Molecular hydrogen suppresses glioblastoma growth via inducing the glioma stem-like cell differentiation 分子氫通過誘導膠質瘤幹細胞樣細胞分化抑製膠質母細胞瘤生長

Editor’s note: Clinical experience indicates that inhalation of hydrogen gas and oxygen gas in the ratio of 67 to 33 for 6 to 9 hours per day can suppress the growth of brain cancer (glioma). Anyone who are interested in buying and testing the hydrogen machine used in the current study (MS-H-3 hydrogen-oxygen nebulizer machine) may contact us at [email protected] We may assist you or may help you get a discounted price.

編者按:臨床經驗表明,以 67 比 33 的比例吸入氫氣和氧氣每天6 到 9 小時可以抑制腦癌(膠質瘤)的生長。 任何有興趣購買和測試當前研究中使用的製氫機 (MS-H-3 hydrogen-oxygen nebulizer machine) 的人都可以通過 [email protected] 與我們聯繫。 我們可以為您提供幫助。或者可以幫助您獲得折扣價。

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Stem Cell Res Ther. 2019; 10: 145.

Published online 2019 May 21. doi: 10.1186/s13287-019-1241-x

PMCID: PMC6528353
PMID: 31113492

Molecular hydrogen suppresses glioblastoma growth via inducing the glioma stem-like cell differentiation

Meng-yu Liu,#1,2 Fei Xie,#1,2 Yan Zhang,#3 Ting-ting Wang,1,2 Sheng-nan Ma,1,2 Peng-xiang Zhao,1,2 Xin Zhang,1,2 Tyler W. Lebaron,4,5 Xin-long Yan,corresponding author1,2 and Xue-mei Macorresponding author1,2

Author information Article notes Copyright and License information Disclaimer

1College of Life Science and Bio-engineering, Beijing University of Technology, Beijing, 100124 China
2Beijing Molecular Hydrogen Research Center, Beijing, 100124 China
3Affiliated Bayi Brain Hospital, The Seventh Medical Center of PLA General Hospital, Beijing, 100700 China
4Correction is Molecular Hydrogen Institute, Enoch, UT USA
5Center of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic
Meng-yu Liu, [email protected].
Contributor Information.
corresponding authorCorresponding author.
#Contributed equally.

Copyright © The Author(s). 2019
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Glioblastoma (GBM) is the most common type of primary malignant brain tumor. Molecular hydrogen has been considered a preventive and therapeutic medical gas in many diseases including cancer. In our study, we sought to assess the potential role of molecular hydrogen on GBM.


The in vivo studies were performed using a rat orthotopic glioma model and a mouse subcutaneous xenograft model. Animals inhaled hydrogen gas (67%) 1 h two times per day. MR imaging studies were performed to determine the tumor volume. Immunohistochemistry (IHC), immunofluorescence staining, and flow cytometry analysis were conducted to determine the expression of surface markers. Sphere formation assay was performed to assess the cancer stem cell self-renewal capacity. Assays for cell migration, invasion, and colony formation were conducted.


The in vivo study showed that hydrogen inhalation could effectively suppress GBM tumor growth and prolong the survival of mice with GBM. IHC and immunofluorescence staining demonstrated that hydrogen treatment markedly downregulated the expression of markers involved in stemness (CD133, Nestin), proliferation (ki67), and angiogenesis (CD34) and also upregulated GFAP expression, a marker of differentiation. Similar results were obtained in the in vitro studies. The sphere-forming ability of glioma cells was also suppressed by hydrogen treatment. Moreover, hydrogen treatment also suppressed the migration, invasion, and colony-forming ability of glioma cells.


Together, these results indicated that molecular hydrogen may serve as a potential anti-tumor agent in the treatment of GBM.

Keywords: Molecular hydrogen, Glioblastoma, Glioma stem-like cell, Cancer cell stemness


Glioblastoma (GBM) is the most common type of primary malignant brain tumor and is characterized by rapid proliferation, diffusive invasion into normal brain tissues, and strong chemoresistance []. The current standard therapy for GBM is maximal resection followed by radiotherapy with concomitant and adjuvant temozolomide []. The invasive properties of GBM are a major obstacle for curative treatment, since it makes complete surgical resection impossible and causes the tumor recurrence after therapy []. Despite years of research investigating potentially new therapies for GBM, current therapeutic strategies are insufficient to control the disease, as is reflected by 1-year relative survival of 37.4% and 5-year survival of 4.9% []. Therefore, the development of new methods for GBM treatment is particularly important.

Molecular hydrogen has been considered as a preventive and therapeutic medical gas since Ohsawa et al. reported that inhalation of 1–4% hydrogen gas markedly reduced the sizes of cerebral infarction in rats []. The potential benefits of H2 have now been demonstrated in over 170 different human and animal disease models []. Recently, the effects of molecular hydrogen in cancer have attracted significant attention. Dole et al. first reported the therapeutic role of hydrogen on cutaneous squamous cell carcinoma in Science in 1975. They found that hyperbaric treatment of 97.5% hydrogen gas at a total pressure of 8 atm for 2 weeks could markedly induce tumor regression in mice []. Due to the special conditions of hyperbaric hydrogen treatment, subsequent research on the anti-cancer effect of hydrogen has been stagnant until the biological effects of low concentration hydrogen were reported on by Ohsawa et al. in 2007. Several years later, Saitoh et al. reported that neutral pH hydrogen-enriched electrolyzed water (NHE water) could achieve inhibition of tumor growth and of tumor invasion in an in vitro cell model []. Subsequent in vitro studies showed that molecular hydrogen could inhibit cancer cell proliferation [], migration, invasion [, ], and colony formation [] and induce cancer cell apoptosis [, ]. Several in vivo studies have demonstrated that molecular hydrogen could prevent carcinogenesis [], inhibit cancer progression [, ], relieve the side effects of chemotherapy or radiotherapy [], and enhance the anti-tumor effects of chemotherapeutic drugs [, ]. Although molecular hydrogen has shown potential in the field of cancer therapy, its anti-cancer properties are limited to only a few tumor types, and the underlying molecular mechanisms remain to be established.

In this study, we investigated the possible therapeutic effects of molecular hydrogen on GBM. Both in vivo and in vitro experimental models were used to evaluate the potential role of molecular hydrogen. The mechanisms underlying the effects of hydrogen have also been investigated.

Materials and methods

Animals and tumor cell lines

Male Wistar rats (8 weeks old, 170–180 g) and female BALB/c nude mice (8 weeks old, 20–24 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were maintained under standard conditions at 22 °C to 25 °C with a 12-h light-dark cycle and were fed a normal diet. All procedures were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China).

Rat C6 glioma cells and human U87 cells were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA). Cells were routinely cultured at 5% CO2 and 37 °C in DMEM/F12 medium (Gibco, NY, USA) supplemented with 10% fetal calf serum (Gibco, NY, USA) and 1% penicillin and streptomycin (Gibco, NY, USA).

Rat C6 glioma model

Rats were fasted for 1 day before the experiments. They were then anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL/kg; Sigma, USA) and fixed in a stereotactic apparatus. The skin of the scalp was then incised in the midline of the skull with surgical scissors. Then, a hole was made in the cranial bone 1 mm posterior to the bregma and 3 mm lateral to the sagittal suture. A 30-gauge needle with a microsyringe was inserted to a depth of 7 mm from the skull surface; C6 glioma cells (1 × 106) in 10 μL phosphate-buffered saline (PBS) were injected stereotactically. The injection was conducted over 10 min, after which the needle was held in position for 5 min and then gradually withdrawn over 3 min to prevent the backward flow of the solutions. After implantation of glioma cells, the rats were randomly divided into two groups including the hydrogen inhalation group (HI) and control group (CTRL).

Mouse U87 subcutaneous model

BALB/c nude mice were injected subcutaneously with 1 × 106 viable U87 cells. After injection, the mice were randomly assigned to two groups including the hydrogen inhalation group (HI) and control group (CTRL). Tumor volumes were measured on a weekly basis using the following formula: volume = width2 × length × 0.4.

Inhalation of hydrogen gas

A transparent closed box (20 cm × 18 cm × 15 cm) was connected to an AMS-H-3 hydrogen-oxygen nebulizer machine (Asclepius Meditec Inc., Shanghai, China), which produces 67% H2 and 33% O2 (V/V). Hydrogen treatment was given on the second day of the rat C6 glioma or mouse U87 subcutaneous model establishment until the end of the experiment. Animals were placed in this box and inhaled the mixed air for 1 h two times per day. During this inhalation, mice were awake and freely moving. Thermal trace GC ultra-gas chromatography (Thermo Fisher, MA, USA) was used to monitor the concentration of hydrogen gas in the closed box.

Hydrogen-rich medium treatment

A hydrogen-rich medium was produced by placing a metallic magnesium stick (Doctor SUISOSUI®; Friendear Inc., Tokyo, Japan) into DMEM/F12 medium (final hydrogen concentration 0.55–0.65 Mm). The hydrogen concentration was monitored by using a needle-type Hydrogen Sensor (Unisense A/S, Aarhus, Denmark).

MR imaging

MR imaging studies were performed with the Bruker 7.0 T ClinScan high-field small-animal MRI system (Bruker BioSpin, Ettlingen, Germany). The tumor-bearing rats were anesthetized with 2% isoflurane in 2 L min−1 of oxygen and maintained at a normal body temperature. The T2-weighted MR images were acquired for each rat on days 17 and 26 after tumor transplantation using a conventional spin-echo sequence with the following parameters: TR = 3140 ms, TE = 37 ms, bandwidth = 130 Hz, flip angle = 180°, FOV = 4 cm × 4 cm, and slice thickness = 1 mm.

Histology and immunohistochemistry

Glioma tissues were embedded in paraffin and then cut into 8-μm-thick sections. Sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases, followed by incubation with 10% normal goat serum. After the blocking serum was removed, sections were incubated overnight at 4 °C with primary antibodies including anti-CD34 (1:200; Abcam), anti-Ki67 (1:200; Abcam), anti-CD133 (1:50; Biobyt), and anti-Nestin (1:100; Millipore). Detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. All data were evaluated by blinded investigators.


Cells were seeded on coverslips, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% BSA for 1 h. Subsequently, cells were incubated overnight at 4 °C with primary antibodies including anti-GFAP (1:200; Abcam) and anti-CD133 (1:50; Biobyt). Cells were then incubated with FITC-conjugated secondary antibody (Abcam) for 1 h. The nuclei were stained with Hoechst, and the fluorescence images obtained with an Olympus IX71 inverted microscope.

Flow cytometry

The expression of CD133 in cultured cells was analyzed by flow cytometry. In brief, cells were incubated for 10 min with 10% horse serum in PBS at 4 °C, followed by incubation with primary anti-CD133 (1:200; Biobyt) at 4 °C for 30 min. After centrifugation, the collected cells were incubated with FITC-conjugated secondary antibody (Abcam) for 20 min. After staining, cells were subjected to flow cytometry for analysis using a flow cytometer (Guava easyCyte 8HT; Millipore).

Sphere formation assay

Cells were collected and washed to remove serum, then suspended in serum-free DMEM or MEM/EBSS medium at a density of 2.0 × 103/mL. Five hundred cells per well were counted and seeded onto ultra-low attachment six-well plates (Corning, Corning, NY) for the sphere formation assay. Cells were cultured in normal serum-free medium or hydrogen-rich serum-free medium with B27 supplement (GIBCO, Grand Island, NY), 20 ng/mL of epidermal growth factor (EGF) (Pepro Tech Inc., Rocky Hill, NJ), and 20 ng/mL of basic fibroblast growth factor (bFGF) (Pepro Tech). The medium was changed every 3 days. Cells were incubated for 10 days, and spheres with a diameter > 50 μm were counted.

Cell migration assay

The cell migration ability was examined using a wound-healing assay. Briefly, cells were seeded in six-well plates at a concentration of 1.0 × 106/well and cultured for 24 h. A plastic pipette tip was used to scratch a line across the cell surface in each plate. The remaining cells were washed three times with PBS to remove the floating cells and debris, followed by incubation for 48 h in normal complete medium or hydrogen-rich medium. The images of the healing process were photographed digitally at the time point of immediately following and 24 h after wounding. The wound-healing assay was performed in three independent experiments.

Cell invasion assay

The cell invasion ability was determined using BD matrigel invasion chambers according to the manufacturer’s protocol. Briefly, the top chambers with polycarbonate filters (8-μm pore size; Costar, Acton, MA) were coated with 50 μL of Matrigel (0.8 μg/μL, 37 °C, 2 h; BD Biosciences, San Diego, CA). 1 × 105 cells in a 100-μL serum-free medium were seeded to the top chamber, and a 650-μL normal complete medium with or without hydrogen was added to the bottom. The cells were allowed to migrate through the porous membrane at 37 °C for 48 h. Then, cells in the upper surface of the chamber were completely removed by cotton swabs. The cells on the lower surface were stained with 0.1% (w/v) crystal violet after fixation, and five random fields from each insert were counted at × 100 magnifications. The invasion assay was conducted in triplicate-independent experiments.

Colony formation assay

One thousand cells per well were counted and seeded in six-well plates. The plates were incubated for 14 days in a normal complete medium either with or without hydrogen, and then the cells were fixed by 4% paraformaldehyde and stained using 0.1% crystal violet. Colonies were counted only if they included at least 15 cells. Triplicate-independent experiments were performed, and all the visible colonies were calculated manually.

Statistical analysis

Groups from cell culture and in vivo experiments were compared using two-tailed Student’s t tests, and results are presented as means ± SEM. All statistical analyses were performed using GraphPad Prism 6.01. A value of p < 0.05 was considered significant.


Hydrogen inhalation inhibited glioma growth in vivo

The effect of molecular hydrogen on tumor growth of glioma cells was first evaluated in a rat C6 glioma model. MR imaging results show no significant difference in tumor size between the HI group (54.76 ± 6.07) and the control group (51.23 ± 9.11) on day 17 after C6 cell implantation. However, on day 26, the tumor volume was significantly decreased in the HI group compared to the control group (223.3 ± 33.83 mm3 vs. 363.3 ± 34.80 mm3, p = 0.045) (Fig. 1a, b). In addition, hydrogen inhalation also induced prolongation of median survival (28.00 days vs. 31.00 days, p = 0.0012) (Fig. 1c).

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