Hydrogen is the smallest molecular weight gas molecule, known as a clean energy source for its high calorific value and pollution-free combustion products. In recent years, hydrogen has gradually become a star molecule in the medical field. Especially, with the incorporation of hydrogen oxygen therapy into the COVID-19 novel coronavirus pneumonia treatment program, the clinical application of hydrogen has reached a new level [1-3]. Hydrogen has weak reducibility and is considered a selective antioxidant that can capture highly oxidizing reactive oxygen species such as hydroxyl radicals (· OH) and peroxynitrite ions (ONOO -). Unlike typical gas signaling molecules such as NO, CO, H2S, etc. [4-6], hydrogen has high biological safety and significant advantages over general antioxidants. No adverse reactions of hydrogen have been found in clinical studies. Hydrogen was first used as a diving auxiliary gas in the medical field, and long-term use of high-pressure hydrogen has not been found to have any safety issues for the human body, indirectly proving the in vivo safety of hydrogen. In fact, hydrogen has been approved for use as a food additive [7].

In 1975, Dole et al. from Baylor University in the United States first reported the medical effects of hydrogen in Science and found that hydrogen has an inhibitory effect on tumor growth in mice. Continuous breathing of high-pressure hydrogen gas (8 atmospheres, 97.5% hydrogen, 2.5% oxygen) for 14 days can significantly regress animal skin tumors or leukemia. This more stringent experimental condition was difficult to replicate, until Roberts et al. [9] conducted similar studies on mouse models of five solid tumors and one leukemia in 1978, but did not achieve similar therapeutic effects as Dole et al. [8]. Due to the different mouse tumor models and modeling methods used in the two studies, and the lack of evidence for the physiological effects of hydrogen, related research has stagnated. In 2007, Ohsawa et al. from Japan Medical University [10] reported in Nature Med ⁃ cine that inhaling 2% hydrogen gas has a therapeutic effect on cerebral ischemia-reperfusion injury. Using electron spin resonance, they found that its therapeutic effect is related to the clearance of hydroxyl radicals (· OH) by hydrogen gas, and confirmed that hydrogen gas has selective antioxidant effects, which can specifically clear harmful reactive oxygen species (ROS) such as · OH and ONOO -, but cannot clear ROS required for maintaining normal physiological activities such as superoxide radicals (O2- ·), nitric oxide radicals (NO ·), and hydrogen peroxide (H2O2). The elucidation of selective antioxidant theory laid the foundation for the development of hydrogen medicine. In addition to the treatment of ischemia-reperfusion injury diseases, extensive research has shown that hydrogen has potential therapeutic effects on over a hundred diseases, such as inflammation, metabolic disorders, and neurodegenerative diseases. Hydrogen has also been extensively studied for the treatment of tumors [11-12].

The anti-tumor effect and mechanism of hydrogen gas

Oxidative stress is closely related to the occurrence and development of malignant tumors. ROS damages DNA, proteins, and cell membrane lipids, which is a trigger for cellular carcinogenesis. Based on its selective antioxidant effect, hydrogen has a significant effect in preventing tumor occurrence. It has been confirmed through animal models of liver cancer that hydrogen molecules can significantly reduce the incidence of liver cancer, shrink tumor volume, and accompanied by a decrease in oxidative stress indicators [13]. Rich hydrogen water can also reduce the incidence of ferro nitrilotriacetate (Fe-NTA) - induced renal cell carcinoma in Wistar rats and inhibit tumor growth in rats [14]. Rich hydrogen water not only inhibits the inflammatory response and macrophage aggregation in kidney tissue, but also suppresses the expression of vascular endothelial growth factor (VEGF), signal transduction and transcription activator 3 (STAT3) phosphorylation levels, and the expression of proliferating cell nuclear antigen (PCNA). Frajese et al. [15] of the University of Rome found that hydrogen rich electrolyzed water treatment can induce apoptosis of breast cancer cells (human MCF ⁃ 7, MDA ⁃ MB ⁃ 453 cells and mouse TUBO cells), reduce the expression of human epidermal growth factor receptor ⁃ 2 (ErbB2/neu), and destroy the phosphorylation of extracellular signal regulated kinase (ERK1/2) and protein kinase B (AKT), and the effect of hydrogen rich electrolyzed water is not affected by p53 tumor suppressor gene, estrogen receptor (ER) and progesterone receptor (progesterone receptor, PR) status. Hydrogen selectively reacts with ROS, reducing chromosome damage in cells and possibly inhibiting abnormal activation of tumor related signaling pathways, allowing ataxia telangiectasia mutant genes to repair chromosome damage in a timely manner, thereby inhibiting the occurrence and development of tumors [16].

Abnormal metabolism of tumor tissue leads to oxidative stress in tumor cells, and high levels of ROS can promote tumor proliferation, migration, and invasion. Therefore, disrupting the redox balance of tumor tissue has become a strategy to inhibit tumor growth and metastasis. Japanese scholars Nishikawa et al. [17] conducted a series of studies using hydrogen rich water containing platinum nanoparticles (with a particle size of about 2nm) and found that platinum containing hydrogen water can inhibit chemically induced malignant transformation of mouse embryonic fibroblasts; At the same time, this platinum hydrogen containing water has a killing effect on human tongue cancer cells HSC-4, human esophageal squamous cell carcinoma cells KYSE70, human promyelocytic leukemia cells HL60, and human gastric adenocarcinoma derived NUGC-4 cells [18-20], but has almost no effect on normal cells, which may be related to the specific endocytosis of colloidal platinum by tumor cells. In vitro experiments have found that colloidal platinum reduces the redox potential of hydrogen water and enhances the capture activity of DPPH free radicals by hydrogen water. However, the intracellular ROS level does not decrease but increases, indicating that colloidal platinum activated hydrogen water can induce redox imbalance in tumor cells [18,21].

In terms of the treatment of lung cancer with hydrogen, Zhang Yu et al. from the Third Hospital of Hebei Medical University [22] conducted extensive research, examining the effect of hydrogen on non-small cell lung cancer. They found that in vitro hydrogen culture (at concentrations ranging from 20% to 60%) can promote apoptosis in lung cancer A549 and NCI-H1975 cells, and can reduce the expression levels of X-link inhibitor of apoptosis protein (XIAP) and baculovirus IAP repeat sequence 3 (BIRC3). High concentrations (60%) of hydrogen gas can also cause an increase in the expression of p21, caspase7, and caspase9 proteins in lung cancer A549 cell lines and A549 cell metastatic tumor tissues, as well as a decrease in the expression of cyclin dependent kinase 4 (CDK4) [23]. Wang et al. [24] also found that hydrogen therapy is associated with chromosome condensation in non-small cell lung cancer. Hydrogen intervention can inhibit the expression of chromosome structure maintenance protein 3 (SMC3) gene and protein in A549 and H1975 cells, thereby altering the cell cycle. Hydrogen gas can also inhibit the expression of Ki-67 antigen, cyclooxygenase-2 (COX-2), and VEGF in lung cancer tissues in mice [25]. Ye et al. [26] found that hydrogen containing electrolyzed water can downregulate the transcription and protein expression of VEGF gene in A549 cells by inhibiting the activation of extracellular signal regulated kinase (ERK). In addition, hydrogen can promote apoptosis or autophagy in lung cancer cells by inhibiting the activation of the STAT3/Bcl2 pathway, and inhibiting autophagy can further enhance apoptosis in lung cancer cells [27]. Hydrogen therapy can also inhibit the deterioration of lung cancer by suppressing the activation of the CD47/CDC42 pathway, and is expected to become an effective treatment for CD47 overexpressing tumor patients [28]. The above research provides a basis for hydrogen therapy for lung cancer, and the efficacy of hydrogen therapy for lung cancer still needs further validation through in vivo studies. There are also individual cases in clinical practice showing that inhaling hydrogen is beneficial for lung cancer patients, but there is still a lack of evidence-based medicine [29].

Zhou Xiao et al. [30] used hydrogen water to culture primary colon cancer cells from 15 patients in vitro. The results showed that hydrogen had an inhibitory effect on the growth of primary human colon adenocarcinoma cells, and patient gender, age, tumor location, and Dukes staging had no significant effect on whether primary cancer cells were inhibited by hydrogen. Zhang Yao et al. [31] found that drinking hydrogen rich water can inhibit tumor growth in mice inoculated with human colorectal cancer SW480 cells. On the 28th day, the tumor volume in the hydrogen intervention group was significantly smaller than that in the control group. Liu et al. [32] conducted in vivo studies and constructed C6 in situ glioma models and U87 subcutaneous tumor models. They found that inhaling 67% hydrogen gas twice a day (1 hour each time) in mice could inhibit glioma growth and prolong lifespan. Hydrogen can downregulate the expression of CD133, Nestin (cell stemness marker), Ki67 (cell proliferation marker), and CD34 (angiogenesis marker), while upregulating the expression of GFAP (cell differentiation marker). Hydrogen also inhibits the in vitro spheroidization ability, migration, invasion, and colony formation ability of glioma cells, indicating that hydrogen can work by reducing the stemness of glioma cells. The study also found that hydrogen cultivation can reduce the stemness of human liver cancer cells Huh7 and downregulate the expression of intermediate filament vimentin, providing evidence for the use of hydrogen in the prevention and treatment of liver cancer [33]. Li Jiawei et al. [34] found that hydrogen can promote the polarization of macrophages to M1, inhibit their polarization to M2, and ultimately promote the apoptosis of breast cancer cells. Research on endometrial cancer has found that hydrogen molecules induce tumor cell pyroptosis through the ROS/NLRP3/caspase-1/GSDMD pathway. After culturing in hydrogen rich water, the levels of ROS and mitochondrial ROS (mtROS) in human endometrial cancer cells (AN3CA, HEC1A, and Ishikawa cells) increased [35].