Hydrogen-Rich Saline Shields Mice from aGVHDScientific Research

Allogeneic hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologic malignant and nonmalignant hematologic diseases, although it is restricted by its severe complications (^{1, 2}). One major complication associated with HSCT is acute graft-versus-host disease (aGVHD), which is always a major cause of death after allo-HSCT (³). The pathophysiology of aGVHD involves complex three stages. Stage I involves tissue damage and cellular activation induced by preconditioning. Stage II involves the activation of donor lymphocytes (T cells), followed by a third stage, in which cellular and inflammatory factors are released, including tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. These cytotoxic molecules directly attack various host tissues and underlie the clinical manifestations of aGVHD (^{4, 5}). The activated cells also produce a large number of harmful free radicals resulting in severe cell damage (³).

Hydrogen has been extensively used in chemical fields, such as fuel processing and fertilizer production (3H₂+N₂→2NH₃). It was considered to be the physiologic inert gas, which is rarely used in the medical field. However, in 2007, Ohsawa et al. (⁶) discovered that H₂ gas has antioxidant and antiapoptotic properties that protect the brain against ischemia–reperfusion injury and stroke by selectively neutralizing hydroxyl and peroxynitrite radicals. Since then, H₂ gas has come to the forefront of therapeutic medical gas research. We also have proposed and proven that hydrogen has radioprotective effects in cultured cells and mice (^7–10).

Recent basic and clinical research has revealed that hydrogen is an important physiologic regulatory factor with anti-inflammatory, antioxidant, and antiapoptotic protective effects on cells and organs, proving that hydrogen could down-regulate cytokines, such as CCL2, IL-1β, IL-6, IL-12, and TNF-α (^11–15), which play important roles on the development of aGVHD. Also, free radicals exert an important role in the occurrence and development of aGVHD (^{16, 17}). Therefore, we reasoned that hydrogen might have therapeutic effects on aGVHD. However, the application of H₂ gas inhalation is not convenient and may be dangerous because it is inflammable and explosive. As we reported previously, H₂ gas saturated saline, which is called hydrogen-rich saline, is easy to apply and is safe (⁷). In the current study, we investigated whether the administration of hydrogen-rich saline exerted a therapeutic effect on aGVHD mice. We demonstrated here that hydrogen treatment could protect mice from lethal GVHD and increase the clinical score of aGVHD mice and also could promote the recovery of white blood cells of aGVHD mice.

RESULTS

Hydrogen Administration After Bone Marrow Transplantation Protects Mice From Lethal Graft-Versus-Host Disease

Initial studies were performed to determine whether hydrogen could protect mice from lethal GVHD in a major histocompatibility complex–incompatible murine bone marrow transplantation (BMT) model. Mice were treated intraperitoneally with physiologic saline or hydrogen-rich saline (5 mL/kg) 24 hr after transplantation every day. Forty percent of GVHD mice without H₂ treatment died by the 16th day after BMT (Fig. 1), whereas 80% of the mice pretreated with H₂ had survived (Fig. 1). Thus, H₂ could protect mice from lethal GVHD.

Hydrogen Reduces Serum Cytokine Levels in Mice Undergoing Graft-Versus-Host Disease

We examined the serum cytokine levels to determine whether hydrogen affected cytokine levels that were increased during the course of GVHD. Mice in all groups were bled on day 7 after BMT and analyzed for the specified cytokines. Untreated mice with GVHD had increased levels of TNF-α, IL-2, and IL-10 compared with control animals (P<0.01 for all three cytokines; Fig. 2). The administration of hydrogen resulted in a significant decrease in serum levels of all three cytokines (Fig. 2) compared with untreated mice with GVHD (P<0.05 for all three cytokines).

Hydrogen Improves Clinic Symptoms of Graft-Versus-Host Disease Mice

To determine the therapeutic effects of hydrogen, the clinical signs of GVHD on the 15th day after BMT were scored on the criteria provided in Table 1. As shown in Figure 3, hydrogen significantly improved clinic symptoms of GVHD mice (P<0.01). Also in Figure 3, the weight of GVHD mice treated with hydrogen was significantly higher than that of GVHD mice treated with hydrogen (P<0.05).

Hydrogen Promotes the Recovery of Peripheral Blood Leukocytes

Leukocyte counts declined rapidly and elevated gradually from day 9 after BMT. Within the whole post-BMT period, the recovery of leukocytes in the H₂ groups was significantly faster than that of the non-H₂ groups (Fig. 4).

On day 28 after BMT, leukocyte counts in the H₂ group returned to 7.8×10⁹/L compared with 4.75×10⁹/L in the non-H₂ group.

DISCUSSION

To our knowledge, hydrogen was firstly applied on hematologic diseases. Also, this is the first study demonstrating that hydrogen has therapeutic effect on aGVHD mice.

Nowadays, the standard initial therapy for GVHD includes the use of high-dose steroids, which results in approximately 40% complete response (CR) rate (¹⁸). This CR rate is unsatisfactory, and in patients who have less than a CR, there is high mortality both from the GVHD itself and from the steroid-related infectious complications. The treatment of steroid-resistant aGVHD is very difficult, and many institutions use monoclonal antibody (mAb), such as anti-TNF-α mAb, anti-CD52 mAb, anti-CDl47 mAb (ABX.CBL), and anti-CD3 mAb to treat steroid-resistant aGVHD patients, but their therapeutic effects are not ideal although with high infection rate. Overall, those treatments of aGVHD did not achieve significant breakthroughs.

In our study, we demonstrated here that hydrogen treatment could protect mice from lethal GVHD and improve clinic syndrome of aGVHD mice and also could promote the recovery of white blood cells of aGVHD mice. Its mechanisms may involve, in part or exclusively, reducing the levels of TNF-α, which is a major cytokine mediator of GVHD-induced tissue damage (^19–21). IL-2 was also reduced in aGVHD mice treated with hydrogen, which also play an important role in the development of aGVHD. Besides, this therapeutic effect may result from the reactive oxygen species scavenging effect of molecular H₂ (⁶), because free radicals exert an important role in the occurrence and development of aGVHD (^{16, 17}). A few side effects of hydrogen were reported. Hydrogen is continuously produced by colonic bacteria in the body and normally circulates in the blood (²²). Inhalation of H₂ gas does not influence physiologic parameters such as body temperature, blood pressure, pH, and pO₂ in the blood, as shown previously (^{6, 23}). Therefore, it is physiologically safe for humans to inhale hydrogen. Hydrogen is a highly diffusible gas and it reacts with hydroxyl radical to produce water (²⁴). Dissolving H₂ in solvent such as physiologic saline, pure water, or medium is easy to apply and is safe. Therefore, it may have great potential for clinical use.

From the results obtained, we conclude that the ability of reducing the levels of cytokines, such as TNF-α, IL-2, and the ability of reducing radical oxygen species play important roles in the therapeutic effects of hydrogen. However, the exact mechanism and the signaling pathway involved in the therapeutic role of hydrogen in GVHD need to be studied in the future.

MATERIALS AND METHODS

Hydrogen-Rich Saline Production

As we reported previously (^{7, 8, 10}), hydrogen was dissolved in physiologic saline 6 hr under high pressure (0.4 MPa) to a supersaturated level using hydrogen-rich water-producing apparatus produced by our department. The saturated hydrogen saline was stored under atmospheric pressure at 4°C in an aluminum bag with no dead volume. Hydrogen-rich saline was freshly prepared every week, which ensured that a concentration of more than 0.6 mM was maintained. Gas chromatography (Biogas Analyzer Systems-1000, Mitleben, Japan) was used to confirm the content of hydrogen in saline by the method described by Ohsawa et al. (⁶).

Mice

All protocols were approved by the Naval General Hospital (Beijing, China) in accordance with the Guide for Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (Publication No. 96-01). Male and female C57BL/6 (C57) (H-2b) and (BALB/c×C57BL/6) F1 (F1) (H-2d/b) mice, 8 weeks old, were kept in top-filtered cages in a standard animal facility. Cages, sawdust, and water bottles were autoclaved once a week.

Graft-Versus-Host Disease Model

The GVHD model was established by the method described previously (¹⁶). In brief, F1 mice were exposed to 750 cGy total body irradiation at a dose rate of 170 cGy/min. Spleen cells from donor mice were suspended in PRMI 1640 medium supplemented with 10% fetal calf serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL; Invitrogen, Carlsbad, CA), washed twice, and resuspended in the same medium. One day after irradiation, F1 mice were infused with 20×10⁶ C57BL/6 spleen cells. As described previously, chimerism was documented by determining the percentage of host- or donor-type cells in blood samples of the transplanted mice (²⁵). As we reported previously (⁷), mice were treated intraperitoneally with physiologic saline or hydrogen-rich saline (5 mL/kg) 24 hr after transplantation every day.

Survival Assays

For the evaluation of the therapeutic effect of hydrogen, mice were returned to the animal facility and routinely cared for 30 days after BMT. Survival was checked and scored daily for 30 days.

Analysis of Cytokine Secretion

Mice in all groups were bled on day 7 after BMT and analyzed for the specified cytokines (IL-2, IL-10, and TNF-α). IL-2, IL-10, and TNF-α were measured with a commercial enzyme-linked immunosorbent assay kit (Biosource, Camarillo, CA) according to the instructions of the manufacturer.

Clinic Score

As described previously (^{26, 27}), mice were monitored for weight loss, posture, activity, fur change, and skin integrity. The clinical signs of GVHD on the 15th day after BMT were scored on the following criteria provided in Table 1.

Leukocyte Counts

For the evaluation of the therapeutic effect of hydrogen, peripheral blood samples were obtained from the tail vein of mice on different days after BMT. The numbers of peripheral blood leukocytes were determined with a hemocytometer as we have described previously (¹⁰).

Statistical Analysis

Survival curves were constructed using the Kaplan-Meier product limit estimator and compared using the log-rank rest. Other results are expressed as mean±SD and compared by the two-sample Student’s t test for differences in means. P<0.05 was deemed to be significant in all experiments.