2.1 Anti-Inflammatory Effect

Inflammation is a protective response of the organism to various types of stimuli, including external damage (such as infection and physical injury) and internal imbalances (such as metabolic disturbances and autoimmune reactions) [39]. The process of inflammation resolution can be divided into stages such as the clearance of stimuli, attenuation of proinflammatory signals, removal of inflammatory cells, macrophage phenotypic transformation, and tissue repair [40-42]. It is important to note that when inappropriate, excessive, or uncontrolled inflammation occurs, it can lead to a range of human diseases, and thus timely suppression of the progression of tissue inflammation and the development of new anti-inflammatory strategies are necessary. Since the Nakao team [43] first confirmed in 2008 that H₂ inhalation could improve transplant-related intestinal injury by inhibiting oxidative stress, numerous studies have subsequently validated the therapeutic potential of molecular H₂ in acute and chronic inflammatory diseases, marking the official entry of H₂ therapy into the field of anti-inflammatory research.

Acute inflammation is the body's direct response to injury or infection, and if acute inflammation is not completely resolved, it can develop into chronic inflammation. The therapeutic effects of H₂ on both types of acute inflammation have been verified. In the alcohol-induced liver injury model, H₂ improves liver tissue pathological damage by reshaping the GM homeostasis and inhibiting the activation of the liver Lipopolysaccharide/Toll-like Receptor 4/Nuclear Factor kappa-light-chain-enhancer of activated B cells （LPS/TLR4/NF-κB） pathway [44]. In a model of diabetes combined with stroke, H₂ intervention downregulates the expression levels of proinflammatory factors (IL-1β, IL-6, Tumor Necrosis Factor-alpha (TNF-α)), while activating the TLR4/NF-κB signaling pathway to achieve neuroprotective effects [45]. In an acute lung injury (ALI) model, H₂ activates the Nrf2 antioxidant pathway and reduces the concentrations of IL-1β, IL-6, and TNF-α, alleviating damage to alveolar epithelial cells [46]. In a sepsis model, H₂ regulates macrophage polarization (inhibiting the M1 phenotype/promoting the M2 phenotype) and inhibits mammalian target of rapamycin (mTOR) phosphorylation, reducing the release of inflammatory mediators such as IL-6, TNF-α, and HMGB1, while increasing the levels of anti-inflammatory factors IL-10 and Transforming Growth Factor-beta (TGF-β) [47]. These findings systematically reveal the multitarget action characteristics of H₂ in the regulation of acute inflammation.

Due to H₂’s clear anti-inflammatory effects and the absence of the severe side effects associated with steroid drugs, it holds greater advantages and application potential in the treatment and prevention of chronic inflammation. It has been found that the activation of NLRP3 plays a role in many chronic inflammatory diseases, and molecular H₂ can effectively inhibit the activation of NLRP3 to ameliorate endometrial cancer [8], kidney inflammation [48], septicemia [49], neuropathic pain [50], and so on. The anti-inflammatory mechanism of H₂ mainly focuses on the regulatory network of inflammatory factors (the balance of proinflammatory/anti-inflammatory factors) and the crosstalk of key signaling pathways (such as NF-κB, Nrf2, mTOR), which is the current consensus in research [51, 52]. For example, NF-κB activation facilitates NLRP3 inflammasome assembly, whereas Nrf2 induction suppresses these pathways via redox homeostasis modulation. Notably, sex-specific differences in patients may influence these mechanisms. An et al. [53] demonstrated that H₂’s therapeutic efficacy is regulated by sex hormones, and exogenous estrogen supplementation enhances H₂-mediated neuroprotection in male mice with intracerebral hemorrhage through inhibition of p-NF-κB and p-IKKβ expression. This finding represents a pivotal consideration for advancing personalized clinical applications of molecular H₂ in inflammation-associated disorders (Figure 1).

2.2 Antioxidant Effect

Recent research progress has revealed the vicious cycle mechanism between persistent oxidative stress and chronic inflammation—persistent oxidative stress is a major trigger of chronic inflammation, and the occurrence of chronic inflammation increases oxidative stress. Therefore, it is necessary to prevent persistent oxidative stress and select effective and safe antioxidants. Oxidative stress is essentially the dynamic imbalance between the generation rate of reactive oxygen species (ROS)/reactive nitrogen species and the clearance capacity of the endogenous antioxidant defense system. This leads to elevated ROS levels, and extensive interaction between free radicals and biomacromolecules, which in turn causes mitochondrial dysfunction, DNA repair mechanism damage, biomacromolecule oxidation modification, and cell programmed death cascade reactions [54]. The antioxidant effect of H₂ has been studied for 17 years. It differs from antioxidant enzymes (superoxide dismutase [SOD], catalase, and glutathione peroxidase [GPX]) and small-molecule antioxidants (vitamins C and E, flavonoids, carotenoids, melatonin, ergothioneine, etc.). H₂ has a selective antioxidant effect, meaning it specifically neutralizes the harmful free radicals •OH and ONOO⁻, but does not affect other superoxide anions and peroxides involved in normal physiological processes [3, 55, 56]. Furthermore, H₂ can activate the endogenous antioxidant system by upregulating the expression and activity of Nrf2, a transcription factor that induces the expression of various antioxidant enzymes [57-59], thereby enhancing the activity of antioxidant enzymes such as SOD, GPX, and glutathione reductase. H₂ mediates ROS regulation through Nrf2, inhibiting NF-κB/NLRP3 inflammasome activation and achieving an antioxidant–anti-inflammatory synergistic effect [60].

Based on its unique antioxidant properties, H₂ has demonstrated significant therapeutic potential in various oxidative damage models. Numerous studies have proven that inhalation of H₂ gas or injection of HRS can be used to treat a range of I/R injuries and other oxidative damage diseases. For example, H₂ can increase the expression of heme oxygenase-1 (HO-1) or activate the phosphatidylinositol-3-kinase (PI3K)–Akt signaling pathway to improve liver I/R injury [61, 62]; reduce myeloperoxidase (MPO) activity and IL-1β/TNF-α levels to alleviate myocardial injury [63]; inhibit the generation of malondialdehyde (MDA) to reduce valve I/R injury [64]; reduce inflammatory cell infiltration to protect skeletal muscle from I/R injury [65]; suppress oxidative stress mediated by NOX2 and angiotensin II type 1 receptor; and reduce thyroid hormone-induced myocardial hypertrophy in rats [66], reducing of lipid peroxidation to prevent ALI [67]; H₂ inhalation reduces serum MDA levels and NOX-1 expression in lung tissue to prevent pulmonary veno-occlusive disease [20]; H₂ alleviates ALI by reducing the levels of MDA in the serum [68], and so on. Notably, oxidative stress and inflammation exhibit bidirectional interaction characteristics in the pathological microenvironment: inflammatory cell infiltration can exacerbate ROS explosion, while oxidative damage products (such as lipid peroxides) can positively regulate proinflammatory signaling cascades. H₂ precisely scavenges toxic free radicals such as •OH, while regulating multiple signaling networks like Nrf2/NF-κB/NLRP3, achieving a synergistic blockade of the oxidative-inflammatory cascade (Figure 1).

2.3 Regulation of Apoptosis

Apoptosis is a form of programmed cell death that efficiently and systematically removes damaged cells, such as those caused by DNA damage or developmental processes [69]. Apoptosis is closely related to other forms of cell death, such as autophagy, necroptosis, and ferroptosis, but according to current literature, the regulatory effect of H₂ on cell death mainly occurs through bidirectional regulation of the apoptosis pathway. The primary mechanism involves regulating the expression of apoptosis-related proteins to inhibit apoptosis, thereby protecting cells from damage and maintaining normal tissue function. For example, H₂ upregulates the expression of the antiapoptotic factor Bcl-2 [70, 71] and downregulates the expression of proapoptotic factors Bax, Caspase-3, Caspase-8, and Caspase-12, thus reducing excessive apoptosis [72-74]. Additionally, H₂ lowers apoptosis levels by inhibiting excessive activation of PARP-1 [75]. Unlike other diseases, the antitumor effect of H₂ involves promoting apoptosis. CDK4 and CDK6 control the cell cycle transition from G1 phase to S phase, and inhibiting their expression halts tumor cell proliferation and induces apoptosis directly. Studies have demonstrated that H₂ inhibits CDK4 and CDK6 to restrict lung cancer progression [76].

In the field of cancer therapy, H₂ demonstrates multidimensional proapoptotic properties. Meng et al. [77] found that H₂ can reverse immune escape in lung cancer cells by inhibiting the expression of CD47 and activating the apoptosis program. Jiang et al. [78] demonstrated that H₂ promotes apoptosis by downregulating Akt phosphorylation and inhibiting the PI3K signaling pathway in non-small cell lung cancer. Chu et al. [79] found that inhalation of H₂ suppresses Hypoxia-Inducible Factor 1 Alpha Subunit (HIF-1α)/NF-κB signaling pathway activation and promotes apoptosis in HeLa cells, counteracting cervical carcinogenesis. This bidirectional regulatory capability allows H₂ to protect normal tissues from excessive apoptosis (such as inflammation-induced cell death) while selectively inducing apoptosis in tumor cells. This microenvironment-dependent regulation provides a theoretical basis for the precise application of H₂ in both tissue protection and cancer therapy (Figure 1).

2.4 Regulation of Energy Metabolism

Mitochondria are the energy factories of cells, responsible for Adenosine Triphosphate (ATP) production, but also participate in important life activities such as cell differentiation, signal transduction, and apoptosis, and can regulate cell growth and the cell cycle. Mitochondrial dysfunction can lead to cardiovascular disease [80], neurodegenerative disease [81], nonalcoholic fatty liver disease (NAFLD) [82], and many other diseases. Therefore, maintaining normal mitochondrial function is crucial. Dumbuya et al.’s study [83] showed that after treating septic rats with HRS, the decline in mitochondrial membrane potential (MMP) and ATP content was improved. HRS protected mitochondrial membrane ultrastructure from damage to some extent and increased the number of mitochondria [83]. Intraperitoneal injection of HRS can prevent mitochondrial lipid peroxidation, thus preventing excessive apoptosis-induced tissue damage, protecting mitochondrial structure, increasing the antioxidant potential of mitochondria, and enhancing ATP levels in obstructive jaundice mice [84]. Dong et al.’s research [85] showed that inhaling 2% H₂ can regulate mitochondrial function and dynamics, increase MMP and ATP levels, and enhance mitochondrial activity in the treatment of sepsis-induced liver injury. Mitochondrial–endoplasmic reticulum (ER) interactions are central to cell metabolism, regulating the exchange of lipids and metabolites between organelles [85]. Cui et al. [86] found that inhalation of high concentrations of H₂ enhances the expression of mitochondrial membrane proteins MFN2 and mitochondrial biogenesis-related factors (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), Nuclear factor E2-related factor 2 (NRF2), and Mitochondrial transcription factor A (TFAM)), while decreasing dystrophin (DRP1) expression, leading to improved mitochondrial function and attenuation of sepsis-associated encephalopathy (SAE) in septic mice.

Mitochondria–ER interactions serve as central hubs of cellular metabolism, playing key roles in lipid and metabolite exchange, thereby influencing overall cell function and energy balance [87]. Molecular H₂ regulates not only mitochondrial energy metabolism but also oxidative stress in the ER. Chen et al. [88] found that ER stress damages the autophagy pathway in septic mice by measuring the expression levels of ER stress-related proteins (such as PERK, IRE1α, and XBP1). H₂ alleviated inflammation and organ damage by inhibiting ER stress and activating the autophagy pathway in septic mice [88]. Sun et al. [89] found that H₂-attenuated ER stress is associated with hyperoxic acute lung injury by reducing the expression of CHOP, GRP78, and XBP1. Guan et al. [73] found that H₂ could downregulate the expression of CHOP, caspase-12, and GRP78, while inhibiting p38 and c-Jun N-terminal kinase (JNK) phosphorylation, and upregulating the LC3-II/I ratio in chronic indirect hypoxia-induced renal injury, suggesting that H₂ reduces ER stress and activates autophagy by inhibiting oxidative stress and JNK/MAPK activation. Shen et al. [90] demonstrated that HRW prevents IBD in mice by reducing levels of p-eIF2α, ATF4, XBP1, and CHOP, key proteins in ER stress.

Summarizing current research, H₂ primarily regulates energy metabolism by acting on mitochondria and the ER. Specifically, H₂ can promote ATP production by enhancing MMP and increasing the activity of mitochondrial respiratory chain complexes. Notably, the functional regulation of the mitochondrial–ER interface may be a key node for H₂’s systemic metabolic regulation, and this discovery provides new research directions for further exploring its cellular protective mechanisms (Figure 1).

2.5 Immunomodulation

The immunomodulatory effects of H₂ exhibit multidimensional characteristics, primarily enhancing immunity by protecting immune organs, reducing free radical damage and oxidative stress to immune cells, and regulating immune cell subtypes to protect the integrity of the immune system and alleviate immunosuppression. Since maternal immune dysregulation can induce preterm birth (PTB), Aoki et al. [91] used anti-CD3 ε to activate effector T cells, leading to PTB and upregulating local and systemic proinflammatory responses to explore the regulatory effects of H₂ on T cells. H₂ treatment inhibited several T-cell effector molecules, such as IFN-γ, IL-4, and GZMB, without affecting the expression of regulatory T (Treg) cells. It decreased IL-26 and IL-22 (Th17 cell effector cytokines), thereby reducing the rate of PTB caused by T cell activation [91]. A series of studies by Chen's team revealed the immunomodulatory effects of H₂. After inhaling H₂ for 2 weeks, patients with advanced non-small cell lung cancer showed significant improvement in T-cell exhaustion. Th cell function returned to normal ranges, and exhausted and senescent cytotoxic T cells gradually decreased to normal levels. The total number of Natural Killer T cells (NKT) and cytotoxic Natural Killer cells (NK) subgroups was higher than the pretreatment percentage. Depleted Vδ2 and Vδ1 cells significantly decreased, while cytotoxic Vδ2 cells significantly increased [92]. Additionally, the total number of γδT cells and NKT subgroups was higher than the pretreatment percentage. In thefollowing year, Chen et al. [93] conducted another study evaluating the effect of inhaling H₂ on the immune function of peripheral blood lymphocyte subgroups in healthy individuals. They unexpectedly found that inhaling high-flow (nonhigh concentration) H₂ gas had an inhibitory effect on immune function in healthy humans [93]. Li et al. [94] found that HRS upregulated Tregs cells in rats with brain I/R injury and downregulated the expression of miR-21, miR-210, and NF-κB. Furthermore, Zhao et al. [95] found that HRS can protect against radiation-induced immune dysfunction by restoring the number of CD4+ T and CD8+ T cells in the spleen. Currently, research on the immunomodulatory functions of H₂ is relatively less explored compared with its anti-inflammatory and antioxidant capabilities. However, it is undeniable that H₂ has a beneficial protective effect on the immune system, and further research is needed to explore the specific mechanisms of how H₂ modulates immune functions and the optimal administration methods (Figure 1).

3.1 Inhalation of H₂

Due to the unique physical properties of H₂, which fall within the explosive range at concentrations ranging from 4 to 74%, it is essential to specify the concentration of H₂ for inhalation therapy. Inhalation of 2–4% H₂ has been chosen in many studies using animal models for disease treatment or clinical treatment to ensure safety [96]. After inhalation, H₂ rapidly permeates all body regions through alveolar ventilation, including inaccessible areas for macromolecules. Importantly, this intervention does not significantly impact physiological parameters such as blood pressure or interfere with red blood cell oxygen-carrying capacity, thereby posing no risk of hemotoxicity even at high concentrations [16].

The inhalation of H₂ through a ventilator, mask, or nasal cannula is primarily used as a therapeutic method or adjunctive measure in clinical practice [97]. Due to the high diffusivity of H₂, it does not consistently act at the target site. Therefore, direct inhalation of H₂ is often clinically employed as an adjunctive treatment for patients requiring long-term hospitalization, such as those with lung and organ problems, diseases of the brain and central nervous system (stroke, Alzheimer's disease [98]), and for cancer treatment and postoperative recovery. In organ transplants, inhalation of H₂ has been shown to reduce intestinal and lung graft damage and prevent organ inflammation [99]. The approval of H₂ inhalation therapy as an emergency treatment for patients in cardiopulmonary arrest by Japan under Advanced Medical Care B on December 1, 2016 represents a significant international advancement in the field of H₂ medicine. In addition, the seventh edition of Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment (7th edition) issued by China National Health Commission recommends the administration of oxygen–H₂ mixture (33.3% O₂ and 66.6% H₂) [100], propelling H₂ to the forefront of current research into therapeutic medical gases. Using statistical analysis and clinical data, Zeng et al. [101] showed that H₂/oxygen might be a helpful therapeutic medicinal gas to raise oxygen saturation (SpO₂) and reduce hospital stays for COVID-19 patients on average (Table 1).

Inhalation of H₂ is clinically more feasible and straightforward compared with other methods of H₂ delivery. Patients can easily self-administer H₂ through inhalation, although this method may not be the most convenient. To date, no side effects have been observed with inhaled H₂ therapy; nevertheless, it necessitates specialized equipment for H₂ production, rendering its inhalation somewhat inconvenient. The concentration of H₂ in the blood and tissues depends on the concentration of inhaled H₂ and the need for continuous inhalation to maintain adequate levels and achieve therapeutic effects. A controversial issue in H₂ inhalation is the choice between inhalation of a H₂–oxygen (HO) mixture or pure H₂, and the dose–effect relationship has not been established. Both HO mixtures and pure H₂ have their advantages. Because H₂ has a low molecular weight, inhalation of H₂ in an HO mixture reduces airway resistance, increases oxygen dispersion, and enhances oxygen flow. At the same time, there is no risk of hypoxia when inhaling the HO mixture, which is usually obtained clinically through water electrolysis (66% H₂ and 33% oxygen) [62]. Inhalation of HO carries a certain level of risk due to the high partial pressure of H₂. The presence of oxygen increases the potential for combustion and explosion, especially when the H₂ concentration exceeds 10%, as it can easily ignite through static electricity without detection, leading to an explosion [33]. Therefore, it is crucial to ensure absolute safety when inhaling HO, often by using H₂ monitors to continuously monitor its concentration [34]. In comparison, inhalation of pure H₂ poses a lower risk for humans as it does not cause explosions but carries a higher risk of oxygen deprivation (Figure 2).

3.2 Drinking HRW and Taking Baths with HRW

HRW is a low-cost, portable, and effective way to transfer H₂. It is safer and more convenient for clinical use compared with inhaled H₂, as it can be consumed freely while the patient is awake without developing tolerance effects over prolonged usage. Drinking HRW exhibits distinct mechanisms of action and effects when compared with inhaling H₂. Consumption of HRW is more likely to exert beneficial effects on the gastrointestinal system, as well as on the liver and brain. The potential impact on the brain may also be attributed to the upregulation of entero-brain axis communication and secondary messenger molecules [102]. The main uptake pathway for drinking HRW is the digestive tract and portal vein system. After entering the pulmonary circulation, most of the ingested H₂ evaporates from the lungs, leaving a small amount for therapeutic utilization. HRW has been extensively studied in the medical field and has a wide range of applications, but most of the research is currently limited to animal testing, such as its potential for preventing liver damage and improving cognitive disorders [103, 104]. Additionally, there are suggestions that HRW may play a role in regulating endogenous H₂ homeostasis and modulating GM; however, further trials are needed to explore the possible mechanisms underlying this interaction.

Recent studies have demonstrated that preworkout intake of HRW can effectively reduce lactic acid levels, thereby enhancing ventilation efficiency and exerting an antifatigue effect [105, 106]. In addition to drinking HRW, HRW baths are widely used as a delivery method for HRW. For example, the application of HRW ankle baths has been shown to reduce joint swelling on pain relief to restore mobility and balance, making it a safe and convenient way to address acute ankle sprains in the field of sports medicine [107]. Clinically, HRW baths exhibit inhibitory effects on inflammation and oxidative stress while demonstrating therapeutic benefits for conditions such as psoriasis [108]. Furthermore, through its anti-inflammatory properties, HRW baths also facilitate the healing process of diabetic foot ulcers [43] (Table 1).

This method is used to produce products such as hydrogen water cups and magnesium sticks110, which enable rapid generation of relatively high concentrations of HRW. However, this process also results in the alkalization of the water and the release of certain metal ions.

Currently, there are two types of electrolytic HRW in the market. One type is based on the traditional method of water electrolysis, where both electrodes are immersed in the water to produce oxygen at the anode and H₂ at the cathode. This method does not affect the acidity of the water; however, it should be noted that the anode may have a strong oxidizing effect on ions present in the water, potentially leading to ozone and hypochlorite production. The other type utilizes proton permeable membrane technology, which separates the two electrodes to produce alkaline water containing H₂ called "electrolytic reduced water" at the cathode and acidic oxygen-containing water at the anode111,112. Although this method of H₂ production has a long history, it yields low concentrations of H₂ in the resulting hydrogen-enriched water (HEW), necessitating urgent efforts to enhance its concentration. With further research and technological improvements, the consumption of HEW could provide an effective means of disease prevention and treatment in the future (Figure 2).

3.3 Injection of HRS and Topical Application

The clinical application of H₂ in hospitals is highly restricted due to safety concerns. Although drinking HRW is generally safe, it is difficult to regulate the amount of H₂ given because the H₂ in the water tends to escape over time and some may be lost in the gastrointestinal tract. HRS provides a safer method of H₂ delivery compared with direct H₂ inhalation, while also providing better control of H₂ concentration.

Molecular H₂ is dissolved into saline at high pressure to produce HRS, a beneficial antioxidant; it has a high H₂ content, weak alkalinity, and negative potential. Moreover, it has potent anti-inflammatory, antioxidative stress, and antiapoptotic properties and is safe and nontoxic [113]. HRS is typically administered to the body through intraperitoneal or intravenous injection, and these two different methods of H₂ delivery elicit distinct changes in tissue H₂ concentration [114, 115]. Following intraperitoneal injection of HRS, the peak concentrations of H₂ in blood and various tissues (including liver, kidney, heart, spleen, pancreas, small intestine, muscle, and brain) were observed at 5 min. In contrast, after intravenous injection of HRS, maximum concentration was achieved within 1 min. Both types of injections demonstrated a dose-dependent increase in H₂ concentrations within the blood as well as liver, spleen, pancreas, and brain tissue [116]. I/R injury is one of the main global causes of high morbidity, disability, and death rates; therefore, it is imperative to find an efficacious treatment strategy. For example, intraperitoneal injection of HRS has been demonstrated to reduce ALI caused by limb I/R [117] injury, attenuate renal I/R injury in rats [118], and exhibit therapeutic amelioration in a rat model of SAE through the inhibition of NLRP3/Caspase-1/TLR4 signaling pathway [119]. Additionally, intravenous administration of HRS has shown potential in mitigating intestinal I/R injury in rats [120] while immersion of lungs in HRS has proven effective in reducing pulmonary I/R injury [121]. However, frequent intraperitoneal injections of HRS pose the risk of cross-infection whereas intravenous administration carries certain dangers [122, 123] (Table 1).

HRS is also widely utilized in the treatment of ocular diseases. It can be injected into the vitreous cavity to reduce retinal excitatory damage and promote recovery of retinal function, or used as a H₂-containing eye drop to protect the retina [124, 125]. In addition, a randomized, double-blind clinical trial demonstrated that HRS nasal rinses were more effective than saline rinses in improving clinical symptoms of chronic rhinitis (CR), particularly in patients with. This highlights the potential effectiveness of HRS in the clinical management of CR patients [126]. Currently, most studies on HRS as an injectable have been conducted in animal trials, and further exploration is needed to promote its clinical application. However, HRS can also be used topically as an organ preservation solution, eye drops, and rinse solution for disease treatment. The common method for producing HRS is to dissolve H₂ in saline at high pressure (0.4–0.6 MPa) for 2 h to achieve a supersaturation level (>0.6 mmol/L) [127]. Saturated HRS is sterilized using gamma radiation and kept in sealed aluminum bags at 4°C and atmospheric pressure. Freshly HRS is prepared weekly to maintain a constant concentration. Gas chromatography is used to confirm the H₂ content of saline using the technique outlined by Ohsawa et al. [3], which is widely used for determining H₂ content in saline (Figure 2).

3.4 Endogenous H₂ Production

Intestinal gases produced by GM include methane (CH₄), H₂, H₂S, and carbon dioxide. Among these gases, H₂ constitutes up to 74% of the total gases produced by GM. For instance, Escherichia coli (E. coli) in the intestine can produce H₂ through its hydrogenase enzyme [128]. However, a significant portion of this H₂ is used by other bacteria, while a minor fraction is absorbed by the intestinal mucosa and subsequently excreted via pulmonary respiration and defecation. Consequently, the overall H₂ production of the GM is determined by both its production and consumption. Differences in GM among individuals contribute to variations in H₂ production, and researchers have conducted GM transplantation experiments across different animals to observe alterations in H₂ production and differences in disease resistance [129]. However, the amount of H₂ generated by GM can be easily disturbed by external factors, and the intake of various nondigestible sugars such as high straight-chain amylose corn starch, pectin, oligofructose, and inulin in mammals can stimulate increased H₂ production by GM [130, 131]. It has been shown that H₂ produced by GM exhibits close associations with certain diseases; for example, H₂ can promote intestinal motility and thus serve as a potential treatment for constipation, while a deficiency of H₂ may be implicated in the development of Parkinson's disease (PD) [132, 133] (Table 1).

Due to its endogenous nature, H₂ is being increasingly explored by researchers for breath testing (BT) to achieve rapid diagnosis of clinical gastrointestinal diseases. BT is an effective and noninvasive diagnostic tool for many gastrointestinal disorders [134]. The H₂ breath test (H₂BT) works on the basis that part of the gas produced by the fermentation of colonic bacteria diffuses into the abdominal venous circulation and travels to the lungs, where it is easily measured while breathing. Therefore, the H₂BT has been employed for various purposes: (a) evaluating carbohydrate malabsorption of different absorbable sugars such as lactose and fructose in the small intestine [135], (b) measuring the time interval between ingestion of nonabsorbable carbohydrates (e.g., lactose) and their interaction with bacteria in the cecum and colon (oral-cecum passage time) [136], and (c) frequently utilizing carbohydrates like glucose or lactulose for H₂ exhalation to identify small GM overgrowth in IBS (SIBO) [137] (Figure 2).

3.5 Nanomaterial-Assisted H₂ Delivery

All of these delivery methods, whether exogenous or endogenous H₂ production, face a common challenge: the accurate delivery of H₂ to the targeted diseased area. The therapeutic effect of H₂ would be greatly improved if it could be targeted at the site of disease and released continuously, as well as stored efficiently. Nanomaterials have garnered considerable attention from researchers due to their unique physicochemical properties. Although the clinical translation of nanotechnology remains a significant challenge, it provides a promising platform for the advancement of H₂ medicine. This has led to the emergence of the concept of nanohydrogen medicine, resulting in the development of numerous nanodelivered H₂ materials (Table 1).

4.1 IBD and Intestinal Injury

IBD, which typically encompasses Crohn's disease and Ulcerative colitis (UC), is characterized by a dysregulated autoimmune response to intestinal ecological dysregulation, triggered by exposure to various irritating environmental factors [163]. The main therapeutic goal for patients with IBD is to reduce inflammation and alleviate symptoms such as abdominal pain and bowel changes. Endoscopy combined with biopsy represents the most efficacious approach for establishing a diagnosis and managing the disease [164]. Current drug treatments for IBD include conventional and biological therapies, with conventional treatments involving anti-inflammatory drugs, immunosuppressants, antibiotics, and probiotics; while biological therapies primarily consist of various anti-TNF-α drugs along with numerous other novel biological agents (e.g., JNK inhibitors) [165]. Due to its high biosafety profile, H₂ has been investigated as a potential treatment option for IBD and intestinal injuries. Increased intestinal inflammation, LPS production, infection risk, and decreased short-chain fatty acid (SCFA) content are the main features of IBD pathogenesis. According to our summary of the mechanism of action of H₂ for the treatment of IBD, it is primarily used to inhibit the production of ROS, reduce the levels of proinflammatory factors, inhibit the activation of relevant inflammatory signaling pathways, and promote the growth of intestinal probiotic bacteria and the metabolites of intestinal flora (SCFAs) through the oral intake of HRW or the injection of HRS, to achieve the goal of treating IBD (Table 3).

4.1.1 H₂ Improves Intestinal Damage by Reducing ROS

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely utilized analgesics and are known to cause gastrointestinal damage, with a greater impact on the small intestine than the stomach. The prevention and treatment of NSAID enteropathy pose significant challenges, as there are currently no viable measures available to prevent initial damage caused by uncoupling of mitochondrial oxidative phosphorylation. Therefore, interventions should primarily focus on downstream inflammatory processes [166]. Considering the potential involvement of ROS in the pathogenesis of this condition, antioxidants hold promise for effectively preventing and treating NSAID-induced enteropathy [167]. Akita et al. [168] discovered that oral HRW inhibited the production of cytokines, MPO, mucosal permeability, and ROS in the small intestinal mucosa induced by indomethacin, an NSAID drug. Additionally, HRW altered the metabolism of intestinal microorganisms and promoted the production of more favorable SCFA [168]. Jin et al. [169] found that HRW significantly activated the Nrf-2 signaling pathway, suppressed ROS expression, attenuated NF-κB signaling pathway activation, and exhibited a protective effect against chronic intestinal inflammation induced by lipopolysaccharide in rats (Figure 3). Hu et al. [170] showed that Electrolyzed Hydrogen Water (ERW) could control colonic inflammation and relieve gastroparesis by inhibiting overproduction of ROS and blocking the communication between oxidative stress and inflammation (Figure 3). Qiu et al. [171] showed that HRS treatment reduced intestinal damage and improved intestinal function in whole-body radiation exposed mice by inhibiting oxidative stress-induced injury and systemic inflammation, suppressing intracellular ROS production and blocking the mitochondrial apoptosis pathway (Figure 3). Chen et al. [172] demonstrated that HRW can reduce ROS production and alleviate intestinal oxidative stress by acting on the SIRT1/Nrf2/HO-1 signalling pathway, thereby alleviating constipation. The above studies have demonstrated that despite the diverse delivery methods of H₂, all of them effectively ameliorated intestinal damage by reducing ROS production (Table 3).

4.2 Intestinal I/R Injury

H₂ has been extensively investigated for its potential to mitigating I/R injury in various organs. For instance, Li et al. [94] demonstrated that HRS exerts a neuroprotective effect against whole brain I/R by upregulating Treg cells and downregulating the expression of miR-21, miR-210, and NF-κB. Liu et al. [183] showed that HRS reduces apoptosis induced by skin I/R and improves flap survival modulation of the Bax/Bcl-2 ratio and inhibition of the activated Apoptosis Signal-Regulating Kinase 1/JNK pathway. Lu et al. [184] revealed that HRS attenuates hepatic I/R injury by inhibiting ER stress-induced apoptosis. Zhang et al. [185] protected rats from liver I/R injury by activating the NF-κB signaling pathway after 1 h of H₂ (2%) inhalation before liver transplantation. Nie et al. [186] identify inhibition of oxidative stress and NLRP3-mediated apoptosis as crucial mechanisms by which H₂ attenuates myocardial I/R injury. Zhai et al. [187] demonstrated that oral administration of lactulose promotes bacterial H₂ production in the gastrointestinal tract, leading to activation of Nrf2 expression and subsequent reduction of brain I/R in rats. These data suggest that both HRS and H₂ may possess therapeutic potential for mitigating organ I/R injury through their anti-inflammatory, antioxidant, apoptosis inhibition, and mediation of various signaling pathways.

The intestine is one of the organs most sensitive to I/R injury, which refers to the exacerbation of intestinal damage following the restoration of blood flow after ischemia [188]. Reduced contractile activity, increased microvascular permeability, and mucosal barrier failure are the hallmarks of intestinal I/R injury. These changes can ultimately lead to systemic inflammatory response syndrome, as well as multiorgan dysfunction and failure [189]. At present, limited research has been conducted on the therapeutic effect of H₂ in treating intestinal I/R injury, and the underlying mechanisms are summarized below (Table 3).

4.2.1 Through Inhibition of the NF-κB/NLRP 3 Pathway, H₂ Reduces Intestinal I/R-Induced Coagulation Problems and Inflammation

Organ and tissue damage are not the only things caused by intestinal I/R injury; it also throws off the balance of coagulation. Under physiological conditions, the coagulation system, anticoagulation system, and fibrinolytic system cooperate to maintain a dynamic balance [190]. MPO serves as an inflammatory biomarker, while TNF-α and IL-6 are classical proinflammatory factors during the inflammatory phase. Wu et al. [188] demonstrated that administration of HRW significantly reduces serum TNF-α levels and mitigates intestinal mucosal injury in I/R rats (Figure 4). Eryilmaz et al. [191] observed that although the administration of HRS does not significantly alter the levels of MPO, MDA, IL-6, and TNF-α in intestinal I/R rats, it results in reduced tissue damage and apoptosis. In contrast, in another study by Shigeta et al. [192], intraluminal injection of H₂-rich glucose saline (HRGS) was found to be effective in inhibiting MDA production and thus attenuating intestinal I/R injury in rats. Protease-activated receptor 1 on the surface of monocytes is activated by tissue factor (TF) expression upregulation, whereas TNF-α initiates the cytokine cascade and encourages monocytes to express TF, creating a harmful cycle of inflammatory cascade and coagulation system activation [193]. One of the main mechanisms tying immunity and inflammation together is NF-κB, which is particularly important for controlling proinflammatory proteins and taking part in the transcription of TF genes [194]. TNF-α, IL-6, and IL-1 β are typical stimulatory signaling molecules of the NF-κB pathway. Buchholz et al. [43] found that inhalation of 2% H₂ attenuated I/R-induced upregulation of the inflammatory mediators CCL2, IL-1β, IL-6, and TNF-α during small intestinal transplantation in rats. Mao et al. [195] found that administration of HRS suppresses the expression of proinflammatory cytokines IL-1β and TNF-α, as well as inhibits the activation of NF-κB in intestinal I/R mice, thereby attenuating the damage caused by intestinal I/R (Figure 4). Yang et al. [120] reported that in a rat model of intestinal I/R injury, elevated levels of TF, p-NF-κB p65, NLRP3, Caspase-1, and inflammatory factors were observed. Intestinal I/R causes coagulation dysfunction and activates NF-κB/NLRP3 signaling pathway in rats. However, treatment with HRS attenuates the inflammatory response and upregulates TF expression to improve coagulation disorders and alleviate intestinal injury induced by I/R [120] (Figure 4).

Oxidative stress plays an important role in intestinal I/R injury and HRS has been shown to protect the intestine from I/R injury by inhibiting lipid oxidation, mitigating intestinal inflammation, reducing oxidative stress, and inhibiting apoptosis [43, 188, 196]. Further research is necessary to determine the exact mechanism and signaling pathway that underlie H₂’s protective effects in intestinal I/R injury (Table 3).

4.3 Colorectal Cancer

Cancer is a significant global public health issue, and CRC ranks as the second leading cause of cancer-related mortality [200]. Current therapeutic approaches for CRC include surgery, radiotherapy, chemotherapy, and targeted therapy [201]. While these anticancer treatments have demonstrated efficacy in enhancing the prognosis of patients with CRC, a significant number of patients encounter unfavorable side effects following surgery and chemotherapy. Growth factors, PI3K/AKT/mTOR, and MAPK are examples of signaling pathways that are crucial to the pathophysiology of CRC [202]. Research has indicated that H₂ inhibits tumor cell activity, proliferation, invasion, and migration through various molecular mechanisms, in a manner that depends on both dose and time. Additionally, it has been shown to mitigate nephrotoxicity in patients undergoing radiotherapy and chemotherapy [203]. According to Wang et al. [76], H₂ dramatically slows tumor growth in vivo and, in a concentration-dependent manner, decreases cell proliferation and induces apoptosis in vitro. Furthermore, H₂ lowers lung tissue levels of ROS, TNF-α, IL-1 β, IL-8, and IL-13 and elevates the SOD expression. Further evidence of the suppression of SMC3 expression in A549 and H1975 cells was provided by immunohistochemical labeling [76]. Yang et al. [9] demonstrated that administration of HRW in a xenograft mouse model results in a significant reduction in both the volume and weight of endometrial tumors, thereby inhibiting the growth of endometrial cancer through ROS/NLRP 3/caspase-1/GSDMD-mediated apoptosis. As a result, H₂ therapy has drawn more attention and is now acknowledged as a potentially effective cancer treatment strategy.

4.3.1 H₂ Improves Prognosis by Restoring Depleted CD8⁺ T Cells in Patients with CRC Cancer

High infiltration of CD8⁺T cells in CRC indicates a favorable prognosis and positive response to immunotherapy [204]. However, prolonged interactions between T cells and tumors impair T-cell competence, while PGC-1α inactivation leads to mitochondrial dysfunction, thereby compromising the immune activity of CD8⁺ T cells. It has been shown that H₂ can activate PGC-1α to restore mitochondrial function and rescue depleted CD8⁺T cells [205]. Programmed cell death 1 (PD-1) is considered a marker of T-cell depletion and is associated with poor prognosis in various cancers. In a clinical study conducted by Akagi and Baba [206], involving 55 patients diagnosed with stage IV CRC, it was observed that inhalation of H₂ for 3 h/day and concurrent chemotherapy in the hospital resulted in a reduction in the abundance of exhausted terminal PD-1⁺CD8⁺ T cells. This depletion of CD8⁺ T cells may be attributed to the activation of mitochondria through PGC-1α, leading to an increase in the population of active terminal PD-1⁻CD8⁺ T cells and ultimately improving patient prognosis [206] (Figure 5 and Table 3).

5.1 Lung Diseases

Lung diseases represent a diverse group of conditions that impact the respiratory system, characterized by distinct etiologies, clinical manifestations, and treatment strategies. These conditions often involve intricate interactions between environmental factors and comorbidities, presenting substantial challenges for accurate diagnosis and effective management. In recent years, H₂ has gained attention as a promising therapeutic intervention, exhibiting significant potential in ameliorating various lung diseases and providing novel insights into their management. Lung injuries comprise a wide array of pathologies resulting from a multitude of etiological factors, including but not limited to mechanical trauma, chemical exposure, and lifestyle-related influences. A comprehensive comprehension of these pulmonary injuries is imperative for accurate diagnosis and effective therapeutic interventions. Studies have shown that H₂ possesses notable therapeutic potential in mitigating various types of lung injuries. In a hyperoxia-induced lung injury model, mice exposed to a high-oxygen (98%) environment were treated with 2% H₂ gas inhalation, which activated the Nrf2 pathway and protected the lungs from oxidative stress-induced damage [215]. Similarly, Nrf2 is a key mediator in the therapeutic effects of H₂ gas on sepsis-induced lung injury. This was demonstrated by Yang et al. [216], who compared the responses of wild-type and Nrf2-deficient mice. Inhalation of 2% H₂ gas for 1 h significantly elevated the levels of SOD, CAT, and HO-1 while reducing HMGB1 levels in wild-type mice. It is noteworthy that these therapeutic effects were not observed in Nrf2-deficient mice, thereby emphasizing the essential role of Nrf2 in mediating the therapeutic properties of H₂ gas [216]. The therapeutic effectiveness of H₂ gas in treating sepsis-induced lung damage in mice has been thoroughly demonstrated; nevertheless, its preventive capabilities require additional experimental investigation. Wang et al. [217] proposed an innovative approach by integrating H₂ gas with engineered microalgae, providing a promising avenue for the prevention of sepsis-associated lung injury.

ALI is a severe and life-threatening clinical condition. Numerous studies have confirmed that H₂ alleviates lung injury through various mechanisms. Nrf2 serves as a critical target in H₂ gas therapy for both hyperoxia-induced lung injury and sepsis-related lung injury. Similarly, in a seawater-induced ALI model, Nrf2 also plays a vital role. Inhalation of 2% H₂ gas for 2 h significantly improved partial pressure of oxygen, reduced inflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as the activities of MDA and MPO, while upregulating the expression of Nrf2 and HO-1 [46]. Li et al. [218] performed an extensive study on the impact of H₂ gas on LPS-induced ALI and found that H₂ gas significantly improved survival rates, enhanced lung function, reduced weight loss, and diminished the levels of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β in mice. Furthermore, H₂ gas offered protection against lung damage by influencing AMPK activity and decreasing the expression of Drp1 and Caspase-3 [218]. Yin et al. [219] conducted both in vivo experiments using an LPS-induced ALI mouse model and in vitro experiments with RAW264.7 macrophages. The results showed that H₂ gas inhalation increased the 72-h survival rate of mice to 80%, reduced lung injury, and suppressed the release of inflammatory cytokines. The in vitro experiments further demonstrated that H₂ gas inhibited the expression of ROS, NO, TNF-α, IL-6, and IL-1β in macrophages, while also reducing TLR4 expression and NF-κB activation [219]. Fu et al. [113], employing an LPS-induced ALI rat model and human pulmonary microvascular endothelial cells, demonstrated that HRS mitigates ALI by downregulating the mTOR/TFEB signaling pathway.

Chronic obstructive pulmonary disease (COPD), the third leading cause of mortality globally, is marked by a high prevalence and significant healthcare burden. Smoking and exposure to toxic particulates represent the primary risk factors. Current therapeutic strategies encompass smoking cessation, inhalation therapy, and pulmonary rehabilitation [220]. Nonetheless, H₂ gas has emerged as a promising therapeutic option for the management of COPD. Animal studies suggest that H₂ gas inhalation may serve as an effective strategy for managing COPD. In a mouse model of cigarette smoke solution-induced COPD-like injury, inhalation of 42% H₂ gas for 75 min twice daily over 9 weeks resulted in a 100% survival rate, compared with 80% in the untreated group. Histopathological evaluations indicated that H₂ gas treatment reduced emphysema-like changes and pulmonary inflammation [221]. Su et al. [222] developed a COPD rat model using cigarette smoke and LPS, observing that a 2-h daily inhalation of H₂ gas significantly enhanced lung function and decreased inflammatory cytokine levels. The therapeutic effects were attributed to the suppression of M1 macrophage polarization and the promotion of M2 macrophage polarization, effectively mitigating pulmonary inflammation. In addition, in a COPD mouse model, inhalation of a gas mixture containing 42% H₂, 21% oxygen, and 37% nitrogen significantly reduced emphysema, collagen deposition, and goblet cell hyperplasia, while inhibiting the activation of ERK1/2 and NF-κB signaling pathways [222].

Molecular H₂ has shown multiple therapeutic advantages in treating pulmonary diseases, including its ability to directly repair lung tissue damage and provide comprehensive protective effects by regulating molecular signaling pathways. Its ability to modulate oxidative stress and inflammatory responses in ALI, along with its role in promoting tissue repair and maintaining immune balance in COPD, underscores its potential to influence disease progression at the level of pathological mechanisms.

5.2 Central Nervous System Diseases

Molecular H₂ has been extensively studied for its potential neuroprotective effects in various neurological disorders, with its therapeutic efficacy primarily attributed to its strong antioxidant, anti-inflammatory, and antiapoptotic properties. These mechanisms help slow the progression of neural damage. H₂ gas has shown significant benefits in neurodegenerative diseases, acute neurological conditions, cognitive impairments, mood disorders, and neuropathic pain. Oxidative stress plays a pivotal role in the pathogenesis of neurodegenerative diseases. It not only triggers lipid peroxidation and DNA damage but also promotes neuroinflammation, further aggravating neuronal injury—key features of various neurodegenerative diseases. Molecular H₂ mitigates neuronal damage through its anti-inflammatory and antioxidant properties, while also regulating cellular signaling pathways to enhance neuronal integrity. Due to its high biosafety, H₂ is regarded as a promising strategy for slowing the progression of neurodegenerative diseases and serving as a long-term therapeutic approach [223]. For example, Hou et al. [104] studied the effects of HRS on cognitive impairment in APP/PS1 mice and found that, although HRW could not clear Aβ, it significantly improved the cognitive abilities of the mice, with this improvement exhibiting gender dependence. In contrast, Zhang et al. [224], using PdH nanoparticles in 3×Tg-AD mice, reported that H₂ gas significantly inhibited the overexpression of APP, BACE1, and sAP, thereby reducing Aβ production. This intervention effectively halted the progression of AD, alleviating cognitive impairment, synaptic deficits, and neuronal death [224]. Moreover, Lin et al. [225] conducted a 7-month treatment with HRW in 3×Tg-AD mice and demonstrated that it prevented synaptic loss and neuronal death, reduced amyloid plaques, hyperphosphorylated tau protein, and neurofibrillary tangles. Additionally, HRW improved disturbances in brain energy metabolism and dysbiosis of GM while mitigating inflammatory responses [225]. The interaction between the gut and brain axis is believed to play a critical role in alleviating neuroinflammation, with the regulation of GM by HRW considered a key mechanism underlying its neuroprotective effects.

In the treatment of acute neurological disorders, rapid diagnosis, and timely intervention are essential. Studies have demonstrated that H₂ therapy provides significant neuroprotective effects in models of acute neurological conditions, including cerebral I/R injury, subarachnoid hemorrhage (SAH), and traumatic brain injury (TBI). For instance, in a cerebral I/R injury model, Lee et al. [226] reported that drinking HRW reduced Bax and TNF-α levels in the hippocampus of mice, decreased ROS levels, and improved anxiety and memory performance. Moreover, intraperitoneal injection of HRS showed notable efficacy by promoting HO-1 expression, increasing SOD levels, and reducing MDA levels, thereby mitigating oxidative stress-induced brain damage [227]. In an SAH model, exposure of mice to a 3% H₂ environment activated Nrf2, upregulated GpX4 expression, and inhibited TLR4 activation, significantly alleviating neurological damage [228]. Similarly, intraperitoneal injection of HRS effectively suppressed the NF-κB pathway and the formation of the NLRP3 inflammasome [50], highlighting the multitarget regulatory effects of H₂ in providing neuroprotection against SAH. Moreover, Wang et al. [229] demonstrated using a microvascular endothelial cell model that H₂ gas activates the PI3K/AKT/GSK3β signaling pathway, thereby enhancing cell survival and providing new insights into the treatment of TBI. In the context of neuropathic pain, molecular H₂ also shown promising therapeutic effects. A 2022 study reported that HRW alleviated inflammatory pain in mice and simultaneously improved associated depressive and anxiety-related behaviors [230]. Additionally, Lian et al. [231] found that in mice with chemotherapy-induced neuropathic pain caused by oxaliplatin, drinking HRW significantly reduced inflammation by inhibiting the LPS–TLR4 pathway and decreasing the expression of TNF-α and IL-6. Furthermore, HRW consumption improved GM composition by increasing the abundance of beneficial bacteria such as Lachnospiraceae, Lactobacillaceae, and Ruminococcaceae, while promoting the production of SCFA. These changes alleviated neuropathic pain through the gut–brain axis [231]. Overall, H₂, by regulating immune responses, protecting neurons, and modulating the gut–brain axis, exhibits multifaceted therapeutic effects and holds significant promise as a potential treatment for central nervous system diseases.

5.3 Cardiovascular System Diseases

Cardiovascular diseases have always been a major factor contributing to global morbidity and mortality, necessitating a comprehensive management strategy that includes drug therapy, lifestyle changes, and innovative treatment approaches. However, existing traditional treatment strategies urgently need innovative breakthroughs. Molecular H₂, as a novel bioactive molecule, has significant therapeutic potential for various cardiovascular conditions, including hypertension, atherosclerosis, myocardial I/R injury, and myocardial infarction.

Hypertension, as a chronic condition, is a critical risk factor for the development of various cardiovascular diseases and, if left uncontrolled, can lead to severe health complications. A study demonstrated that intermittent hypoxia rats inhaling 3% H₂ gas for 2 h daily over 35 days showed significantly reduced levels of 8-hydroxy-2-deoxyguanosine and increased SOD levels. These findings indicate that H₂ gas alleviates oxidative stress, improves vascular dysfunction, and effectively lowers blood pressure without noticeable side effects [232]. Furthermore, in a rat model of hypertension induced by nephrectomy, inhalation of 1.3% H₂ gas successfully controlled blood pressure by reducing sympathetic nervous system activity and enhancing parasympathetic nervous system activity [233]. Atherosclerosis is a disease characterized by persistent inflammation of the arterial walls, with its progression being heavily influenced by inflammatory responses. To overcome the limitations of current therapeutic approaches, researchers have developed nanomaterial-assisted H₂ delivery systems, which have significantly enhanced antioxidant and anti-inflammatory efficacy. In ApoE−/− mice models, intraperitoneal injection of HRS was shown to inhibit the expression of Lectin-like Oxidized Low-Density Lipoprotein Receptor 1 (LOX-1) and the activation of NF-κB, effectively preventing the onset of atherosclerosis [234]. While existing medications can alleviate inflammation and reduce lesions, their limited efficacy and stability hinder broader application. To address these challenges, Xu and colleagues [235] developed a Palladium Hydride-Tellurium (PDH-Te) nanozyme that not only exhibits potent ROS-scavenging abilities but also continuously releases H₂ gas to suppress proinflammatory cytokine release, thereby significantly improving atherosclerosis. Additionally, H₂ has been shown to regulate hypercholesterolemia, a key risk factor for atherosclerosis. For instance, intravenous injection of H₂ nanobubble solution slowed disease progression with good safety and no adverse effects on animal survival rates [236].

In a myocardial ischemia model, intraperitoneal injection of 10 mL/kg HRS prior to reperfusion significantly reduced oxidative stress by modulating the total oxidative status and total antioxidant status, thereby minimizing myocardial cell damage and degeneration [237]. Another study showed that perfusion of H₂-rich Krebs-Ringer solution (H₂ concentration: 0.6 mmol/L, pH: 7.3) in rat hearts activated the JAK2–STAT3 signaling pathway while downregulating the p-STAT1/STAT1 pathway, thus reducing myocardial apoptosis [238]. Additionally, HRS mitigated I/R injury by promoting mitochondrial autophagy via the PINK1/Parkin pathway to eliminate damaged mitochondria [239]. Nie et al. [151] developed H2-PFOB NEs with a high H₂-loading capacity that deeply penetrated ischemic myocardium, significantly enhancing antioxidant and anti-inflammatory effects and demonstrating superior therapeutic efficacy. When combined with low-intensity focused ultrasound-induced H₂ release, the therapeutic effects of H2-PFOB NEs were further enhanced [151].

In cardiac arrest (CA) and cardiopulmonary resuscitation models, H₂ gas has also demonstrated protective effects on cardiac tissue by reducing injury markers and inflammation. Gong et al. [240] reported that inhalation of 2% H₂ gas significantly reduced levels of myocardial injury markers, such as creatine kinase-MB and cardiac troponin-T, while protecting myocardial tissue from further damage by inhibiting autophagy. Furthermore, in a myocardial infarction model, daily inhalation of 2% H₂ gas for 3 h over 28 days effectively suppressed the activation of the NLRP3 inflammasome, reduced cardiac fibrosis, and improved cardiac function [241]. These studies suggest that H₂ therapy may serve as a viable adjunct to conventional treatments, offering new avenues for myocardial protection and recovery. The integration of nanomaterial-assisted H₂ delivery technologies has demonstrated significant potential to enhance therapeutic efficacy and offers promising prospects for achieving precision treatment of cardiovascular diseases.

5.4 Liver and Kidney Diseases

The liver and kidneys, as vital organs responsible for metabolism and detoxification, are crucial for maintaining overall health. Liver and kidney diseases are often interrelated, frequently cooccurring due to shared pathophysiological mechanisms and common risk factors. Therefore, preserving the health of these organs and identifying associated risk factors at an early stage are essential for safeguarding overall well-being. NAFLD not only affects the liver but may also have systemic impacts, including on the kidneys and other organs. Studies have demonstrated that administering 0.6 mmol of HRS to NAFLD rats improves insulin sensitivity, enhances glucose tolerance, reduces liver enzyme and lipid levels, and achieves therapeutic effects by inhibiting TNF-α and IL-1β in the liver while upregulating PPARα and PPARγ expression [242]. Furthermore, Liu et al. [243] found that the therapeutic effects of H₂ gas on NAFLD are dose dependent, with 4% H₂ outperforming 67% H₂ in reducing liver enzyme levels Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) and lipid accumulation. In another study, Xue et al. [44] demonstrated that inhalation of 4% H₂ in an NAFLD rat model significantly lowered plasma LPS levels, inhibited the LPS/TLR4/NF-κB signaling pathway to reduce liver inflammation, and enhanced intestinal barrier function by upregulating Zo-1 and occludin expression. It also positively regulated the GM, increasing the Bacteroidetes/Firmicutes ratio, thereby highlighting the crucial role of the gut–liver axis in H₂’s therapeutic effects on NAFLD [44]. H₂ has also shown potential in mitigating chemotherapy-induced liver injury. For instance, Yang et al. [244] observed that CRC patients receiving mFOLFOX6 chemotherapy exhibited stable liver function tests (ALT, AST, Alkaline Phosphatase (ALP)) after drinking HRW, indicating its hepatoprotective effects. Similarly, Gao et al. [245] demonstrated that injecting HRS in rats effectively reduced ALT and AST levels caused by doxorubicin, decreased ROS and MDA production, and regulated the Bax/Bcl-2 ratio to alleviate inflammation and apoptosis. Li et al. [246] also proposed the potential protective effects of H₂ against cisplatin-induced side effects, although further research is needed to validate this hypothesis.

Chronic kidney disease (CKD) is a progressive disease whose major contributing factors include diabetes and hypertension. Studies suggest that H₂ not only holds therapeutic potential for managing diabetes and hypertension but also effectively aids in the prevention and treatment of CKD. For instance, in CKD rats, drinking HRW significantly reduced plasma Monocyte Chemoattractant Protein-1 (MCP-1) levels [247]. In a mouse model of kidney injury induced by a high-oxalate diet, HRW consumption markedly improved serum creatinine, blood urea nitrogen, and kidney injury markers such as kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), while reducing proinflammatory cytokines and oxidative stress markers. HRW has been shown to mitigate renal injury by inhibiting key signaling pathways, including PI3K/AKT, NF-κB, and TGF-β [48]. Meanwhile, H₂ has also demonstrated potential in the prevention of CKD [248]. In glycerol-induced acute kidney injury models, inhalation of both 4 and 67% H₂ gas demonstrated enhanced antioxidant, anti-inflammatory, and antiapoptotic effects. Although no clear dose-response relationship was observed, higher concentrations of H₂ gas (67%) produced more pronounced improvements in kidney histology and morphology compared with lower concentrations (4%) [249]. Similarly, in I/R injury models, HRS treatment significantly reduced renal fibrosis and preserved kidney function [250]. Another study comparing short-term and long-term HRS injections revealed that intraperitoneal HRS increased the Bcl-2/Bax ratio and inhibited the expression of apoptotic proteins such as caspase-3, caspase-8, and caspase-9, but long-term HRS treatment was more effective in promoting renal recovery than short-term treatment [251], as it sustained the suppression of inflammation and apoptosis. These findings underscore the dose-dependent nature of H₂ therapy in liver and kidney diseases. Moreover, prolonged treatment may further enhance therapeutic outcomes, providing theoretical support for the application of H₂ gas in metabolic diseases.

5.5 Cancer

Cancer encompasses a group of complex diseases characterized by uncontrolled cell growth and proliferation, with diverse manifestations and underlying mechanisms. While traditional approaches such as surgery, chemotherapy, and radiotherapy remain the cornerstone of cancer treatment—particularly effective for early-stage cancers—their limitations have prompted researchers to explore innovative therapies. As a result, there has been a growing interest in developing novel adjunctive therapies with high safety profiles and minimal side effects. Advances in targeted therapy, immunotherapy, gene therapy, nanomedicine, and stem cell therapy have opened new avenues for cancer treatment. However, managing side effects continues to pose a significant challenge. Molecular H₂, with its excellent safety profile and broad therapeutic potential, presents a promising complementary approach to cancer treatment. Preliminary progress has already been achieved in its application to various types of cancer.

As noted earlier, the therapeutic effects of H₂ were initially identified through hyperbaric H₂ therapy for treating skin cancer [2]. Since then, its therapeutic efficacy has been validated in various cancers, including CRC, lung cancer, gastric cancer, and breast cancer. For example, Zhang et al. [252] reported that H₂ gas increased apoptosis in A549 cells while reducing the expression of XIAP and BIRC3 proteins in studies on A549 cells and their nude mouse models. Furthermore, inhalation of 60% H₂ gas significantly reduced tumor volume in experimental mice [252]. Meng et al. [77] extended these findings by investigating the effects of H₂ gas on A549 and H1975 lung cancer cells. They discovered that H₂ gas significantly enhanced apoptosis by inhibiting CD47 expression and suppressed cell growth, invasion, and migration [77]. These findings further reinforce the potential role of H₂ therapy in lung cancer treatment, particularly in modulating apoptosis-related pathways.

In gastric cancer research, Zhu et al. [10] found that H₂ gas downregulated the expression of lncRNA MALAT1 and EZH2 while upregulating miR-124-3p in Human Gastric Cancer Cell Line MGC-803 (MGC-803), Human Gastric Cancer Cell Line BGC-823 (BGC-823) gastric cancer cell lines and gastric cancer mouse models, thereby significantly inhibiting tumor growth and reducing cancer cell proliferation and migration. The potential of combination therapies has also been validated. For instance, combining platinum nanocolloid (Pt-nc) with H₂ gas effectively inhibited the growth of human promyelocytic leukemia HL60 cells and NUGC-4 cells derived from human gastric adenocarcinoma [253]. Chu et al. [79] demonstrated that H₂ gas exerts selective effects on HeLa cervical cancer cells and HaCaT keratinocytes. In a HeLa xenograft mouse model, continuous H₂ gas inhalation significantly reduced tumor growth. Cell-based experiments revealed that H₂ gas selectively increased apoptosis in HeLa tumor cells while having minimal impact on nontumor cells such as HaCaT keratinocytes [79]. This selective action highlights the potential of H₂ gas as a safer option for cancer therapy.

Additionally, as a metabolite produced by GM, H₂ gas plays an important role in maintaining tumor resistance. Current studies have demonstrated that H₂ therapy is effective in treating various cancers with no significant side effects. Moreover, recent research has explored the potential for combining H₂ therapy with conventional treatments such as chemotherapy and radiotherapy, demonstrating improved efficacy and reduced side effects [254]. However, despite its promising potential as an adjunctive cancer therapy, H₂ gas alone may not achieve sufficient therapeutic effects. At this stage, H₂ therapy should not replace traditional treatments such as chemotherapy or radiotherapy. Future research should focus on the combined application of H₂ gas with other emerging therapies to fully harness its potential.

6.1 Clinical Applications of Molecular H₂ in the Treatment of Gastrointestinal Diseases or Through Modulation of GM

It should be acknowledged that there are currently few clinical trials of H₂ for the treatment of intestinal disorders, but it is worth noting that the measurement of exhaled H₂ and CH₄ gases following the ingestion of readily metabolizable carbohydrates in humans has emerged as an important noninvasive breath test to aid in the diagnosis of SIBO [255, 256]. This is the most widespread clinical application of H₂ to assist in the treatment of intestinal disorders. Furthermore, noninvasive detection of head and neck squamous cell carcinoma [257], gestational diabetes mellitus [258], heart failure [259], and autism (ASD) [260], based on the ratio of exhaled H₂ to CH₄, has also shown potential, because SIBO is associated with the development of some diseases, and alterations in the composition of the GM of the patient result in changes in the amount of exhaled H₂ gas. Additionally, there are several clinical uses of H₂ in the treatment of other diseases that have been demonstrated to work by acting on the GM, such as Xie et al. [261] who found that H₂ could alter the composition of the GM in people with opioid addiction and thus act on the gut–brain axis to improve the symptoms of depression and anxiety in people addicted to opioids. In a randomized double-blind controlled clinical study, Liang et al. [262] found that HRS could improve metabolism by modulating GM in patients with impaired fasting glucose (Table 4).

6.2 Clinical Applications of Molecular H₂ in Respiratory Diseases

H₂ is particularly effective in treating respiratory diseases, as it is primarily administered by inhalation, and clinical studies have demonstrated the potential of H₂ for diseases such as asthma, COPD, and COVID-19. Wang et al. [263] found that a single 45-min inhalation of 2.4% H₂-containing steam mixed gas reduced airway inflammation in both asthmatics and COPD patients in a prospective study of 10 asthmatics and COPD patients. Niu et al. [264] assessed glycolytic and mitochondrial oxidative phosphorylation activity in asthmatics and a mouse model of allergic airway inflammation, showing that H₂ increased ATP production as well as the activity of mitochondrial respiratory chain complexes I and III. Zheng et al. [265] conducted a prospective clinical trial to evaluate whether HO mixtures are superior to oxygen in improving symptoms in patients with acute exacerbation of COPD (AECOPD). The results showed a significant reduction in cough assessment test scores in the H₂/oxygen group, with no adverse events reported, indicating that H₂/oxygen therapy was more effective and safe compared with standard oxygen therapy [265]. Similarly, Zeng et al. [101] treated 33 patients with H₂/oxygen therapy and found that it improved dyspnea and disease progression in COVID-19 patients, resulting in higher SpO₂ levels and shorter hospital stays. Tan et al. [266] conducted a randomized, single-blind, placebo-controlled trial with 32 participants and found that although H₂-enriched water did not improve dyspnea in patients with long-COVID compared with acute COVID-19, it alleviated fatigue and improved cardiorespiratory endurance, musculoskeletal function, and sleep quality (Table 4).

6.3 Clinical Applications of Molecular H₂ in Neurological Diseases

Animal experiments have demonstrated that H₂ plays a beneficial role in treating neurological disorders, while human trials suggest its potential therapeutic value for neurodegenerative conditions. Yoritaka et al. [267] conducted a randomized, double-blind, placebo-controlled pilot study to investigate the ameliorative effects of inhaled H₂ on PD, but their findings indicated that although inhalation of H₂ was safe, it had no significant beneficial effect on PD patients. Although molecular H₂ demonstrates limited therapeutic efficacy in PD, its antioxidant effects and mitochondrial function modulation in acute brain injury and neurodegenerative diseases remain scientifically noteworthy. Ono et al. [268] recruited 34 patients with cerebral infarction due to atherosclerotic disease, dividing them into two groups: one with 26 patients receiving edaravone alone, and the other with eight patients receiving a combination of edaravone and HRS. Surprisingly, the combination group showed a more pronounced improvement in MRI markers [268]. Nagata et al. [269] recruited 38 patients hospitalized for acute ischemic stroke in an open-label, prospective, nonrandomized study to assess the safety of intravenous H₂-enriched glucose-electrolyte solution combined with t-PA. The results indicated that the therapy was relatively safe for patients with acute cerebral infarction, though the study lacked a separate H₂-only group due to insufficient patient numbers, and one patient experienced diarrhea, which could not be definitively linked to the treatment [269]. In a 2014 publication by Takeuchi et al. [270], they announced that they had initiated a double-blind randomized controlled trial (RCT) in a large study population, enrolling 450 patients with high levels of SAH, in order to evaluate the efficacy of H₂ therapy in the treatment of early brain injury, delayed cerebral ischemia and vasospasm, but unfortunately, the results of this clinical study have not been published so far. Following the discovery of the synergistic effects of H₂ with other therapies, Ono et al. [271] later recruited 50 patients with acute cerebral infarction to study the effects of H₂ alone. The patients were divided into two groups, one receiving H₂ treatment and the other receiving gas without H₂, and the results showed that H₂ treatment stabilized vital signs and improved patients' ability to perform daily activities, with no adverse reactions observed. No adverse effects were found during the experiment, and safety was guaranteed [271]. In 2023, Ono et al. [272] published another article demonstrating that inhalation of 3% H₂ alone for 1 h for 6 months provided symptomatic relief and improvement in patients with AD (Table 4).

6.4 Clinical Applications of Molecular H₂ in Cardiovascular System Diseases

At present, the ameliorative effect of H₂ on cardiovascular disease in humans still needs to be confirmed by large-scale study populations. However, some clinical studies have already demonstrated the high safety and feasibility of H₂ in treating cardiovascular disease. The first human pilot study of H₂ for post-CA syndrome was conducted in 2016, showing that inhalation of H₂ led to a favorable neurological prognosis. However, the study was limited by a small sample size and the short duration of H₂ inhalation due to the capacity limitations of the gas cylinders [273]. In another study, 24 patients undergoing cardiopulmonary bypass (CPB) surgery were randomized into two groups: 12 patients received inhaled H₂ at a concentration of 1.5–2.0%, while the control group did not receive H₂, and the results showed that H₂-treated patients had improved erythrocyte function and reduced oxidative stress [274]. Song et al. [275] found beneficial lipid-lowering effects of H₂ in animal studies, and thus they included 20 patients with potential metabolic syndrome to investigate the ameliorative effects of H₂-enriched water in humans, showing that supplementation with H₂-enriched water appeared to reduce serum low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB) levels, and to improve the function of dyslipidemia-impaired high-density lipoprotein cholesterol, reducing oxidative stress. This means that H₂ may play a beneficial role in the prevention of potential metabolic syndrome [275]. In a recent posthoc analysis of a RCT, Tamura et al. [276] found that the combination of H₂ inhalation and Targeted Temperature Management (TTM) (TTM32–TTM34) improved neurological prognosis after cardiogenic out-of-hospital cardiac arrest (OHCA) compared with TTM only (TTM32–TTM34) at 32–34°C. However, the limitations of this study also need to be taken into account; this was a posthoc analysis with sample size, and the results of this study could be used as a reference to design a larger sample size to be explored in subsequent studies (Table 4).

6.5 Clinical Applications of Molecular H₂ in Chronic Diseases

As early as 2008, 1 year after the anti-inflammatory and antioxidant effects of H₂ were first identified, a randomized, double-blind, placebo-controlled crossover trial involving 30 patients with type 2 diabetes mellitus (T2DM) demonstrated the beneficial effects of HRW consumption in preventing T2DM and insulin resistance [277]. Another small exploratory study revealed the protective effect of H₂ on the lacrimal gland. This study, which involved both mice and 10 human subjects, showed that H2-producing supplements significantly increased the concentration of exhaled H₂ and improved tear stability and symptoms of dry eye [278]. Tao et al. [279] conducted a 13-week clinical trial of H₂/oxygen inhalation in 43 subjects with NAFLD. Their findings showed that H₂/oxygen inhalation improved blood lipid profiles and liver enzyme levels, with significant improvements in hepatic fat content in moderate-to-severe cases as detected by ultrasound and CT scanning [279]. In another study, Asada et al. [280] selected two men and two women to investigate the effects of HRW on oxidative stress-related skin issues and lipid metabolism markers, as well as to determine whether dissolved H₂ remained effective after boiling. The results indicated that HRW baths improved visceral fat and skin spots and that dissolved H₂ showed resistance to high temperatures [280]. Zhu et al. [281] found that HRW baths had a therapeutic effect on psoriasis, with patients receiving HRW baths experiencing a reduction in the size of their psoriasis and a reduction in itching. In another clinical study on psoriasis treatment, H₂ was administered via HRS injection, HRW ingestion, and inhalation of 3% H₂. The results suggested a reduction in IL-6 and IL-17 levels in individual patients following H₂ treatment [96]. Nanobubble HRW baths provide better solubility of H₂ in water than HRW baths alone and reduce H₂ escape to a certain extent. Tanaka et al. [282] found that nanobubble HRW baths not only alleviated the inflammatory symptoms and skin appearance of patients with connective tissue diseases but also improved the serum antioxidant capacity of healthy patients. In 2022, a clinical study demonstrated the anti-inflammatory and antioxidant effects of orally administered solid H₂ capsules in individuals with chronic inflammation, highlighting the potential long-term health benefits of H₂ [283] (Table 4).

6.6 Clinical Applications of Molecular H₂ in Cancer

Recent years have witnessed groundbreaking advancements in the application research of H₂ within cancer therapeutics. Chen et al. [284] conducted a prospective follow-up study involving 82 patients with stage III and IV cancers receiving H₂ inhalation therapy. They found that H₂ inhalation improved the quality of life and helped control cancer progression, with effects varying across different cancers. The best outcomes were observed in lung cancer patients, whereas pancreatic and gynecological cancers showed the least improvement [284]. Chen et al. [92] included 20 patients with non-small cell lung cancer to study the ameliorative effects of H₂ and found that the superior therapeutic effect of H₂ in lung cancer patients was mainly through the improvement of immune senescence in lymphocyte populations. Beyond its direct antitumor effects, molecular H₂’s capacity to mitigate treatment-related adverse effects provides a strategic advantage for its integration into comprehensive cancer care protocols. In another clinical trial the following year, Chen et al. [254] recruited 58 adult patients to investigate the effects of H₂ inhalation alone versus H₂ in combination with chemotherapy, targeted therapy, and immunotherapy, respectively, for the treatment of advanced non-small-cell lung cancer. Sixteen months of follow-up found that progression-free survival in the control group was lower than that in the H₂ inhalation group alone, and significantly lower than that in the other three combination therapy groups. In this study, not only the therapeutic effect of H₂ alone but also the synergistic effect of H₂ in combination with other therapies was highlighted [254]. In a retrospective observational study, Hirano et al. [285] found that H₂ inhalation showed promising results in cancer treatment and also ameliorated bone marrow damage induced by intensity modulated radiation therapy (IMRT) in cancer patients, without compromising the antitumor effects of IMRT, thereby underscoring its potential in adjuvant therapy. These findings provide multilayered evidence for the integration of H₂ in comprehensive cancer management. The clinical value of molecular H₂ has become increasingly prominent across multiple systemic diseases mentioned above. Future research necessitates sustained breakthroughs in three dimensions: in-depth exploration of mechanisms (e.g., epigenetic regulation), optimization of intervention strategies (e.g., precision delivery systems), and interdisciplinary integration (e.g., H₂ medicine with artificial intelligence (Table 4).