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. 2026 Feb 18;15(2):260. doi: 10.3390/antiox15020260

Long-Term Consumption of Hydrogen-Rich Water Mitigates Oxidative Stress, Hepatic Inflammation, and Apoptosis in Rats with LPS-Induced Chronic Liver Injury

Luyao Zhang 1,2, Hanyu Wang 1,2, Yingxuan Mai 1,2, Qi He 1,2, Tao Liu 1,2, Na Zhang 3,*, Jiantao Zhang 1,2,*
Editor: Montserrat Marí
PMCID: PMC12938009  PMID: 41750640

Abstract

(1) Background: Chronic inflammation is considered an important pathological basis underlying the development and progression of multiple metabolic liver diseases; although hydrogen-rich water (HRW) has shown beneficial effects in acute inflammation, its long-term impact on chronic hepatic inflammation remains unclear. (2) Methods: Sprague–Dawley rats were pretreated with HRW for 8 months, after which a lipopolysaccharide (LPS)-induced rat model of chronic hepatic inflammation was established, with continuous HRW administration throughout the experimental period. (3) Results: Long-term HRW consumption significantly reduced LPS-induced inflammatory cell infiltration in liver tissue, suppressed the abnormal elevation of pro-inflammatory factors, and maintained relatively stable expression levels of anti-inflammatory factors. In addition, HRW attenuated pro-apoptotic signaling associated with the death receptor pathway, mitochondrial pathway, and endoplasmic reticulum stress (including Bax, Cyt c, Caspase-3, Caspase-8, Caspase-9, Caspase-12, CHOP, and GRP78), while preserving the expression of the anti-apoptotic protein Bcl-2, thereby partially preventing the tendency toward hepatocyte apoptosis induced by chronic inflammation. (4) Conclusions: Long-term consumption of HRW may delay the onset and progression of chronic inflammation-related liver injury by alleviating inflammatory responses, oxidative stress, mitochondrial dysfunction, and apoptotic activity, thus exerting a preventive protective effect on the liver.

Keywords: hydrogen-rich water, chronic inflammation, oxidative stress, apoptosis, mitochondria, endoplasmic reticulum stress

1. Introduction

The liver is a central organ responsible for metabolism, detoxification, and immune regulation, and plays a critical role in maintaining systemic homeostasis. Owing to its pivotal position in metabolic processing and immune surveillance, the liver is particularly susceptible to a wide range of endogenous and exogenous stimuli [1]. In this context, inflammation represents an essential defensive response of the liver to harmful insults, contributing to the limitation of tissue damage and the promotion of repair processes. However, when acute inflammatory responses fail to resolve in a timely manner, or when low-level injurious stimuli persist over prolonged periods, the inflammatory state may gradually become chronic [2]. Such a chronic inflammatory condition is considered one of the important early pathological foundations underlying the development and progression of chronic diseases [3,4].

During the persistent inflammatory response, the continuous release of inflammatory mediators and alterations in local microcirculatory function may lead to the abnormal accumulation of reactive oxygen species (ROS), thereby inducing oxidative stress-related damage. Excessive ROS can disrupt cellular membrane integrity, subsequently impair mitochondrial structure and function and suppress adenosine triphosphate (ATP) synthesis [5]. Under these conditions, the mitochondria-mediated intrinsic apoptotic pathway may be activated, characterized by the release of cytochrome c (Cyt c) from mitochondria under the regulation of Bcl-2 family proteins, which promotes apoptosome formation and sequential activation of initiator Caspases (such as Caspase-9) and effector Caspases (such as Caspase-3) [6,7,8,9]. Meanwhile, in the extrinsic apoptotic pathway, members of the tumor necrosis factor receptor (TNFR) family can induce Caspase-8 activation by facilitating the assembly and activation of Caspase complexes, thereby further amplifying apoptotic signaling [10]. In addition, when intracellular calcium overload causes damage to the endoplasmic reticulum membrane, an endoplasmic reticulum stress response can be triggered, accompanied by activation of Caspase-12, which likewise initiates downstream Caspase cascade reactions and contributes to the apoptotic process [9,11,12]. Overall, under conditions of sustained inflammatory stimulation, pathological alterations—including dysregulated expression of inflammatory mediators, exacerbated oxidative stress–related injury, and increased levels of cellular apoptosis—often occur concurrently, collectively reflecting a disruption of hepatocellular homeostasis. If such a state persists without appropriate modulation, it may, under certain conditions, contribute to the development of structural liver alterations such as fibrosis and cirrhosis, and potentially progress to hepatic functional failure [13,14]. Long-term regulation of inflammation-associated pathological processes, particularly through mild interventions implemented before or during the early stages of tissue injury, has been suggested to help preserve hepatic homeostasis and reduce the risk of subsequent damage. Compared with therapeutic strategies targeting established structural lesions, preventive and health-oriented interventions aimed at maintaining tissue function and homeostasis are considered more consistent with the long-term management paradigm of chronic inflammation-related diseases and may offer broader practical applicability.

In recent years, hydrogen-rich water (HRW) has attracted increasing attention as an emerging therapeutic approach, and its protective effects have been reported in various models of acute inflammation-related diseases [15,16,17]. Unlike conventional antioxidants, molecular hydrogen has a very small molecular weight, allowing it to readily diffuse across biological membranes and remain dissolved in water [18]. Under inflammation-associated pathological conditions, HRW is thought to reduce oxidative stress burden, thereby alleviating inflammatory responses at the systemic level and limiting tissue damage, which highlights its favorable antioxidant properties [19]. Further studies suggest that HRW may also exert comprehensive protective effects in functional disorders by modulating apoptosis-related processes [20]. These findings provide a theoretical basis for the application of HRW in disease states in which inflammation and oxidative stress represent key pathological features.

Given that current research on the effects of long-term consumption of HRW on chronic liver inflammation remains limited, and that its associated biological effects and underlying mechanisms have not yet been systematically elucidated, the present study first simulated a lifestyle background of sustained HRW intake in experimental rats under normal feeding conditions. Subsequently, lipopolysaccharide (LPS), a commonly used inflammatory stimulus, was employed to establish a rat model of chronic liver inflammation, during which HRW administration was continuously maintained throughout model induction and the subsequent experimental period. This study was designed with a primary focus on preventive intervention, aiming to exploratorily evaluate the hepatic response characteristics and associated pathological changes following chronic inflammatory stimulation under conditions of long-term HRW intake, and to provide a preliminary analysis of the potential regulatory processes involved.

2. Materials and Methods

2.1. Animals

Sprague–Dawley rats (SD, n = 48; 24 males and 24 females; 220 ± 20 g) were obtained from Liaoning Changsheng Biotechnology Co., Ltd, Benxi, China. and housed at Northeast Agricultural University. Animals were maintained under controlled conditions (25 ± 1 °C, 55% relative humidity, 12 h light/dark cycle) and were free of pre-existing health conditions prior to experimentation. All procedures complied with the ARRIVE guidelines and the Regulations on the Management of Laboratory Animals of the People’s Republic of China and were approved by the Animal Ethics Committee of Northeast Agricultural University (approval No. NEAUEC2022-3-21).

2.2. Experimental Scheme

Rats had free access to a standard diet and were acclimatized before the experiment. Animals were randomly assigned to four groups: control (C, normal water, n = 12), HRW (H, HRW), LPS (L), and HRW + LPS (HL). Rats in the H and HL groups received HRW for 8 months, whereas those in the C and L groups received normal water. At the beginning of the eighth month, rats in the L and HL groups were injected with LPS (200 μg/kg; Xinle Biotechnology, Shanghai, China) via the tail vein, while rats in the C and H groups received sterile saline. LPS or saline was administered once weekly for four weeks. Blood samples were collected at the end of each week. At the end of the experiment, rats were anesthetized with isoflurane, and blood was collected from the inferior vena cava. Livers were excised, rinsed with saline, snap-frozen in liquid nitrogen, and stored at −80 °C for further analyses.

2.3. Preparation of HRW

A hydrogen generator (QL-500, Saikesaisi Hydrogen Energy Co., Jinan, China) was used to bubble hydrogen gas into water to prepare HRW. Hydrogen gas was introduced into a water bottle at an output flow rate of 500 mL/min. After 15 min of aeration, HRW was freshly prepared twice daily and immediately transferred to aluminum drinking bottles for administration to the rats (H and HL groups), and the water was replaced every 12 h. To minimize hydrogen loss during storage and use, the bottles were filled with minimal headspace and kept sealed during the feeding period. The dissolved hydrogen concentration in freshly prepared HRW was measured using a dissolved hydrogen meter (ENH-1000, Beijing PKSAIR Technology Development Co., Ltd., Beijing, China) and was designed to be higher than 500 ppb at the time of preparation.

2.4. Liver Index Measurement

After excision, fresh liver tissues were carefully isolated, with surrounding connective tissue and residual blood removed. The livers were gently rinsed with physiological saline, blotted dry to remove surface moisture, and then weighed to obtain the wet liver weight. The terminal body weight of each rat was recorded simultaneously. The liver index was calculated using the following formula:

Liver index = liver weight (g)/body weight (g) × 100%.

2.5. Blood Cell Count

The blood routine indexes of the rats in each group, mainly white blood cells, neutrophils, and lymphocytes (WBC, NE, and LY), were detected using a blood cell counter.

2.6. Aminotransferases Activities

A 10% liver tissue homogenate was prepared by homogenization, and the ratio of normal saline to liver tissue was 9:1. The homogenate was centrifuged at 560× g for 10 min, and the supernatant was collected. Alkaline phosphatase (ALP) test kit, alanine transaminase (ALT) test kit, aspartate transaminase (AST) test kit (A059-2, C009-2-1, C010-2-1, Jiancheng, Nanjing, China) were used to measure the changes in the content of ALP, ALT, and AST.

2.7. Histological Study

Liver tissues were fixed in 4% paraformaldehyde for over 24 h, followed by routine paraffin embedding and hematoxylin–eosin (H&E) staining. Histopathological changes after LPS treatment, including degeneration, necrosis, and inflammatory cell infiltration, were evaluated under a light microscope. Liver pathological scores were determined according to a previous study based on the extent of immune cell infiltration and structural damage (0, none; 1, mild; 2, moderate; 3, severe) [21].

For transmission electron microscopy, liver samples (1.0 mm × 1.0 mm × 1.0 mm) were immediately fixed in 2.5% glutaraldehyde (pH 7.2), post-fixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and ultrastructural images were acquired using a transmission electron microscope.

2.8. Immunofluorescence

For the TUNEL assay, paraffin-embedded tissue sections were dewaxed and rehydrated, followed by rinsing with distilled water. Sections were treated with proteinase K to enhance membrane permeability and then incubated with the TUNEL reaction mixture. Nuclei were counterstained with DAPI. Slides were mounted using an anti-fade mounting medium, and images were captured using a fluorescence microscope.

For ROS detection, frozen sections were equilibrated to room temperature, air-dried, and circled with a hydrophobic pen. After treatment with an autofluorescence quencher and washing, sections were incubated with the ROS-sensitive fluorescent probe. Nuclei were counterstained with DAPI, and fluorescence images were acquired using a fluorescence microscope.

2.9. ELISA

IL-1β, TNF-α, IL-10, IL-6, SOD, and ATP ELISA kits (Jingmei Biotechnology, Yancheng, China) were used to determine 10% homogenate of serum and liver tissue samples as per the manufacturer’s instructions. The absorbance of samples was recorded at 450 nm by an Epoch microplate reader (BioTek, Winooski, VT, USA). The expression of IL-1β, TNF-α, IL-10, IL-6, SOD and ATP was calculated using a standard curve.

2.10. Test Kit

The CAT, GSH-px, and MDA use kits (A007-1-1, A005-1-2, A003-1, Jiancheng, Nanjing, China) to measure the activities of CAT and GSH-px and the content of MDA. The absorbance of each sample was recorded according to the manufacturer’s instructions.

2.11. Mitochondrial Protein Extraction

The instructions follow the steps of using tissue mitochondria isolation kit (C3606, Beyotime Biotechnology Co., Ltd., Shanghai, China) to extract mitochondrial proteins and use them for subsequent ATP and related mitochondrial protein expression tests.

2.12. Western Blot

Total proteins were separated using a 6–15% gradient SDS-PAGE gel (Shanghai Epizyme Biomedical Technology Co., Ltd, Shanghai, China) and subsequently transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin at room temperature for 2 h. The membrane was incubated with a suitable primary antibody and incubated overnight at 4 °C. The antibodies used are listed in Table S1. Afterward, the membrane was incubated with goat anti-rabbit IgG (dilution ratio 1:10,000) for 1 h, following it was washed with TBST buffer. Next, the protein bands were observed by ECL luminescent fluid (Biyuntian Biotechnology, Shanghai, China). We used a Tanon-5200 (Tianneng Technology, Shanghai, China) chemiluminescent imaging system to capture the images. The gray value was calculated using the ImageJ 1.54g (NIH, Bethesda, MD, USA) software for each band. We standardized the expression of protein using a corresponding internal control (β-actin and β-tubulin).

2.13. qRT-PCR

Total RNA was isolated from liver tissues using TRIzol reagent (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using the HiScript® RT SuperMix (Vazyme Biotech Co., Ltd.) for qPCR (+gDNA wiper). Quantitative real-time PCR was performed with 2× Taq SYBR Green qPCR Mix on a LightCycler 480 real-time PCR system (Roche Applied Science, Penzberg, Germany). The thermal cycling program consisted of an initial denaturation at 105 °C, 40 cycles of denaturation at 95.0 °C for 5 s and annealing/extension at 60.0 °C for 30 s, during which fluorescence data were collected. Subsequently, a melting curve analysis was conducted by incubating the samples at 95.0 °C for 15 s, followed by 60.0 °C for 1 min. Fluorescence acquisition was then performed at 60.0 °C with a temperature increment of 0.5 °C per step and a ramp rate of 0.2 °C/s for 70 cycles to generate the melting curve. Relative gene expression was calculated using the 2−ΔΔCt method, with β-actin used as the internal control. Primer information is provided in Supplementary Table S2.

2.14. Statistical Analysis

Data analysis and graphing were performed using GraphPad Prism 9.5. All experimental data are presented as the mean ± standard deviation (mean ± SD). Statistical comparisons among groups were conducted using one-way analysis of variance, followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant, and a p value < 0.01 was considered highly significant.

3. Results

3.1. HRW Improves Liver Function and Preserves Hepatic Structural Integrity in Chronic Hepatic Inflammation

A hematology analyzer was used to assess WBC counts, NE%, LY%, and Mon% (Figure 1a–d). No significant differences in routine blood parameters were observed between the C and H groups. In contrast, WBC counts were significantly elevated in the L group compared with the C and H groups throughout the experimental period, confirming LPS-induced inflammation. Compared with the L group, the HL group exhibited significantly reduced WBC counts, accompanied by decreased NE% and Mon% and an increased LY%. The liver index is shown in Figure S1. The liver index in the L group was significantly higher than that in the C and H groups, whereas no significant difference was observed between the HL and C groups. Analysis of hepatic biochemical function revealed that ALT and AST levels were markedly increased in the L group relative to the C and H groups (Figure 1e,f). In contrast, ALT, AST, and ALP levels (Figure S2) were significantly reduced in the HL group compared with the L group. Regarding fibrosis-related markers (Figure 1g), TGF-β expression (Figure 1h) was significantly elevated in the L group and markedly higher than in the C, H, and HL groups, whereas no significant differences were observed among the latter three groups. A similar expression pattern was observed for α-SMA (Figure 1i), which was significantly increased only in the L group. In contrast, COL1A1 expression (Figure 1j) did not differ significantly among the experimental groups.

Figure 1.

Figure 1

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Effects of HRW on blood routine parameters, hepatic biochemical indices and fibrosis indicators in rats with chronic inflammation. (a) WBC count in blood; (b) NE percentage in blood; (c) LY percentage in blood; (d) Mon percentage in blood. (e) Hepatic ALT activity; (f) Hepatic AST activity. (g) Western blot bands of liver fibrosis-related proteins; (h) quantitative analysis of TGF-β protein expression; (i) quantitative analysis of α-SMA protein expression; (j) quantitative analysis of COL1A1 protein expression. Data are expressed as mean ± SD. Compared with the C group, * p < 0.05, ** p < 0.01; compared with the H group, # p < 0.05, ## p < 0.01; compared with the HL group, ! p < 0.05, !! p < 0.01.

Histopathological changes in liver tissue were evaluated by H&E staining (Figure 2a–d). The C and H groups displayed intact hepatic lobular architecture, with hepatocytes arranged in regular plates and exhibiting normal morphology, primarily distributed around the central vein. In the HL group, mild inflammatory cell infiltration was observed in the liver tissue, accompanied by partial loosening of hepatocyte cytoplasm, sinusoidal dilation, and mild congestion (Figure 2c). In contrast, liver injury was most pronounced in the L group, characterized by marked hepatocyte swelling, disruption of lobular architecture, disorganized hepatic cords, prominent inflammatory cell infiltration, occasional hepatocyte nuclear dissolution, and evident sinusoidal congestion (Figure 2d). Histological scoring results are shown in Figure S3. No significant difference in pathological scores was observed between the H and C groups, whereas a significant difference was detected between the HL and L groups. Ultrastructural alterations of hepatocytes are shown in Figure 2e–h. In the C and H groups, hepatocytes exhibited normal ultrastructure, with intact nuclear morphology, well-defined nucleoli and nuclear membranes, evenly distributed chromatin, orderly endoplasmic reticulum, and abundant glycogen granules (Figure 2e–h). In the L group, hepatocytes displayed nuclear shrinkage with partial dissolution, peripheral chromatin condensation, mitochondrial swelling, severe endoplasmic reticulum dilation, and a marked reduction in glycogen granules. In comparison, ultrastructural damage in the HL group was markedly attenuated relative to the L group.

Figure 2.

Figure 2

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Effects of HRW on hepatic morphology in rats with chronic inflammation. (a–d) H&E-stained liver Sections (400×): (a) C group; (b) H group; (c) HL group; (a) L group. (e–h) Transmission electron microscopy images showing hepatocyte ultrastructure (10,000× and 20,000×): (e) C group; (f) H group; (g) HL group; (h) L group. Data are expressed as mean ± SD.

3.2. HRW Attenuates Hepatic Inflammatory Responses in Chronic Hepatic Inflammation

As shown in Figure 3a–d, no significant differences were observed in the levels of IL-1β, TNF-α, IL-6, or IL-10 between the C and H groups. Compared with the L group, hepatic levels of IL-1β, TNF-α, and IL-6 were significantly reduced in the HL group, whereas IL-10 levels were significantly increased. Figure 3e,f presented the expression of CRP protein and the corresponding quantitative analysis. No significant difference in CRP protein levels was detected between the C and H groups. In contrast, CRP protein expression in the HL group was significantly lower than that in the L group.

Figure 3.

Figure 3

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Effects of HRW on inflammation-related factors in the liver of rats with chronic inflammation. (a) TNF-α levels; (b) IL-1β levels; (c) IL-6 levels; (d) IL-10 levels. (e) Western blot of CRP expression in the liver; (f) Quantitative analysis of CRP protein expression. Data are expressed as mean ± SD. Compared with the C group, * p < 0.05, ** p < 0.01; compared with the H group, # p < 0.05, ## p < 0.01; compared with the HL group, !! p < 0.01.

3.3. HRW Alleviates Oxidative Stress in the Liver in Chronic Hepatic Inflammation

We further evaluated oxidative stress-related injury in the liver. Changes in ROS levels are shown in Figure 4a. No significant differences in ROS content were observed between the C and H groups. ROS levels in both the L and HL groups were significantly higher than those in the C and H groups; however, compared with the L group, hepatic ROS levels were significantly reduced in the HL group. Alterations in antioxidant enzyme activities and lipid peroxidation are presented in Figure 4b–f. No significant differences were observed in CAT, SOD, GSH-px activities or MDA levels between the H and C groups. Compared with the L group, antioxidant enzyme activities, including SOD, CAT, and GSH-px, were significantly increased in the HL group, accompanied by a marked reduction in MDA content.

Figure 4.

Figure 4

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Effects of HRW on oxidative stress-related parameters in the livers of rats with chronic inflammation. (a) ROS fluorescence levels in the liver; (b) quantitative analysis of hepatic ROS levels. (c) SOD activity; (d) CAT activity; (e) GSH-Px activity; (f) MDA content. Data are expressed as mean ± SD. Compared with the C group, ** p < 0.01; compared with the H group, ## p < 0.01; compared with the HL group, !! p < 0.01.

3.4. HRW Suppresses Hepatocyte Apoptosis in Chronic Hepatic Inflammation

As shown in Figure 5a,b, hepatocyte apoptosis was evaluated by TUNEL staining. No obvious differences were observed between the C and H groups. Compared with the C and H groups, levels of hepatocyte apoptosis were significantly increased in both the L and HL groups. However, compared with the L group, the extent of hepatocyte apoptosis was significantly reduced in the HL group. We further examined the expression of proteins associated with the death receptor-mediated apoptotic pathway, and the results are shown in Figure 5c–g. No significant differences in the expression levels of TNFR1, FAS, caspase-8, or caspase-3 were observed between the C and H groups. In contrast, the expression levels of TNFR1, FAS, Caspase-8, and Caspase-3 were significantly elevated in both the L and HL groups compared with the C and H groups. Notably, compared with the L group, the expression of TNFR1, FAS, Caspase-8, and Caspase-3 was significantly downregulated in the HL group.

Figure 5.

Figure 5

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Effects of HRW on hepatocyte apoptosis in rats with chronic inflammation. (a) TUNEL assay for the detection of apoptosis. Green fluorescence indicates TUNEL-positive apoptotic cells, DAPI (blue) stains cell nuclei, and merged images represent the overlay of TUNEL and DAPI signals. (b) Quantitative analysis of TUNEL-positive cells. (c) Western blot bands of death receptor pathway-related proteins; (d) quantitative analysis of TNFR1 protein expression; (e) quantitative analysis of FAS protein expression; (f) quantitative analysis of Caspase-8 protein expression; (g) quantitative analysis of Caspase-3 protein expression. Data are expressed as mean ± SD. Compared with the C group, * p < 0.05, ** p < 0.01; compared with the H group, # p < 0.05, ## p < 0.01; compared with the HL group, !! p < 0.01.

3.5. HRW Alleviates Mitochondrial Dysfunction and Energy Metabolic Impairment Under Chronic Hepatic Inflammation

The expression of proteins associated with mitochondrial biogenesis is shown in Figure 6a–d. No significant differences in NRF1, TFAM, or PGC-1α protein expression were observed between the C and H groups. Compared with the C and H groups, the expression of these proteins was significantly reduced in both the L and HL groups. However, relative to the L group, PGC-1α expression was significantly increased in the HL group, accompanied by marked elevations in NRF1 and TFAM levels. To further assess mitochondrial functional status, the mRNA expression levels of the mitochondrial DNA-encoded genes ND1, ND4, and Cyt-b were measured (Figure S4). Compared with the C and H groups, ND1, ND4, and Cyt-b expression levels were significantly reduced in the HL group, but remained significantly higher than those in the L group. Hepatic ATP levels are shown in Figure 6e. No significant differences were observed between the C and H groups, whereas ATP levels were significantly increased in the HL group compared with the L group. The expression of proteins involved in mitochondrial fusion and fission is presented in Figure 6f–k. No significant differences were detected between the C and H groups. Compared with the L group, the HL group exhibited significantly increased expression of the fusion-related proteins MFN1, MFN2, and OPA1, along with markedly reduced expression of the fission-related proteins DRP1 and FIS1. Finally, mitochondrial apoptosis-related markers are shown in Figure 6l–p. Compared with the L group, the HL group showed significantly lower expression levels of Bax, Cyt c, and Caspase-9, while Bcl-2 expression was significantly increased.

Figure 6.

Figure 6

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Effects of HRW on mitochondrial biogenesis (a–d), ATP levels (e), mitochondrial dynamics (f–k), and key proteins involved in the mitochondrial apoptotic pathway in the livers of rats with chronic inflammation. (a) Western blot bands of key proteins related to mitochondrial biogenesis; (b) quantification of NRF1 protein expression; (c) quantification of TFAM protein expression; (d) quantification of PGC-1α protein expression. (e) Hepatic ATP levels. (f) Western blot bands of proteins involved in mitochondrial dynamics; (g) quantification of MFN1 protein expression; (h) quantification of MFN2 protein expression; (i) quantification of OPA1 protein expression; (j) quantification of DRP1 protein expression; (k) Densitometric quantification of FIS1 protein expression. (l) Western blot bands of key proteins in the mitochondrial apoptotic pathway; (m) quantification of Bax protein expression; (n) quantification of Cyt c protein expression; (o) quantification of Caspase-9 protein expression; (p) quantification of Bcl-2 protein expression. Data are expressed as mean ± SD. Compared with the C group, * p < 0.05, ** p < 0.01; compared with the H group, # p < 0.05, ## p < 0.01; compared with the HL group, ! p < 0.05, !! p < 0.01.

3.6. HRW Attenuates Endoplasmic Reticulum Stress-Associated Pro-Apoptotic Signaling Under Chronic Hepatic Inflammation

Changes in endoplasmic reticulum (ER) stress-associated apoptotic signaling are shown in Figure 7a–h. No significant differences in ER stress-related protein expression were observed between the C and H groups. Following LPS stimulation, GRP78 expression was significantly increased in the L group (Figure 7c) and was also elevated in the HL group compared with the C and H groups, but remained significantly lower than that in the L group. Among the canonical unfolded protein response (UPR) sensors (Figure 7d–f), PERK expression was significantly higher in the HL group than in the C group but significantly lower than in the L group, with IRE1 showing a similar trend. In contrast, ATF6 expression was significantly increased only in the L group, whereas no significant differences were observed between the HL, C, and H groups. Consistently, ER stress-related pro-apoptotic markers CHOP and Caspase-12 were markedly increased in the L group (Figure 7g,h) and, although still elevated in the HL group relative to the C and H groups, were significantly reduced compared with the L group.

Figure 7.

Figure 7

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Effects of HRW on ER stress-related proteins in the livers of rats with chronic inflammation. (a,b) Western blot bands of ER stress-related proteins protein expression; (c) quantification of GRP78 protein expression; (d) quantification of PERK protein expression; (e) quantification of ATF6 protein expression; (f) quantification of IRE1 protein expression; (g) quantification of CHOP protein expression; (h) quantification of Caspase-12 protein expression. Data are expressed as mean ± SD. Compared with the C group, * p < 0.05, ** p < 0.01; compared with the H group, # p < 0.05, ## p < 0.01; compared with the HL group, ! p < 0.05, !! p < 0.01.

4. Discussion

In an LPS-induced rat model of chronic liver inflammation, we systematically evaluated the preventive effects of long-term consumption of HRW. The results suggest that, under conditions of sustained HRW intake, the extent of liver injury elicited by subsequent chronic inflammatory stimulation may be relatively attenuated. This preventive effect may be associated with the coordinated modulation of multiple pathological processes, including inflammatory responses, oxidative stress status, apoptotic activity, and mitochondrial function.

After LPS treatment, serum levels of ALT, AST, and ALP in rats were significantly increased, indicating impaired liver function. This pattern is consistent with the findings reported by Melini et al., suggesting that liver function-related enzymes commonly show similar elevations under inflammatory conditions [22]. Histological and ultrastructural analyses further revealed evident inflammatory responses and characteristic ultrastructural damage in the livers of LPS-treated rats, supporting the reliability of the chronic hepatic inflammation model from both morphological and functional perspectives. Regarding fibrosis-related markers, the expression levels of TGF-β and α-SMA were increased, indicating partial activation of profibrotic signaling. However, no significant differences in COL1A1 expression were observed among the groups. This finding suggests the absence of evident collagen deposition or established fibrotic lesions, indicating that fibrotic injury had not yet developed. Taken together, the present model mainly reflects the early, inflammation-driven initiation of hepatic pathological changes rather than established liver fibrosis or advanced chronic liver disease [23,24]. Further discrimination between inflammation-associated early remodeling and definitive fibrotic formation requires evaluation using histological staining for collagen deposition, such as Sirius Red or Masson staining. Although these assessments were not performed in the present study, the available molecular and biochemical indicators support the conclusion that this model predominantly reflects inflammation-driven early hepatic injury responses.

At the level of inflammatory responses, we observed that under long-term consumption of HRW, the levels of IL-1β, IL-6, TNF-α, and CRP were relatively reduced in rats with chronic inflammation. These findings suggest that HRW may, to some extent, be involved in modulating inflammation-related processes under chronic inflammatory conditions. This observation is consistent with the anti-inflammatory effects of HRW reported by Hu et al. in a colitis model [25]. Previous studies have shown that during inflammatory processes, the excessive release of pro-inflammatory cytokines can induce the generation of ROS [26]. The accumulation of ROS can, in turn, further exacerbate inflammatory responses by amplifying the activation of inflammation-related signaling pathways, thereby forming a vicious cycle between inflammation and oxidative stress [27]. In our study, HRW intervention was associated with reduced levels of MDA and ROS, along with increased activities of SOD, CAT, and GSH-Px. These results suggest that long-term consumption of HRW may help prevent imbalance of the antioxidant defense system under chronic inflammatory conditions, which is consistent with the findings reported by Chen et al. [28].

Under conditions of persistent chronic inflammation and oxidative stress, hepatocytes gradually shift from functional impairment to programmed cell death, which has become a non-negligible pathological feature in the progression of chronic liver injury. In the mitochondria-mediated intrinsic apoptotic pathway, mitochondrial homeostasis plays a central regulatory role, and mitochondrial biogenesis as well as the dynamic balance between fusion and fission are essential for maintaining mitochondrial integrity [29,30,31,32]. Under long-term consumption of HRW, the expression levels of mitochondrial biogenesis-related proteins PGC-1α, NRF1, and TFAM were increased. Meanwhile, the expression of mitochondrial fusion-related proteins Mfn1, Mfn2, and OPA1 was elevated, whereas the levels of mitochondrial fission-related proteins DRP1 and Fis1 were decreased. These findings suggest that HRW may help prevent or alleviate mitochondrial homeostasis imbalance under chronic inflammatory conditions. Mitochondria contain circular double-stranded DNA (mtDNA), which carries genetic information that can be transcribed and translated to synthesize proteins required for ATP production. Previous studies have shown that LPS reduces the expression of the ND1, ND4, and Cyt b genes [33]. In our study, at the mRNA level, the expression of mitochondrial DNA-encoded genes ND1, ND4, and Cyt b in the HRW intervention group remained lower than that in the control group but was markedly restored compared with the model group. These findings suggest that preventive HRW intake may, to some extent, alleviate the impairment of mitochondrial transcriptional function under prolonged inflammatory stimulation. Concurrently, protein levels of Bax, Cyt C, and Caspase-9 were significantly reduced, while Bcl-2 expression was markedly increased in the livers of rats subjected to long-term HRW intervention. These results indicate that HRW may attenuate the activation tendency of mitochondria-related apoptotic signaling under chronic inflammatory conditions by maintaining mitochondrial homeostasis. In addition, within the extrinsic apoptotic pathway, protein expression levels of TNFR1, FAS, Caspase-8, and Caspase-3 were lower in rats receiving long-term HRW compared with the model group. This suggests that HRW may participate in the regulation of hepatocyte apoptosis by modulating death receptor-mediated extrinsic apoptotic signaling. Given that apoptosis is often accompanied by the establishment of ER stress responses, we further examined ER stress-related markers. The results showed that under long-term HRW intervention, the expression levels of the ER chaperone protein GRP78 and the pro-apoptotic transcription factor CHOP were relatively reduced, and no evident activation of Caspase-12 was observed. Moreover, analysis of classical UPR sensors revealed decreased expression levels of PERK and IRE1 compared with the model group, while ATF6 did not show a clear activation trend. These findings suggest that HRW may, to some extent, alleviate ER stress burden and its associated pro-apoptotic signaling under chronic inflammatory conditions.

Our study indicates that under long-term consumption of HRW, the systemic response to chronic inflammation-associated liver injury may be attenuated. This observation is consistent with the findings reported by Kawai et al. in a nonalcoholic steatohepatitis model [34]. Importantly, the effects of HRW observed in this study appear to reflect a preventive, multifaceted modulation rather than a direct reversal of established pathological alterations. From a mechanistic perspective, HRW may contribute to maintaining mitochondrial structural and functional stability, thereby limiting excessive activation of apoptosis-related signaling pathways, including mitochondria-dependent intrinsic pathways, death receptor-mediated extrinsic pathways, and endoplasmic reticulum stress-associated pathways. Through these coordinated regulatory effects, HRW may reduce hepatocyte susceptibility to programmed cell death under chronic inflammatory conditions, consistent with a preventive role centered on the preservation of cellular homeostasis. Under the experimental conditions employed, long-term oral administration of HRW did not result in apparent adverse effects, providing preliminary experimental evidence supporting its safety as a long-term preventive intervention. Nevertheless, it should be noted that the present study primarily focused on the preventive modulatory effects of HRW. Whether HRW also exerts therapeutic efficacy once liver inflammation or injury has been established remains to be clarified. Future studies may address this issue by incorporating different intervention time points or by including therapeutic treatment groups. Moreover, although a relatively long-term preventive administration protocol was adopted to better simulate sustained lifestyle exposure, future investigations may explore whether shorter preventive intervention periods could achieve comparable effects while improving model reproducibility and experimental efficiency. Finally, while the present study systematically examined the effects of long-term HRW consumption across multiple signaling pathways, the precise regulatory hierarchy and interactions among these pathways remain incompletely understood. Future studies integrating pharmacological inhibitors or genetic approaches, such as targeted knockdown or knockout of key molecules, will be required to delineate pathway-specific contributions and to further elucidate the molecular mechanisms underlying the effects of HRW.

5. Conclusions

Long-term consumption of hydrogen-rich water, as a preventive intervention, may help maintain hepatic structure and function under chronic inflammatory conditions by alleviating inflammatory responses, reducing oxidative stress, and delaying apoptosis-related processes.

Acknowledgments

During the preparation of this manuscript, the author used ChatGPT (GPT-5.2, OpenAI) for the purposes of polishing the English grammar and correct improper word usage. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox15020260/s1, Figure S1: Effects of HRW on relative liver weight in rats with chronic inflammation; Figure S2: Effects of HRW on hepatic ALP activity in rats with chronic inflammation; Figure S3: Effects of HRW on hepatic pathological scores in rats with chronic inflammation; Figure S4: Effects of HRW on hepatic mitochondrial gene expression in rats with chronic inflammation; Table S1: Antibody description; Table S2: The primer sequences used in this study; Table S3: Abbreviations.

antioxidants-15-00260-s001.zip (353.2KB, zip)

Author Contributions

Conceptualization, L.Z., H.W. and Q.H.; methodology, L.Z., Y.M. and H.W.; software, Q.H. and L.Z.; validation, T.L., H.W., J.Z., N.Z. and Q.H.; formal analysis, L.Z., H.W. and Y.M.; investigation, Q.H., H.W. and J.Z.; resources, J.Z., T.L. and Y.M.; data curation, Y.M., L.Z., H.W. and T.L.; writing—original draft preparation, L.Z.; writing—review and editing, L.Z., H.W., Q.H. and Y.M.; visualization, J.Z., T.L. and N.Z.; supervision, J.Z., T.L. and N.Z.; project administration, J.Z., T.L. and N.Z.; funding acquisition, J.Z. and N.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Review Board of the Institutional Animal Care and Use Committee of Northeast Agricultural University (protocol code NEAUEC2022 03 21 and date 21 March 2022 of approval).

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors declare that all data supporting the findings of this study are available in the article or Supplementary Materials File.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the National Natural Science Foundation of China (grant no. 32473107).

Footnotes

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Associated Data

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Supplementary Materials

antioxidants-15-00260-s001.zip (353.2KB, zip)

Data Availability Statement

The authors declare that all data supporting the findings of this study are available in the article or Supplementary Materials File.

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