Therapeutic Effect And Mechanism Of H2in Aplastic Anemia Mice

BackgroundAplastic animia (AA) is a hematologic disease caused by the disorder of the functionand the number of hemopoietic stem cell. The major clinical symptom is peripheralcytopenia. Now it is considered that the disorder of immune system especially T cellmediated immune reaction play an important role in the developing of AA. Dendriticcells (DCs), the most potent antigen-presenting cells (APCs) active T cells byrecognition/processing and presenting antigens. T cell proliferation and activationwere promoted and up-regulated Th1/Th17/CD8+T cells and down-regulatedTh2/Treg cells. On the one hand, Th1cells can induce bone marrwo failure bysecreting cytokines; on the other hand, Th1cells can induce the apoptosis andnecrosis of hemopoietic stem cells by promoting the proliferation and activation ofCD8+T cells. Th17damage the hemopoietic stem cells by secreting such asIL-10/17/21/22. CD8+T cells kill the hemopoietic stem cells by mediating signalingpathway like perforiin/granzyme and TNF. Treg, a kind of regulatory T cell, play animportant role in AA patient. Because of the down-regulated in AA, the immunereactions were overexpressed and lose the regulatory function to cellular immunity.Finally, the disease of AA developed.At present, the clinical treatment methods for aplastic anemia include bone marrowtransplantation/immunosuppressive therapy/estrogenandrogen therapy etc. Althoughthese methods can recovery some patients, its side effects are still no ignore. Solooking for a new high efficiency drug has important significance.In2007，Ohsawa etc found that hydrogen can exerts its antioxidation by scavengingfree radical selectively. From then, the hydrogen biology is evoking heat discussionworldwide. Current studies showed that hydrogen can treat many diseases bydown-regulated cytokines such as TNF-α/IFN-γ/IL. Our datas showed that hydrogencan play an important role in radioprotection. Our research also showed thathydrogen-rich saline can cure acute graft-versus-host disease, on the basis of micemodel. But there is no report about the treatment effect of hydrogen-rich saline toaplastic anemia. In this study, we research the treatment effect of hydrogen-rich salineto aplastic anemia and the possible mechanism on the basis of aplastic anemia micemodel. Research content1. The establishment of aplastic anemia in mice2. The effect of hydrogen-rich saline to weight in aplastic anemia mice3. The effect of hydrogen-rich saline to peripheral blood in aplastic anemia miceThe effect of hydrogen-rich saline to white blood cell/red blood cell/platelet/hemoglobin4. The effect of hydrogen-rich saline to bone marrow in aplastic anemia mice4.1The effect of hydrogen-rich saline to bone marrow nucleated cells4.2The effect of hydrogen-rich saline to bone marrow microenvironment4.3The effect of hydrogen-rich saline to bone marrow cells apoptosis5. The effect of hydrogen-rich saline to pluripotential hematopoietic stem cells5.1The effect of hydrogen-rich saline to colony forming unite of spleen.5.2The effect of hydrogen-rich saline to colony forming unite of granulocytemacrophage6. The effect of hydrogen-rich saline to immune system in aplastic anemia mice6.1The effect of hydrogen-rich saline to T cells6.2The effect of hydrogen-rich saline to TNF-α/IFN-γ/IL-6Research methods1. The preparation of hydrogen-rich saline: H2was dissloved iin physiological salinefor2h under high pressure (0.4MPa) to a supersaturated level using hydrogen-richwater-producing apparatus produced by our faculty.2. The establishment of aplastic anemia model: recipient mice were preirradiated with5Gy total body irradiation (TBI) and were injected with MHC-mismatched LN cells(5×106) through the lateral tail vein4hours after irradiation.3. Measure the weight: the weight of each groups were measured by electronicbalance for28days.4. The detection of peripheral blood: peripheral blood was collected after enucleationof eyeball. The peripheral blood indexes of each group were detected using bloodtesting instrument.5. The detection of bone marrow:5.1The detection of bone marrow nucleated cells: the femurs of the mice wereprocured and the numbers of the bone marrow nucleated cells were counted undermicroscope.5.2The detection of bone marrow microenvironment: the femurs of the mice were procured and the bone marrow microenvironment was detected using H-E histologicalsection.5.3The detection of bone marrow cells apoptosis: the femurs of the mice wereprocured and the cell apoptosis was detected using Tunel histological section.6. The detection of hematopoietic stem cell:6.1The detection of CFU-S: the bone marrow cells were extracted and injected tomice which were preirradiated with5Gy total body irradiation through the lateral tailvein.14days later, the mice were killed and the numbers of CFU-S were countedunder microscope.6.2The detection of CFU-GM: the bone marrow cells were extracted and culturedin methylcellulose culture.12days later, the CFU-GM were stained using trypan blueand the numbers of the CFU-GM were counted under microscope.7. The detection of immune system in aplastic anemia mice7.1The detection of T cells: the spleen was removed, hemogenized with a tissuegrinder in Iscove modified Eagle media, filtered through100um nylon mesh, washedin Iscove midified media, and counted by the use of a counter. For flow cytometry,cells were incubated in Geys solution twice for10mins each to lyse red blood cells.After washing in flow buffer, cells were incubated with premixed antibody cocktailfor30mins on ice, washed, resuspended in flow buffer, and analyzed by the use of aLSR flow cytometer (Beckman).7.2The detection of the subset of CD4+cells: the spleen was removed,hemogenized with a tissue grinder in Iscove modified Eagle media, filtered through100um nylon mesh, washed in Iscove midified media, and counted by the use of acounter. For Real-time PCR, RNA was extracted and the transcription factors ofTh1/2/17and Treg were detected.7.3The detection of cytokines related to the subset of CD4+cells: the spleen wasremoved, hemogenized with a tissue grinder in Iscove modified Eagle media, filteredthrough100um nylon mesh, washed in Iscove midified media, and counted by the useof a counter. For Real-time PCR, RNA was extracted andIFN-γ/IL-12/TGF-β1/IL-4/IL-5/IL-10/IL-17/IL-23were detected.7.4The detection of TNF-α/IFN-γ/IL-6: Blood was collected by orbital sinusbleeding into tubes containing50ul of0.5M EDTA and centrifuged at3000rpm for10mins. Plasma was removed and stored at-20. Plasma cytokine concentrations weremeasured by use of ELISE Array Kits. Results1. The effect of hydrogen-rich saline to weight in aplastic anemia miceHydrogen-rich saline was administered to aplastic anemia mice for28days.28dayslater, the group with hydrogen-rich saline is much heavier than that of the AA group.The average growth is1.90±0.25g in AA mice and3.01±0.15g in experimental group(P<0.05). The data showed that hydrogen-rich saline can improve the quality of life.2. The effect of hydrogen-rich saline to peripheral blood in aplastic anemia miceHydrogen-rich saline was administered to aplastic anemia mice for28days. Aftertreated with hydrogen-rich solution for28days, the RBC, WBC, HGB and Plt countsof the experimental group were all increased significantly compared to the AA group.All these results showed that hydrogen-rich solution indeed had an effect onalleviating AA peripheral blood injury.3. The effect of hydrogen-rich saline to bone marrow in aplastic anemia mice3.1Hydrogen-rich saline was administered to aplastic anemia mice for28days.Compared to the AA group, the experimental group significantly acceleratedhematopoietic recovery by increasing the numbers of BMNC.28days later, thenumber of BMNC in the experimental group returned to8.8×106/femur, as comparedto4.35×10~6/femur in AA group (P<0.05).3.2Hydrogen-rich saline was administered to aplastic anemia mice for28days. Atday28after modeling AA, the numbers of bone marrow cells were significantlyhigher in the experimental group, compared to the AA group.3.3Hydrogen-rich saline was administered to aplastic anemia mice for28days. Atday28after modeling AA, the numbers of apoptotic cells were significantly lower inthe experimental group, compared to the AA group.4. The effect of hydrogen-rich saline to pluripotential hematopoietic stem cells4.1Hydrogen-rich saline was administered to aplastic anemia mice for28days.After28days of hydrogen-rich solution treatment, the colony formation numbers ofthe AA group was markedly lower than that of the experimental group. The numbersof CFU-S are9/spleen in experimental group compared to5.3/spleen in AA group(P<0.05).4.2Hydrogen-rich saline was administered to aplastic anemia mice for28days.After28days of hydrogen-rich solution treatment, the colony formation numbers ofthe AA group was markedly lower than that of the experimental group. The numbersof CFU-GM were63/vessel in experimental group and32.1/vessel in AA group (P<0.05). All those data indicated a direct proliferation due to hydrogen-rich solutionon committed hematopoietic progenitor cells.5. The effect of hydrogen-rich saline to immune system in aplastic anemia mice5.1Hydrogen-rich saline was administered to aplastic anemia mice for14days.After14days of hydrogen-rich solution treatment, the percent of CD4+was increasedto8.31%in experimental group compared to3.96%in AA group. While, the percentof CD8+was decreased to4.51%in experimental group compared to4.77%in AAgroup. The ratio of CD4/CD8was1.84in the experimental group compared to0.83inthe AA group (P<0.05).5.2Hydrogen-rich saline was administered to aplastic anemia mice for14days.After14days of hydrogen-rich solution treatment, the transcription factors of Th1(T-bet)/Treg (Foxp3) and Th17(RORγt) were decreased significantly in experimentalgroup compared to that in AA group, while the transcription factors of Th2(GATA3)was increased in experimental group compared to that in AA group.5.3Hydrogen-rich saline was administered to aplastic anemia mice for14days.After14days of hydrogen-rich solution treatment, the expression of IFN-γ、IL-12、TGF-β1、IL-4、IL-17and IL-23were down-regulated in experimental group，whileIL-5and IL-10were up-regulated。5.4Hydrogen-rich saline was administered to aplastic anemia mice for14days.After14days of hydrogen-rich solution treatment, the level of TNF-α in experimentalgroup (73.1±1) was far below the AA group (114.3±12.6); the level of IFN-γ inexperimental group (63.3±13.7) were reduced dramatically, while the level of IFN-γin AA group (107.1±15.1) was still at high level; the level of IL-6in experimentalgroup (27.2±4.9) were reduced while the level of IL-6in AA group was33.9±1.7(Fig.5E). There was significant difference between AA group and experimental group(P<0.05).DiscussionAA is characterized by hypocellular bone marrow resulting from damage tohemopoietic stem cells. This produces pancytopenia with risk of severe anemia, majorhemorrhage, and life-threatening infections. Abnormal immunity is the major factormediating the pathogenesis of AA. Standard therapies, such as bone marrowtransplantation, corticosteroids, and/or antithymocyte globulin, have a clinical benefitin approximately50%of patients. However, many patients fail to respond to thesetreatments or relapse after treatment. Recently, our data showed that hydrogen-rich solution can protect the immune system and accelerate the recovery ofimmunodeficiency induced by irradiation. Other data showed that hydrogen canvarious of cytokines, such as TNF-α, IFN-γ, IL-1, IL-6, IL-12in different models.Meantime, hydrogen is produced by colonic bacteria in our body continuously and hasno side effects to our body. It can be as an ideal drug for AA.The peripheral blood profile is the evidence of bone marrow failure that has beentermed as “residual injury”. And in AA patients, the injury which leads to impair ofstem cell proliferation is permanent. The values of hematologic parameters in AApatients are far below than normal values. The improvement of peripheral bloodparameters is an indicator that AA is relieved. In our study, AA mice which weretreated with hydrogen-rich solution appear to be sensitive to hydrogen-rich solution,and changes were analyzed. The results demonstrated that the hematologic parametersof AA mice treated with hydrogen-rich solution increased much higer than thechanges seen in AA mice. The counts of RBC, WBC, Plt and HGB were significantlyincreased in experimental group. This result is consistent with other observations thatcan accelerate rehabilitation of hematologic parameters.The bone marrow which consists of hematopoietic stem cells (HSCs),mesenchymal stem cells (MSCs), and endothelial progenitor cells (EPCs) isconsidered as the major hematopoietic tissue. Bone marrow is damaged severelyduring the development of AA. The cavum ossis was filled with a great deal of fatcells and plasma cells. As a result, the hematopoietic capacity is diminished. In thepresent study, bone marrow cell counts and bone marrow histological examination,as indexes of bone morrow damage, showed that the recovery speed of bone marrowin AA mice with hydrogen-rich solution faster than that in AA mice. CFU-S andCFU-GM are good indications of bone marrow cell viability, proliferation. Thenumbers of CFU-S and CFU-GM can reflect the situation of hematopoietic cells.CFU-S and CFU-GM were measured to detect the sensitivity of bone morrowhematopoietic cells to hydrogen-rich solution. Hematopoietic cells from the AA micewere highly sensitive to hydrogen-rich solution. The numbers of CFU-S andCFU-GM in experimental group are much more compared to AA group. All thoseresults showed that hydrogen-rich solution can improve the bone marrowmicroenvironment, promote the nucleated cells proliferation, and colony formation.AA is an immune-mediated disease. The disorder of immune system especiallyT cell plays an important role in the development of AA. Antigens are presented to T cells by antigen-presenting cells (APCs), which trigger T cells to activate andproliferate. Activated T cells were differentiated into CD4+cells which containTh1/Th2/Th17/Treg typies and CD8+cells. All those cells play important roles inaplastic anemia. On the one hand, Th1cells can secrete a variety of immunemolecules including IFN-γ, TNF-α directly; On the other hand, Th1cells canpromote the activation of NK cells, CD8+T cells, and macrophage cells which couldsecrete various of cytokine including IFN-γ, TNF-α, IL-6, IL-2and mediateapoptosis. Abnormal proliferation of CD8+T cells induced the imbalance of the ratioof CD4/CD8. IFN-γ and TNF-α up-regulate other T cells’ cellular receptors and alsothe Fas receptor. Increased IL-2leads to the expansion of T cells. Activation of Fasreceptors by the Fas ligand induces the apoptosis of target cells. All those immunecytokine cells compose a cytokine network to destruct stem cells as well asmesenchymal stem cells and endothelial progenitor cells. As a result, stem cells aresignificantly impaired and lose the capacity of proliferation and differentiation. In thepresent study, our data showed a return to normal of the CD4/CD8in AA mice aftertreatment with hydrogen-rich solution for28days. It is consistent with the results ofother AA researches. Meanwhile, we also observed that IFN-γ, TNF-α, and IL-6were decreased after treatment with hydrogen-rich solution14days later. All together,we deduced that hydrogen-rich solution may heal AA by mediating immune system.