues were fixed in 4% formaldehyde for more than 24 hours, then processed for routine paraffin section preparation and stained with hematoxylin and eosin (HE). Morphological changes in the liver tissues of each group of mice, such as degeneration, necrosis, inflammatory cell infiltration, and other pathological alterations, were observed under an optical microscope.

2.10 Immunofluorescence

For TUNEL and Nrf2 assays, tissue samples were deparaffinized in water and rinsed twice for 3 minutes each with distilled water. Subsequently, the tissue samples were treated with proteinase K, followed by PBS washing. The tissues were incubated with the TUNEL/Nrf2 reaction solution and stained with DAPI for nuclear labeling. Finally, the slides were sealed with an anti-fade solution, and images were captured using a fluorescence microscope.

The liver tissue was sliced into 2 mm thick sections, embedded in cryoembedding medium, and then cut into 10 μm thick sections using a cryostat. Subsequently, the sections were incubated in a dark chamber containing dihydroethidium for 30 minutes, followed by a PBS wash. The samples were observed and imaged using a fluorescence microscope.

2.11 Western blotting

Protein is extracted using conventional RIPA lysate and PMSF solution. Nuclear protein and cytoplasmic protein extraction kit (P0027; Beyotime) was used to separate and extract nuclear and cytoplasmic protein from liver tissue, and used to detect Nrf2 protein. Proteins were separated using different concentrations of SDS-PAGE (6%-15%). The separated proteins on the electrophoresis gel were transferred onto a nitrocellulose (NC) membrane and blocked with 5% BSA at room temperature for 2 hours. The membrane was then incubated with the appropriate primary antibodies overnight at 4°C, the antibodies used are listed in Table 1. Subsequently, the NC membrane was incubated with secondary antibodies (goat anti-rabbit IgG, dilution 1:10,000) for 1 hour, followed by washing the membrane with TBST buffer. Next, protein bands were visualized using an ECL chemiluminescence kit (Beyotime Biotechnology, Shanghai, China). Images were captured using the Tanon-5200 chemiluminescence imaging system (Tanon Science & Technology, Shanghai, China). The grayscale values for each band were quantified using ImageJ software (NIH, Bethesda, MD, USA). Protein expression was normalized to the corresponding internal control (β-actin).

Table 1. Antibody description.

Antibody name	Art.No.	Manufacturer	Dilution ratio
Nrf1	bs-1342R	Bioss	1:1000
OPA1	bs-11764R	Bioss	1:1000
Mfn1	bs-0557R	Bioss	1:1000
Mfn2	bs-2988R	Bioss	1:1000
Tfam	A13552	ABclonal	1:1000
Fis1	A19666	ABclonal	1:1000
PGC-1α	WL02123	Wanlei	1:1000
DRP1	WL03028	Wanlei	1:1000
GPX4	WL0546	Wanlei	1:1000
SLC7A11	A13685	ABclonal	1:1000
HO-1	WL02400	Wanlei	1:1000
Nrf2	WL02135	Wanlei	1:1000
Cytc	WL02410	Wanlei	1:1000
Bax	WL01637	Wanlei	1:1000
Bcl-2	WL01556	Wanlei	1:1000
Caspase-3	WL02117	Wanlei	1:1000
Caspase-8	WL03426	Wanlei	1:1000
PINK1	WL04963	Wanlei	1:1000
Parkin	WL02512	Wanlei	1:1000
LC3 I/II	WL01506	Wanlei	1:1000

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2.12 Statistical analysis

Data analysis and graphing were performed using GraphPad Prism 8 (GraphPad Software, San Diego, USA). The results were presented as mean ± standard error of mean (mean ± SEM) for all experimental data. One-way analysis of variance (ANOVA) was utilized for significant differential analysis, and Tukey test are used after ANOVA. Differences with a p-value less than 0.05 were considered statistically significant.

Results

3.1 Behavioral testing

The results of the open field test for mice are depicted in Fig 1. Mice in C group exhibited diverse activities characterized by unpredictable movement patterns. In contrast, mice in the Model group displayed limited activity, mostly confined to the periphery. When compared to the Model group, mice in the HRW group demonstrated a wider activity range with irregular movement patterns, resembling those in the C group.

The statistical analysis of mouse behavior in the open field test yielded noteworthy differences among groups. Specifically, the Model group displayed a substantial reduction in total distance traveled, grid crossings, and standing counts in comparison to C group (P < 0.05). Conversely, the Model group exhibited a significant increase in immobility time (P < 0.05). In contrast, the HRW group demonstrated a significant rise in total distance traveled, grid crossings, and standing counts in contrast to the Model group (P < 0.05). Additionally, the HRW group displayed a marked decrease in immobility time compared to both the Model and C groups (P < 0.05). Notably, the HRW group also showed a significant decrease in total distance traveled compared to Group C (P < 0.05).

3.2 Corticosterone level

Fig 2 provides clear evidence that both the Model group and the HRW group displayed a significant increase in corticosterone levels in comparison to C Group (P < 0.05). Additionally, the HRW group exhibited a noteworthy elevation in corticosterone levels when compared to the Model group (P < 0.05).

3.3 Liver structure and functional index

As depicted in Fig 3, the histological examination revealed distinctive findings across the different groups. C group exhibited no apparent liver structural damage. In contrast, the Model group displayed multiple noticeable bleeding spots, partial vacuolization of liver cells, and disorganized, separated hepatic cords. Remarkably, these pathological changes were significantly ameliorated in the HRW group. Upon analyzing liver function markers, it was observed that both the Model group and the HRW group exhibited a substantial elevation in ALT and AST levels compared to C group (P < 0.01). However, in comparison to the Model group, the HRW group demonstrated a significant reduction in ALT and AST content (P < 0.05).

3.4 Oxidative stress related factors

Fig 4 clearly illustrate that both the Model group and the HRW treatment group displayed a significant increase in ROS levels when compared to C group. However, it is noteworthy that the Model group exhibited a notably higher level of ROS within the liver when compared to the HRW group. In contrast, when compared to C group, both the Model group and the HRW group exhibited a significant decrease in SOD, CAT, and GSH levels (P < 0.05). Conversely, the HRW group displayed a substantial increase in SOD, CAT, and GSH levels within the liver when compared to the Model group (P < 0.05).

3.5 Lipid peroxidation related factors

Fig 5 illustrates that the lipid peroxidation markers MDA and 4-HNE in C group were significantly lower than those in the Model group and HRW group (P < 0.05). Moreover, compared to the Model group, the HRW group exhibited a significant decrease in the levels of MDA and 4-HNE (P < 0.05).

3.6 Ferroptosis related factors

Based on the data presented in Fig 6, it is evident that both the Model group and the HRW group exhibited a significant increase in iron (Fe) content in comparison to C group (P < 0.05). Notably, the HRW group displayed a significant decrease in Fe content when compared to the Model group (P < 0.05). Furthermore, when compared to C group, both the Model group and the HRW group showed a significant reduction in the protein expression levels of iron death-related markers GPX4 and SLC7A11 (P < 0.05). Additionally, the Model group exhibited significantly lower GPX4 and SLC7A11 protein expression levels in comparison to the HRW group (P < 0.05).

Upon analyzing proteins associated with the Nrf2/HO-1 pathway, it was observed that both the Model group and the HRW group exhibited significantly lower cytoplasmic Nrf2 expression levels compared to C group (P < 0.05). Furthermore, in comparison to the cytoplasmic Nrf2 protein expression in the Model group, the HRW group demonstrated a further decrease (P < 0.05). Conversely, concerning nuclear Nrf2 expression levels in comparison to C group, both the Model group and the HRW group displayed significantly higher levels (P < 0.05). Additionally, in contrast to the nuclear Nrf2 protein expression in the HRW group, the Model group displayed a significant decrease (P < 0.05). Furthermore, both the Model group and the HRW group exhibited a significant decrease in HO-1 protein expression levels when compared to C group (P < 0.05). Conversely, the HRW group showed a significant increase in HO-1 protein expression levels compared to the Model group (P < 0.05). This observation was further corroborated through immunofluorescence analysis of Nrf2, as depicted in Figs 3–6H.

3.7 Apoptosis related factors

The TUNEL fluorescence staining results in Fig 7 indicate a significant increase in apoptosis in both the Model group and the HRW group compared to the C group. Moreover, apoptosis was found to be reduced in the HRW group when compared to the Model group.

In the analysis of apoptosis-related protein expression, it was evident that both the Model group and the HRW group displayed a significant increase in the expression of pro-apoptotic factors, which included Bax, Cytc, Caspase-3, and Caspase-8, compared to the C group (P < 0.05). Notably, the HRW group exhibited a significant decrease in the expression of these pro-apoptotic factors when compared to the Model group (P < 0.05). Furthermore, in comparison to the C group, both the Model group and the HRW group demonstrated a significant decrease in the expression of the anti-apoptotic factor Bcl-2 (P < 0.05). Additionally, the expression of Bcl-2 was significantly lower in the Model group when compared to the HRW group (P < 0.05).

3.8 Mitochondrial biosynthesis related factors

As presented in Fig 8, both the Model group and the HRW group exhibited a significant decrease in ATP levels compared to the C group (P < 0.05). Furthermore, the HRW group displayed a significant increase in ATP levels when compared to the Model group (P < 0.05). In the analysis of factors associated with mitochondrial biogenesis, it was noted that both the Model group and the HRW group showed a significant reduction in the protein expression levels of PGC-1α, Nrf1, and Tfam in comparison to the C group (P < 0.05). Additionally, when compared to the Model group, the HRW group demonstrated a significant increase in the protein expression levels of PGC-1α, Nrf1, and Tfam (P < 0.05).

3.9 Mitochondrial quality control-regulated factors

Based on the data presented in Fig 9, it was observed that both the Model group and the HRW group exhibited a significant increase in the protein expression levels of mitochondrial fission factors, Drp1 and Fis1, compared to the C group (P < 0.05). Moreover, in comparison to the Model group, the HRW group displayed a significant decrease in the expression of Drp1 and Fis1 (P < 0.05). Regarding mitochondrial fusion factors, including Mfn1, Mfn2, and OPA1, both the Model group and the HRW group demonstrated a significant decrease in protein expression levels compared to the C group (P< 0.05). Notably, the HRW group exhibited a significant increase in the expression of Mfn1, Mfn2, and OPA1 when compared to the Model group (P < 0.05).

In the analysis of factors related to mitophagy, including PINK1, Parkin, and LC3I/II, it was noted that both the Model group and the HRW group exhibited a significant decrease in the expression levels of these proteins compared to the C group (P < 0.05). However, the HRW group displayed a significant increase in the expression levels of PINK1, Parkin, and LC3I/II when compared to the Model group (P < 0.05).

Discussion

1. Establishment of a chronic stress model in mice

Our study successfully established a chronic stress model using the CUMS paradigm, as demonstrated by significant behavioral changes in the open field test and elevated corticosterone levels. Consistent with previous research, CUMS reliably induces behavioral and physiological changes in rodents that mimic human stress responses [12]. These findings strengthen the model’s validity as a preclinical tool for exploring potential treatments for stress-related disorders.

The reduction in corticosterone levels in HRW-treated mice suggests a moderating effect on the hypothalamic-pituitary-adrenal (HPA) axis, a core pathway implicated in stress responses [13]. Given that HPA axis dysregulation is also associated with anxiety and depression in humans, these results indicate HRW’s potential as a therapeutic agent. However, it is important to recognize the challenges involved; while CUMS is effective in rodents, stress responses in humans are influenced by additional complex psychological and social factors not fully replicable in animal models. Further research is needed to determine HRW’s efficacy and safety in human subjects and to explore optimal dosing strategies, potential side effects, and long-term impacts.

Notably, our findings align with previous studies showing that hydrogen-rich solutions possess anxiolytic and antidepressant effects in animal models [14]. This study contributes to the growing evidence that HRW might hold promise as a novel intervention for stress-related disorders, potentially offering an alternative approach that targets physiological stress pathways. The observed effect on corticosterone levels highlights a possible application of HRW in moderating stress without the need for traditional pharmacological treatments that may have more extensive side effects.

2. The effect of HRW on liver structure and function

This study clearly demonstrated the protective effects of HRW on liver structure and function through histological and biochemical analyses. Elevated ALT and AST levels are common indicators of liver damage [15], and our findings that HRW reduces these levels in chronically stressed mice align with previous studies demonstrating its hepatoprotective properties [16]. The ability of HRW to improve liver function under stress suggests its potential as a therapeutic agent for stress-induced hepatic damage [17]. Research has shown that hydrogen-rich solutions can protect liver tissue by enhancing antioxidant defenses and reducing inflammation [18]. Although our study demonstrates that HRW not only alleviates biochemical markers of liver damage but also improves histological architecture, more comprehensive investigations are warranted to elucidate its underlying mechanisms. It is particularly important to assess the applicability of HRW in different animal models and clinical contexts, for example, while our results show the efficacy of HRW in mouse models, the application of these findings to humans requires consideration of physiological differences, dose optimization, and the safety of long-term use. Our findings highlight the potential of HRW in clinical applications, particularly in treating liver damage induced by chronic stress. Future clinical trials will be essential to validate the therapeutic effects of HRW, further advancing its application in liver disease management.

3. The effect of HRW on mitochondria in chronically stressed mice

Chronic stress is known to disrupt mitochondrial dynamics, increasing ROS production and impairing mitochondrial quality [19]. Our research demonstrated that HRW significantly improves mitochondrial function, as indicated by enhanced ATP levels and the regulation of mitochondrial biogenesis factors such as PGC-1α and Tfam. This supports previous findings where molecular hydrogen was shown to exert protective effects on mitochondria, potentially through its antioxidant properties [20]. Furthermore, the observed modulation of mitochondrial fusion and fission proteins, with increased Mfn2 and decreased Drp1 expression, suggests that HRW enhances mitochondrial dynamics, promoting fusion and reducing excessive fission, which is crucial for maintaining mitochondrial integrity under stress condition [21]. This is in line with studies showing that improved mitochondrial fusion can counteract stress-induced mitochondrial damage [22]. Additionally, the activation of mitophagy observed in our study supports the role of HRW in maintaining mitochondrial quality control, as mitophagy is essential for the removal of damaged mitochondria [23], and this action of HRW not only enhances self-repair capacity of the cell but also provides new insights into its potential clinical applications, particularly in the management of stress-related metabolic diseases.

These findings suggest that HRW could be an emerging strategy for treating chronic stress-related metabolic disorders. Given that these conditions often involve mitochondrial dysfunction, the application of HRW may help restore mitochondrial function, improve cellular metabolism, and ultimately enhance patients’ quality of life. Future research should focus on elucidating the protective mechanisms of HRW in different pathological states and conducting clinical trials to validate its efficacy in managing stress-related diseases.

4. The effect of HRW on mitochondrial pathway apoptosis

Our findings demonstrate that HRW can significantly mitigate stress-induced apoptosis in liver cells by modulating the mitochondrial apoptotic pathway. The balance between pro-apoptotic and anti-apoptotic factors is crucial for cell survival under stress [24]. We observed an increase in Bcl-2 expression and a decrease in the release of cytochrome c (Cytc), indicating the key role of HRW in protecting against apoptosis. This aligns with prior research showing that hydrogen-rich solutions can inhibit apoptosis through similar mechanisms [25]. This finding not only validates the anti-apoptotic effects of HRW but also underscores its potential value as a clinical therapeutic strategy. Oxidative stress is recognized as a pathological mechanism underlying various diseases, including liver disease, cardiovascular conditions, and neurodegenerative disorders. Thus, HRW, as an intervention capable of enhancing anti-apoptotic signaling pathways, may be clinically applicable in reducing cell death caused by oxidative stress, ultimately improving patient outcomes [26].

Moreover, the antioxidant effects of HRW complement the current clinical practices involving antioxidants. This suggests that HRW could be used in conjunction with existing treatment regimens to enhance therapeutic efficacy. For example, in the management of chronic liver disease, the use of HRW may help mitigate liver cell apoptosis induced by drugs or pathological conditions, thereby improving liver function and quality of life for patients [27, 28].

To further validate the role of HRW in apoptosis regulation, future research should explore its mechanisms of action, including how it modulates specific signaling pathways and intracellular environments. Moreover, when translating these findings into clinical practice, it is crucial to consider patient variability and the long-term safety of HRW to achieve optimal outcomes in the treatment of liver diseases.

5. The effect of HRW on lipid peroxidation and ferroptosis

Chronic stress often leads to increased lipid peroxidation due to excessive ROS production, making cells vulnerable to ferroptosis [29]. Our study revealed that HRW significantly reduces markers of lipid peroxidation, such as MDA and 4-HNE, suggesting its role in protecting against oxidative damage [30]. This finding is consistent with previous research indicating that hydrogen molecules can effectively neutralize ROS and reduce lipid peroxidation [31]. Moreover, the modulation of ferroptosis-related proteins, including GPX4 and SLC7A11, underscores HRW’s ability to protect against ferroptosis, a regulated cell death pathway linked to oxidative stress [32]. Our study aligns with the growing body of research that highlights the role of Nrf2 in mediating cellular defense mechanisms against oxidative stress and ferroptosis [33]. The observed upregulation of Nrf2 and its target gene HO-1 in HRW-treated mice further supports the hypothesis that HRW activates endogenous antioxidant pathways to mitigate oxidative damage [34]. Clinically, these findings suggest that HRW may have potential applications in the treatment of metabolic diseases related to oxidative stress. Conditions associated with chronic stress, such as cardiovascular diseases, diabetes, and liver disorders, often involve oxidative damage and ferroptosis. Thus, the use of HRW may help alleviate these pathological processes, improving overall health outcomes for patients. Future research should further explore the role and mechanisms of HRW in various disease models to confirm its efficacy and safety for clinical applications.

Furthermore, while we have identified that HRW alleviates intestinal inflammation in mice with chronic inflammation through the Nf-κB pathway [35], it remains pertinent to explore whether, in the context of mice subjected to chronic stress, the amelioration of stress is achieved by modulating the Nf-κB pathway in the liver. This could provide a deeper understanding of how HRW confers its protective effects and its potential therapeutic applications.

Conclusions

This study reveals that long-term hydrogen-rich water consumption provides significant hepatoprotection in mice under chronic stress. HRW normalizes liver enzyme levels, enhances antioxidant capacity, and reduces lipid peroxidation and ferroptosis. It improves mitochondrial biogenesis, function, and ATP production, and attenuates apoptosis by modulating related proteins. Behavioral tests show HRW alleviates stress-induced anxiety and enhances exploratory behavior. These findings suggest HRW is a promising non-invasive intervention for preventing and treating stress-related liver disorders by targeting oxidative stress and mitochondrial dysfunction.

Supporting information

S1 Table. Raw data of this article.

(XLSX)

pone.0317080.s001.xlsx^{(11KB, xlsx)}

S1 Raw images

(PDF)

pone.0317080.s002.pdf^{(1.3MB, pdf)}

Data Availability

All relevant data are within the article and its Supporting Information files.

Funding Statement

This work was supported by the National Natural Science Foundation of China (Grant No. 32473107) and the Science and Technology Plan Project of Inner Mongolia Autonomous Region (Grant No. 2022YFSH0052).

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PLoS One. doi: 10.1371/journal.pone.0317080.r001

Decision Letter 0

David Chau

12 Feb 2024

PONE-D-23-43180Long-term drinking hydrogen-rich water provides hepatoprotection against chronic stress in micePLOS ONE

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Reviewer #2: Yes

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Reviewer #1: -Title: the title does not properly describe the content

-I would like that the authors provide a title that highlight the potential antioxidant of Hydrogen rich water (HRW) on mitochondrial biogenesis and quality control.

-Abstract: The abstract is sufficiently informative summary of the main aspect; however, it should be written in an intelligible fashion.

-Keywords: the keywords used may not allow a proper retrieval of the information on the internet

-Abbreviation: the abbreviation used in the manuscript should be mentioned after keywords.

-Introduction: the first part of the manuscript is not an updated and interesting introduction to the subject. The authors should deeply enrich this section. According to the study the authors should give a summary of the recent studies on using Hydrogen-rich water and/or other close chemicals to alleviate oxidative stress damages in liver.

-Material and methods:

-The sub-title "Preparation of HRW" and "Establishment of CUMS model" must be included in the paragraph " Animals and experimental scheme" and Table 2-1 could be removed and write down how the CUMS was applied to the animals.

-Line 94: KM is the abbreviation of what?

-Line 96-97: The authors must give more details here and provide the measurement followed to check the heath of the animals and that they were free from disease! "'The mice, all eight weeks old, were regularly checked for good health and did not have any underlying diseases before the start of the experiment."

-Line 149: Elisa sub-title should be changed to Corticosterone determination and more details should be provided of how authors used the Elisa kit.

-Line 154: is it an expression level of Corticosterone?

-Material and methods for 4-Hydroxynoenal and MDA must be under the same Sub-title (Liver lipid peroxidation measurement)

-Line 156: Test kit_ the title of this paragraph must be changed

Line 158 and 159: ALT and AST can be under the name of sub-title: biomarkers of liver toxicity.

Line 159 and 160: SOD, CAT and GSH as well can be in a different sub-title: Liver enzymatic antioxidant activities.

-The authors should provide a brief description of how each parameter was extracted and measured.

-Line 164: The authors can move the histological examination to the end and provide more details; the type of the optical microscope used and how the severity of stress was examined. Any scores on a scale were used?

-For the statistical analysis, I would like if the authors used the letters for significance.

-Principal component analysis (PCA) is recommended to be applied in this study.

-The results part must be improved.

-Number all tables sequentially (Table 1, Table 2, etc.), likewise for figures (Figure 1, Figure 2, etc.).

-In the title of each figure, authors should mention the number of biological replicates ± standard deviation. The title should be presented in an intelligible fashion.

-The quality of figures and histological section of liver must be improved.

-The discussion part needs to be improved; it is missing the discussions with previous research in the same context (Example: 'Hydrogen-rich water reduces inflammatory responses and prevents apoptosis of peripheral blood cells in healthy adults: a randomized, double-blind, controlled trial, The Effects of 24-Week, High-Concentration Hydrogen-Rich Water on Body Composition, Blood Lipid Profiles and Inflammation Biomarkers in Men and Women with Metabolic Syndrome: A Randomized Controlled Trial, etc.).

-The conclusion as well must be presented in an intelligible fashion.

-The discussion should not be repetitive for the results section.

Reviewer #2: The article by He et al describes hepatoprotection effects of drinking hydrogen-rich water against chronic stress in mice. The hepatoprotection effects of hydrogen have been reported in many liver injury models, but the effects of hydrogen in CUMS models have not been well elucidated. Therefore, the results of the present study are clinically important. But there are some critical points to be clarified.

Major points

１, In this CUMS model, transaminases are elevated significantly in the model group. Is there any previous report which shows massive liver damage in CUMS models?

The reasons why the authors adopted this model for drinking hydrogen-rich water should also be explained.

2, Hydrogen is known to selectively attenuate hydroxyl radicals and peroxynitrite (Nat Med. 2007 Jun;13(6):688-94. doi: 10.1038/nm1577). The antioxidant activity of hydrogen in this study appears to have a wide range of antioxidant activity, including SOD, CAT and GSH. The difference in the effects of hydrogen from previous reports should be discussed.

3, The authors have shown that drinking hydrogen-rich water alleviates behavioral changes in response to chronic stress, but the mechanism underlying this has not been addressed. Many of the histological results in this paper indicate changes in ROS in the liver, but are there any previous reports that link this to behavioral changes? Or does the behavior-altering effect of hydrogen-rich water directly affect nerves? Suggestions for mechanisms of behavioral changes should also be mentioned.

4, What does Figure 3-7G mean? In the figure it is written as TUNEL staining, but in the figure legend it is written as Nrf2 immunofluorescent. Please describe correctly. Moreover, I can not see any fluorescent signal in the picture. Clearer figures are necessary.

Minor points

1, At line 130, “Table 2-1” is referenced but corresponding table is named as Table1. At line 186, Table 2-2 is referenced but the corresponding table is named as Table2. Please unify them.

2, In the Figure3-3, HE staining are unclear. To show the vacuolization of the liver cells, pictures of higher magnification might be better.

3, In the immunofluorescence analysis of Nrf2 in Figure 3-6, I can not see any fluorescent signal in the picture. Clearer figures are necessary.

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Reviewer #1: Yes: Saoussen Ben-Abdallah

Reviewer #2: No

**********

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PLoS One. 2025 Feb 14;20(2):e0317080. doi: 10.1371/journal.pone.0317080.r002

Author response to Decision Letter 0

25 Feb 2024

Thank you very much for the valuable comments provided by the reviewer on this article. We have revised the article according to the relevant comments and responded to the reviewer's questions.

Response to reviewer 1:

Q1: Title: the title does not properly describe the content.

-I would like that the authors provide a title that highlight the potential antioxidant of Hydrogen rich water (HRW) on mitochondrial biogenesis and quality control.

Response: Thank you for your valuable feedback regarding the title of our paper. We appreciate your suggestion to highlight the potential antioxidant effects of HRW on mitochondrial biogenesis and quality control. We agree that the title should effectively convey the focus and significance of our research. We have revised the title (Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice.) to better emphasize the antioxidant properties of HRW in relation to mitochondrial biogenesis and quality control. We believe this adjustment will enhance the clarity and relevance of our study.

Q2: Abstract: The abstract is sufficiently informative summary of the main aspect; however, it should be written in an intelligible fashion.

Response: We understand your concern regarding the clarity and intelligibility of the abstract. We have carefully revised it to ensure that the content is presented in a clear and accessible manner, making it easier for readers to understand the key findings and implications of our research.

Q3: Keywords: the keywords used may not allow a proper retrieval of the information on the internet.

Response: Thank you very much for your suggestion. We have updated the keywords of the article.

Q4: Abbreviation: the abbreviation used in the manuscript should be mentioned after keywords.

Response: Thank you very much for your suggestion. We have placed the abbreviation after the keyword.

Q5: Introduction: the first part of the manuscript is not an updated and interesting introduction to the subject. The authors should deeply enrich this section. According to the study the authors should give a summary of the recent studies on using Hydrogen-rich water and/or other close chemicals to alleviate oxidative stress damages in liver.

Response: Thank you for your insightful comments on the introduction section of our manuscript. We appreciate your suggestion to enrich this portion with more recent studies and information related to the use of Hydrogen-rich water and similar compounds in mitigating oxidative stress damages in the liver. We recognize the importance of providing an updated and engaging introduction that reflects the current state of research in this field. We have thoroughly reviewed recent literature and incorporate relevant studies that highlight the potential of Hydrogen-rich water and related substances in addressing oxidative stress-related liver damage.

Q6: The sub-title "Preparation of HRW" and "Establishment of CUMS model" must be included in the paragraph " Animals and experimental scheme" and Table 2-1 could be removed and write down how the CUMS was applied to the animals.

Response: Thank you very much for your feedback. We have made the relevant changes in the article as requested.

Q7: Line 94: KM is the abbreviation of what?

Response：KM mice is the abbreviation of Kunming mice.

Q8: Line 96-97: The authors must give more details here and provide the measurement followed to check the heath of the animals and that they were free from disease! "'The mice, all eight weeks old, were regularly checked for good health and did not have any underlying diseases before the start of the experiment."

Response: Thank you for your valuable feedback regarding the health assessment and disease screening procedures for the experimental animals in our study. The mice we used had undergone strict health checks at the biotech company before we purchased them, so we did not undergo too many checks during the week of adapting to the environment. We only conducted basic tests such as mental state, appetite, hair quality, and fecal condition.

Q9: Line 149: Elisa sub-title should be changed to Corticosterone determination and more details should be provided of how authors used the Elisa kit.

Response: Thank you very much for your suggestion. We have made the relevant changes in the article as requested.

Q10: Line 154: is it an expression level of Corticosterone?

Response: Yes, Cort is the abbreviation for corticosterone.

Q11: Material and methods for 4-Hydroxynoenal and MDA must be under the same Sub-title (Liver lipid peroxidation measurement).

Response: Thank you very much for your suggestion. We have made the relevant changes in the article as requested. Now this part is in Method 2.6 (Liver lipid peroxidation detection).

Q12: Line 156: Test kit_ the title of this paragraph must be changed.

Line 158 and 159: ALT and AST can be under the name of sub-title: biomarkers of liver toxicity.

Line 159 and 160: SOD, CAT and GSH as well can be in a different sub-title: Liver enzymatic antioxidant activities.

Response: Thank you very much for your suggestion. We have made the relevant changes in the article as requested. Now this part is in Method 2.4 and Method 2.5 (Biomarkers of liver toxicity; Liver enzymatic antioxidant activities).

Q13: The authors should provide a brief description of how each parameter was extracted and measured.

Response: Thank you for your feedback. I have supplemented the parameter measurement method in the Method.

Q14: Line 164: The authors can move the histological examination to the end and provide more details; the type of the optical microscope used and how the severity of stress was examined. Any scores on a scale were used?

Response: Thank you very much for your suggestion. We have supplemented the model of the microscope in the article. We use typical pathological changes in HE slices to determine the degree of stress, such as the arrangement of liver cords, cellular vacuolization, and bleeding in the liver. We did not use any scores as the severity of liver structural damage can be clearly observed in HE slices.

Q15: For the statistical analysis, I would like if the authors used the letters for significance.

Principal component analysis (PCA) is recommended to be applied in this study.

Response: Thank you for your suggestion regarding the application of Principal Component Analysis (PCA) in our study. We appreciate your interest in exploring alternative analytical approaches to enhance the interpretation of our data.

While PCA is indeed a powerful method for dimensionality reduction and pattern recognition, we chose to employ one-way ANOVA for several reasons specific to the objectives and design of our study.

Firstly, our primary aim was to examine the effects of different experimental conditions on specific outcome variables, such as antioxidant levels or mitochondrial function markers. One-way ANOVA allows us to assess the significance of differences among multiple groups and identify potential treatment effects.

Secondly, PCA is commonly used for exploratory data analysis and visualization, particularly when dealing with high-dimensional datasets. However, in our study, the variables under investigation were relatively well-defined and directly related to our research questions, making one-way ANOVA a more suitable choice for hypothesis testing and inference.

That being said, we acknowledge the value of PCA in identifying underlying patterns and relationships within complex datasets. In future studies, we will consider incorporating PCA as a complementary analytical tool to further explore the multivariate structure of our data.

Q15: Number all tables sequentially (Table 1, Table 2, etc.), likewise for figures (Figure 1, Figure 2, etc.).

In the title of each figure, authors should mention the number of biological replicates ± standard deviation. The title should be presented in an intelligible fashion.

-The quality of figures and histological section of liver must be improved.

Response: Thank you very much for your feedback. We have made the changes according to the relevant requirements.

Q16: The discussion part needs to be improved; it is missing the discussions with previous research in the same context (Example: 'Hydrogen-rich water reduces inflammatory responses and prevents apoptosis of peripheral blood cells in healthy adults: a randomized, double-blind, controlled trial, The Effects of 24-Week, High-Concentration Hydrogen-Rich Water on Body Composition, Blood Lipid Profiles and Inflammation Biomarkers in Men and Women with Metabolic Syndrome: A Randomized Controlled Trial, etc.).

Response: Thank you for your valuable feedback regarding the discussion section of our manuscript. We appreciate your insight into the need for a more comprehensive discussion, including discussions with previous research in the same context.

We understand the importance of contextualizing our findings within the broader body of literature and engaging in meaningful discussions with relevant previous research. In response to your suggestion, we have enhanced the discussion section by thoroughly integrating relevant studies and comparing our results with those reported in the existing literature.

Q17: The conclusion as well must be presented in an intelligible fashion.

Response: Thank you for your feedback regarding the conclusion section of our manuscript. We appreciate your emphasis on the need for clarity and intelligibility in presenting the conclusion. we have revised the conclusion section to ensure that it effectively communicates the main takeaways from our research in a manner that is easily understandable to our readers.

Q18: The discussion should not be repetitive for the results section.

Response: Thank you for your valuable feedback regarding the discussion section of our manuscript. We carefully reviewed and revised the discussion to ensure that it complements the results without repeating them verbatim.

Response to reviewer 2:

Q1: In this CUMS model, transaminases are elevated significantly in the model group. Is there any previous report which shows massive liver damage in CUMS models?

The reasons why the authors adopted this model for drinking hydrogen-rich water should also be explained.

Response: Thank you for your insightful questions regarding our choice of the Chronic Unpredictable Mild Stress (CUMS) model and the rationale behind studying the effects of hydrogen-rich water in this context.

Regarding previous reports demonstrating liver damage in CUMS models, while extensive literature exists on the behavioral and neurobiological effects of CUMS, reports specifically focusing on massive liver damage in CUMS models are relatively limited. However, there are studies suggesting that chronic stress can lead to hepatic dysfunction and alterations in liver enzymes, albeit the extent of liver damage may vary depending on the experimental conditions and duration of stress exposure. We will thoroughly review existing literature to provide a comprehensive discussion on the potential impact of CUMS on liver function and its relevance to our study. I have listed several relevant literature on CUMS induced liver injury.

(1) Shuxie-1 Decoction Alleviated CUMS -Induced Liver Injury via IL-6/JAK2/STAT3 Signaling (DOI: 10.3389/fphar.2022.848355). (2) Dose-Effect/Toxicity of Bupleuri Radix on Chronic Unpredictable Mild Stress and Normal Rats Based on Liver Metabolomics (DOI: 10.3389/fphar.2021.627451).

As for the rationale behind adopting the chronic stress model for studying the effects of hydrogen-rich water, chronic stress is known to trigger oxidative stress and inflammation, which are implicated in the pathophysiology of various stress-related disorders, including liver dysfunction. Hydrogen-rich water has gained attention for its potential antioxidant properties, which could counteract oxidative stress and mitigate its detrimental effects on liver function.

Q2: Hydrogen is known to selectively attenuate hydroxyl radicals and peroxynitrite (Nat Med. 2007 Jun;13(6):688-94. doi: 10.1038/nm1577). The antioxidant activity of hydrogen in this study appears to have a wide range of antioxidant activity, including SOD, CAT and GSH. The difference in the effects of hydrogen from previous reports should be discussed.

Response: Thank you for highlighting the comprehensive antioxidant activity of hydrogen. While Ohsawa et al.'s study provides valuable insights into the antioxidant properties of hydrogen, it's essential to recognize that the biological effects of hydrogen molecules on SOD/CAT/GSH.

(1) The effect of hydrogen-rich water on letrozole-induced polycystic ovary syndrome in rats (DOI: 10.1016/j.rbmo.2023.103332).

(2) Hydrogen-rich water ameliorates rat placental stress induced by water restriction (DOI: 10.4103/2045-9912.241064).

(3) Local Treatment of Hydrogen-Rich Saline Promotes Wound Healing In Vivo by Inhibiting Oxidative Stress via Nrf-2/HO-1 Pathway(DOI: 10.1155/2022/2949824)

In our study, we observed [insert findings related to hydrogen's effects on liver damage and antioxidant enzyme activity]. These results are consistent with the notion that hydrogen exerts potent antioxidant effects, which may help mitigate oxidative stress and preserve liver function in the context of chronic stress exposure.

Q3: The authors have shown that drinking hydrogen-rich water alleviates behavioral changes in response to chronic stress, but the mechanism underlying this has not been addressed. Many of the histological results in this paper indicate changes in ROS in the liver, but are there any previous reports that link this to behavioral changes? Or does the behavior-altering effect of hydrogen-rich water directly affect nerves? Suggestions for mechanisms of behavioral changes should also be mentioned.

Response: Thank you for your insightful comments regarding the mechanism underlying the behavioral changes observed in response to chronic stress and the potential link between liver ROS changes and behavioral alterations in our study.

We appreciate your acknowledgment of the focus of our experiment, which primarily aims to investigate the protective mechanism of hydrogen-rich water on liver damage induced by chronic stress. While behavioral changes are indeed a part of the validation data for establishing a chronic stress mouse model, we understand the importance of addressing the broader implications of our findings, including their impact on behavior.

While the precise mechanisms through which hydrogen-rich water influences behavior warrant further investigation, our study provides valuable insights into the potential therapeutic effects of hydrogen-rich water in mitigating the adverse effects of chronic stress on both liver and behavioral health.

Thank you for highlighting these important aspects, and we appreciate the opportunity to further explore the mechanistic underpinnings of our research findings.

Q4: What does Figure 3-7G mean? In the figure it is written as TUNEL staining, but in the figure legend it is written as Nrf2 immunofluorescent. Please describe correctly. Moreover, I can not see any fluorescent signal in the picture. Clearer figures are necessary.

Response：Thank you very much for your correction. I apologize for my basic mistake. I have changed the NRF2 in the legend to TUNEL and greatly improved the quality of the fluorescent images for your better viewing.

Attachment

Submitted filename: Response to reviewers.docx

pone.0317080.s003.docx^{(28.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0317080.r003

Decision Letter 1

David Chau

23 Apr 2024

PONE-D-23-43180R1Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice.PLOS ONE

Dear Dr. Zhang,

Please submit your revised manuscript by Jun 07 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

David Chau

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: -Thank you for the update on the manuscript revision. While there has been notable progress in improving the Material and Methods section, the manuscript, particularly the Discussion and Conclusion sections, still require significant attention and improvement. Ensuring a thorough interpretation of the study's findings and accurate conclusions drawn from them is paramount, to enhance the manuscript's overall quality and impact.

-The manuscript requires significant improvement in adhering to standard English conventions. It's imperative to rectify this to uphold the manuscript's clarity, accuracy, and overall quality.

-Addressing these issues will ensure that the manuscript meets the necessary standards for publication.

Reviewer #2: The results of the present study are clinically important.

The authors did revised manuscript according to the Reviewer the request, appropriately.

Reviewer #3: The authors have done an excellent job in addressing the research questions regarding the hepatoprotective properties of hydrogen-rich water (HRW) in mice subjected to chronic stress. Their methods were well-detailed, allowing for a clear understanding of how the study was conducted. The results presented compelling evidence of HRW's beneficial effects on liver health, including histological improvements, normalization of liver function indicators, and modulation of various biochemical markers associated with oxidative stress, ferroptosis, and apoptosis.

However, while the content of the manuscript is comprehensive and scientifically sound, there is room for improvement in the flow of the presentation. The transition between sections could be smoother to enhance the coherence and readability of the paper. For instance, integrating a brief overview of the methods at the beginning of the results section could help readers better understand the context of the findings. Additionally, organizing the results in a more structured manner, perhaps grouping them based on the specific mechanisms or pathways examined, could improve clarity and facilitate the interpretation of the data. Please have a look at this article to discuss specific mechanism (https://www.mdpi.com/1422-0067/25/2/973)

Furthermore, the discussion section could benefit from a more nuanced exploration of the implications of the findings and their significance in the broader context of liver health research. While the conclusions succinctly summarize the key findings of the study, elaborating on the potential clinical relevance of HRW as a hepatoprotective intervention and discussing its limitations and future directions would provide a more comprehensive understanding of the research implications.

In summary, while the authors have made significant contributions to the understanding of HRW's hepatoprotective effects, refining the content flow and enhancing the depth of discussion would further strengthen the manuscript's impact and readability.

Here are a few points?

1.Can you please cite a few studies for this?

Moreover, numerous studies have reported that drinking hydrogen-rich water can increase hydrogen concentration in the liver, allowing hydrogen molecules to exert their effects within the liver.

2. Please rewrite the conclusion in 4-5 sentences without numbering them.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Saoussen Ben Abdallah

Reviewer #2: No

Reviewer #3: Yes: Gagandeep Dhillon

**********

PLoS One. 2025 Feb 14;20(2):e0317080. doi: 10.1371/journal.pone.0317080.r004

Author response to Decision Letter 1

25 Apr 2024

Response to reviewers:

Reviewer 1: Thank you for the update on the manuscript revision. While there has been notable progress in improving the Material and Methods section, the manuscript, particularly the Discussion and Conclusion sections, still require significant attention and improvement. Ensuring a thorough interpretation of the study's findings and accurate conclusions drawn from them is paramount, to enhance the manuscript's overall quality and impact.

-The manuscript requires significant improvement in adhering to standard English conventions. It's imperative to rectify this to uphold the manuscript's clarity, accuracy, and overall quality.

-Addressing these issues will ensure that the manuscript meets the necessary standards for publication.

Response: Thank you for your feedback on the revised manuscript. We appreciate your thorough evaluation and constructive comments.

We acknowledge your observations regarding the Discussion and Conclusion sections and recognize the importance of ensuring a thorough interpretation of the study's findings. We have reexamined these parts to provide a more comprehensive and accurate analysis, aligning the conclusions more closely with the study's results to enhance the manuscript's overall quality and impact.

Furthermore, we understand the significance of adhering to standard English conventions for clarity, accuracy, and overall quality. We have made efforts to solve this problem to ensure that the manuscript meets the necessary standards for publication.

Thank you once again for your valuable feedback. We are committed to making the required revisions to improve the manuscript in accordance with your suggestions.

Reviewer 2: The results of the present study are clinically important.

The authors did revised manuscript according to the Reviewer the request, appropriately.

Response: Thank you for acknowledging the clinical importance of the study's results. We greatly appreciate your recognition of the revisions made in response to the reviewer's feedback. We have strived to incorporate the suggestions appropriately to enhance the manuscript's quality and clarity.

If there are any further areas you believe require attention or if you have any additional feedback, please do not hesitate to let us know. We are committed to ensuring that the manuscript meets the necessary standards for publication.

Thank you for your continued support and guidance throughout this process.

Reviewer 3: The authors have done an excellent job in addressing the research questions regarding the hepatoprotective properties of hydrogen-rich water (HRW) in mice subjected to chronic stress. Their methods were well-detailed, allowing for a clear understanding of how the study was conducted. The results presented compelling evidence of HRW's beneficial effects on liver health, including histological improvements, normalization of liver function indicators, and modulation of various biochemical markers associated with oxidative stress, ferroptosis, and apoptosis.

Q1: Can you please cite a few studies for this?

Moreover, numerous studies have reported that drinking hydrogen-rich water can increase hydrogen concentration in the liver, allowing hydrogen molecules to exert their effects within the liver.

Q2: Please rewrite the conclusion in 4-5 sentences without numbering them.

Response: Thank you for providing detailed feedback on our manuscript regarding the hepatoprotective properties of HRW in mice subjected to chronic stress. We appreciate your recognition of the thoroughness of our methods and the compelling evidence presented in the results section.

We agree with your assessment regarding the flow of the presentation and acknowledge the importance of enhancing coherence and readability. Integrating a brief overview of the methods at the beginning of the results section and organizing the results in a more structured manner, possibly grouping them based on specific mechanisms or pathways examined, are excellent suggestions. We have carefully reviewed the suggested article and conducted group discussions from a mechanistic perspective.

Additionally, we acknowledge the need for a more nuanced exploration of the implications of our findings in the discussion section. We have already discussed the limitations and future research directions of this study in the last paragraph, in order to provide deeper insights into the protective effects of hydrogen rich water on the liver.

In summary, we are grateful for your feedback and will work diligently to refine the content flow and enhance the depth of discussion to further strengthen the manuscript's impact and readability. Your guidance is invaluable in this process, and we are committed to addressing these suggestions to improve the overall quality of the manuscript.

Thank you once again for your thorough evaluation and constructive feedback.

Reply to Q1: We have cited relevant research articles at the end of this sentence.

Reply to Q2: We have removed the numbers and consolidated them into one paragraph.

Attachment

Submitted filename: Response to reviewers.docx

pone.0317080.s004.docx^{(22.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0317080.r005

Decision Letter 2

David Chau

31 Jul 2024

PONE-D-23-43180R2Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice.PLOS ONE

Dear Dr. Zhang,

Please submit your revised manuscript by Sep 14 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

David Chau

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

**********

6. Review Comments to the Author

Reviewer #1: 1. The method outlined in the abstract is overly verbose.

2. Ensure that the titles of tables and graphs are concise and descriptive, avoiding narrative language (e.i Table 1 As shown in the table, the type, brand, item number and dilution ratio of the first antibody factor used in the WB test)

3. Certain sections of the discussion reiterate the results unnecessarily. A discussion should primarily focus on comparing your findings with existing research. Please revise the discussion section to incorporate more references to other studies, rather than merely narrating the results.

4. The conclusion remains inadequate and requires rewriting.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Saoussen Ben Abdallah

**********

PLoS One. 2025 Feb 14;20(2):e0317080. doi: 10.1371/journal.pone.0317080.r006

Author response to Decision Letter 2

23 Aug 2024

Reviewer #1:

Comment 1：The method outlined in the abstract is overly verbose.

Response:

Thank you to the reviewer for reviewing our work and providing valuable feedback. Regarding the issue you raised about the excessive length of the methods section in the abstract, we have streamlined the content multiple times while retaining the most essential information. However, due to the inclusion of multiple measurement indicators in this study, in order to ensure accurate communication of research methods, we have to provide a detailed description of the main method steps in the abstract.

We understand that the abstract needs to be concise, but at the same time, we also hope to provide sufficient information in this section so that readers can have a comprehensive understanding of the research design and main measurement indicators. Therefore, while minimizing non essential content, we chose to retain key methodological information related to the research.

We believe that this information is crucial for understanding the rigor of the research and the validity of the results, but we are also willing to optimize the expression as much as possible in further revisions to present the methodology section in a more concise and clear manner.

Thank you again for your feedback. We look forward to your further guidance and suggestions.

Comment 2: Ensure that the titles of tables and graphs are concise and descriptive, avoiding narrative language (e.i Table 1 As shown in the table, the type, brand, item number and dilution ratio of the first antibody factor used in the WB test)

Response：

Thank you to the reviewer for reviewing and providing suggestions for our work. Based on your feedback, we have simplified the titles of the tables and charts. After receiving your feedback, we specifically checked the title of "Table 1" and simplified it to "Table 1 Antibody Description”， To ensure that the title is both concise and descriptive, avoiding narrative language.

We believe that this modification can better meet your requirements and make the table content more intuitive and clear.

Thank you again for your valuable feedback. We look forward to your further guidance

Comment 3: Certain sections of the discussion reiterate the results unnecessarily. A discussion should primarily focus on comparing your findings with existing research. Please revise the discussion section to incorporate more references to other studies, rather than merely narrating the results.

Response:

Thank you to the reviewer for their careful review and feedback on our discussion section. Based on your suggestion, we have comprehensively rewritten the discussion section, with a focus on comparing the findings of this study with existing research. During the revision process, we removed redundant descriptions of the results to ensure that the discussion focused more on comparison and analysis with existing literature.

Through these adjustments, we hope that the discussion section can more effectively showcase the position of our research within the existing knowledge system, as well as the contribution of our results to related fields. Thank you again for your valuable feedback. We look forward to your further feedback.

Comment 4: The conclusion remains inadequate and requires rewriting.

Response:

Thank you to the reviewer for reviewing and providing feedback on our work. Based on your suggestion, we have rewritten the conclusion section. The new conclusion provides a clearer summary of the main findings of the study and further emphasizes the contribution and significance of these findings to related fields.

We believe that these modifications make the conclusion section more comprehensive and in-depth, better aligning with the overall goals and outcomes of the research.

Thank you again for your valuable feedback. We look forward to your further guidance.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0317080.s005.docx^{(22.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0317080.r007

Decision Letter 3

David Chau

22 Oct 2024

PONE-D-23-43180R3Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice.PLOS ONE

Dear Dr. Zhang,

Please submit your revised manuscript by Dec 06 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

David Chau

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: Thank you for submitting the revised Manuscript.

-In the result part, the figures names should not be written in the text. Each figure should be followed by its name.

-While the manuscript was significantly improved and it present a solid foundation, the discussion part it is not strongly made in its current form.

-The discussion part lacks the depth, critical analysis, and clinical relevance needed (Strong discussions connect animal model findings to potential human applications, or at least acknowledge the challenges of translation) to make it truly strong. Addressing these aspects would significantly enhance the quality of this Manuscript.

Thank you!

**********

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PLoS One. 2025 Feb 14;20(2):e0317080. doi: 10.1371/journal.pone.0317080.r008

Author response to Decision Letter 3

21 Nov 2024

Reviewer #1:

Comment 1：In the result part, the figures names should not be written in the text. Each figure should be followed by its name.

Response: Thank you for your thorough review and valuable feedback on my manuscript. Regarding your comment about “the figure names should not be written in the text; each figure should be followed by its name,” I will carefully consider this and make the necessary revisions. In the revised manuscript, I will ensure that figure names are not mentioned in the text and that each figure is followed by its corresponding name. Thank you again for your constructive suggestions; I will work to enhance the quality of the manuscript.

Comment 2&3: While the manuscript was significantly improved and it present a solid foundation, the discussion part it is not strongly made in its current form.

The discussion part lacks the depth, critical analysis, and clinical relevance needed (Strong discussions connect animal model findings to potential human applications, or at least acknowledge the challenges of translation) to make it truly strong. Addressing these aspects would significantly enhance the quality of this Manuscript.

Response：Thank you for your thorough review and constructive feedback on my manuscript. I greatly appreciate your comments regarding the discussion section. I recognize that the discussion lacks depth, critical analysis, and clinical relevance, and I will take this feedback seriously as I work on the revisions.

In the revised manuscript, I will enhance the discussion section, particularly by making stronger connections between findings from animal models and potential human applications, as well as addressing the challenges associated with translation. I believe these modifications will significantly improve the overall quality of the manuscript.

Thank you once again for your valuable insights; I will strive to make the manuscript more robust and clinically relevant.

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PLoS One. doi: 10.1371/journal.pone.0317080.r009

Decision Letter 4

David Chau

2 Dec 2024

PONE-D-23-43180R4Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice.PLOS ONE

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6. Review Comments to the Author

Reviewer #1: 1. Thank you for addressing the feedback and improving the manuscript. However, the manuscript still does not meet the standards of fluent, native-level English writing. Issues such as repetition, word choice, clarity, phrasing, and grammatical structure persist. We strongly recommend enlisting the help of a native English-speaking PhD editor to refine the manuscript further.

2. The abstract should be refined to avoid repetition and the use of the term "mechanisms" might be overstated given the data presented.

2. Although the discussion section has been improved, it could benefit from a deeper analysis that includes more detailed comparisons with studies evaluating alternative treatments. This would help strengthen the manuscript's position and demonstrate the relative significance of your findings within the broader context of similar research.

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PLoS One. 2025 Feb 14;20(2):e0317080. doi: 10.1371/journal.pone.0317080.r010

Author response to Decision Letter 4

3 Dec 2024

Reviewer #1:

Comment 1：In the result part, the figures names should not be written in the text. Each figure should be followed by its name.

Comment 2&3: While the manuscript was significantly improved and it present a solid foundation, the discussion part it is not strongly made in its current form.

Thank you once again for your valuable insights; I will strive to make the manuscript more robust and clinically relevant.

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PLoS One. doi: 10.1371/journal.pone.0317080.r011

Decision Letter 5

David Chau

22 Dec 2024

Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice.

PONE-D-23-43180R5

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

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6. Review Comments to the Author

Reviewer #1: The manuscript is now presented in a clear and coherent manner, written in Standard English, effectively conveying the importance of this work.

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PLoS One. doi: 10.1371/journal.pone.0317080.r012

Acceptance letter

David Chau

5 Jan 2025

PONE-D-23-43180R5

PLOS ONE

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Supplementary Materials

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Data Availability Statement

All relevant data are within the article and its Supporting Information files.

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PERMALINK

Long-term consumption of hydrogen-rich water provides hepatoprotection by improving mitochondrial biology and quality control in chronically stressed mice

Qi He

Xiang Lan

Mengyuan Ding

Na Zhang

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials & methods

2.1 Animals and experimental scheme

2.1.1 Experimental animals grouping and experimental process

2.1.2 Preparation of HRW

2.1.3 Establishment of CUMS model

2.2 Behavioral testing

2.3 Corticosterone determination

2.4 Biomarkers of liver toxicity

2.5 Liver enzymatic antioxidant activities

2.6 Liver lipid peroxidation detection

2.7 ATP content detection

2.8 Tissue iron content detection

2.9 Histological examination

2.10 Immunofluorescence

2.11 Western blotting

Table 1. Antibody description.

2.12 Statistical analysis

Results

3.1 Behavioral testing

Fig 1. Movement analysis of mice in open field experiments.

3.2 Corticosterone level

Fig 2. Chronic stress mice drinking HRW decreased corticosterone levels.

3.3 Liver structure and functional index

Fig 3. The consumption of HRW by mice experiencing chronic stress resulted in a notable reduction in liver damage.

3.4 Oxidative stress related factors

Fig 4. Mice subjected to chronic stress and provided with HRW exhibited a significant reduction in oxidative stress levels.

3.5 Lipid peroxidation related factors

Fig 5. Mice subjected to chronic stress and provided with HRW exhibited a significant reduction in lipid peroxidation levels.

3.6 Ferroptosis related factors

Fig 6. Activation of Nrf2/HO-1 pathway by HRW alleviates ferroptosis in chronically stressed mice.

3.7 Apoptosis related factors

Fig 7. HRW consumption reduces apoptosis in the liver of chronically stressed mice.

3.8 Mitochondrial biosynthesis related factors

Fig 8. HRW enhances mitochondrial function and biogenesis in chronically stressed mice.

3.9 Mitochondrial quality control-regulated factors

Fig 9. HRW improves mitochondrial quality control in chronically stressed mice.

Discussion

1. Establishment of a chronic stress model in mice

2. The effect of HRW on liver structure and function

3. The effect of HRW on mitochondria in chronically stressed mice

4. The effect of HRW on mitochondrial pathway apoptosis

5. The effect of HRW on lipid peroxidation and ferroptosis

Conclusions

Supporting information

Data Availability

Funding Statement

References

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David Chau

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Author response to Decision Letter 0

Decision Letter 1

David Chau

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Decision Letter 3

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Decision Letter 4

David Chau

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