| Aplastic anemia (AA), a common clinical malignant blood disease, is anacquired bone marrow failure syndrome caused by chemical, physics, biologi-cal, heredity and some unknown causes. The main clinical manifestations areanemia, bleeding tendency and infection symptoms. The pathogenesis of AA iscomplicated and heterogeneity. Up till now, there are no effective methods forAA treatment. So it is significant to explore the mechanism and developtherapeutic methods theoretically and practically.A great number of researches have shown that chronic intermittenthypobaric hypoxia (CIHH) has protective effect on tissue and organ of the body.For example, CIHH enhances the tolerance of body to ischemia and hypoxia,protects heart, brain, liver and kidney against ischemia/reperfusion injury, andantagonizes apoptosis and oxidation. Also it was reported that CIHH regulatesimmune function and vasomotoricity, keeps blood pressure stable throughfacilitating baroreflex. Our previous studies showed that CIHH had no effectson basic cardiac function, but protected heart against ischemia/reperfusioninjury. Recently, our research demonstrated that CIHH had effective preventiveand therapeutic effects on the renal vascular hypertension, collagen-inducedarthritis and fructose feeding-induced metabolic syndrome rats.It is well known that the plateau hypoxia promotes the production of redblood cells. Researches in vitro showed that hypoxia could promote theproliferation of hemopoietic progenitor cells (HPCs) or hemopoietic stem cells(HSCs), bone marrow mesenchymal stem cells (BMMSCs), and could inhibitthe differentiation of HSCs and BMMSCs. Hypoxia has been proved toimprove hematopoietic microenviroment (HIM) and affect the hematopoieticprocess. However, the effect of CIHH on blood system and hemopoiesis is notclear yet and the effect of CIHH on AA has not been reported. The objective of this study was to investigate the effect of CIHH on AArats induced by combination of5-fluorouracil (5-FU) and busulfan (BU)systemically in whole body, tissue, cellular and molecular levels usingexperimental zoology, cellular biology, morphology and molecular biologymethods, and explore the underlying mechanism. The study consists of fourparts:(1) To confirm the preventive and therapeutic effect of CIHH on AA ratswith hematopoietic stem cell failure induced by combination of5-FU and BU;(2) To investigate the immunological mechanism for protective effect of CIHHagainst AA through observing the effect of CIHH on T lymphocyte subpopula-tions of peripheral blood in AA rats;(3) To investigate the anti-apoptosismechanism for protective effect of CIHH against AA through observing effectof CIHH on bone marrow cells apoptosis in AA rats;(4) To investigate thehematopoietic HIM for protective effect of CIHH against AA throughobserving effect of CIHH on BMMSCs and its adhesiveness.Partâ… The protective effects of chronic intermittent hypobaric hypoxiaagainst aplastic anemia ratsObjective: To investigate the preventive and therapeutic effect of CIHHon AA rats with hematopoietic stem cell failure induced by combination of5-FU and BU.Methods: The adult male Sprague-Dawley rats were randomly dividedinto the six groups: control group (Con), CIHH treatment group (CIHH), CIHHpre-treatment group (Pre-T), pre-aplastic anemia group (Pre-AA), CIHH post-treatment group (Post-T), post-aplastic anemia group (Post-AA). Pre-AAå'ŒPost-AA rats were given an intraperitoneal injection with combination of5-FUand BU to induce AA; Pre-T and Post-T rats were exposed to hypobarichypoxia simulating3,000m altitude(barometric pressure:525mmHg, PO2:108.8mmHg) in a hypobaric chamb-er for28days,5hours per day before andafter AA induction, respectively. CIHH rats accepted intermittent hypobarichypoxia treatment only and Con rats were not experienced neither AAinduction nor CIHH treatment. The pathologic morphology of bone marrowwas observed by hematoxylin-eosin (H-E) staining. The HPCs of colony-forming unit-granulocyte-macrophage (CFU-GM), colony-formingunit-erythroid (CFU-E), burst-forming unit-eryth-roid (BFU-E) andcolony-forming forming unit-megakaiyocyte (CFU-MK) were assayed by themethod of methylcellulose-based semisolid cultures. The serum interleukin-3(IL-3), erythropoietin (EPO), tumor necrosis factor-alpha (TNF-α) andinterleukin-2(IL-2) was measured by enzyme linked immuno-sorbent assay(ELISA).Results:1. Compared with Con rats, the body weight of CIHH and Pre-Trats were not significantly different (P>0.05);The body weight of Pre-AA andPost-AA rats significantly decreased (P<0.01); The body weight of Post-T ratssignificantly decreased (P<0.01).2. The incidence rate of AA in Pre-T rats (25%) was significantly lowerthan that in Pre-AA rats (80%, P<0.01).The initial onset time of AA in Pre-Trats (24.9±4.4d) was significantly later than that in Pre-AA rats (16.2±5.3d,P<0.01).The mority in Post-T rats (30%) was significantly lower than that inPost-AA rats (70%, P<0.01).3. Compared with Con rats, the value of red blood cells (RBCs), whiteblood cells (WBCs), platelets (PLAs), retieulocytes (Rets), hemoglobin (HGB),hematocrit (HCT), the percentage of neutrophils (NEU), lymphocyte (LYM)and monocyte (MON) in peripheral blood of CIHH and Pre-T rats were notsignificantly different (P>0.05); Except percentage of LYM was significantlyincreased (P<0.01), other indexes of peripheral blood in Pre-AA and Post-AArats were obviously decreased (P<0.01); Except the value of RBCs, WBCs,PLA, Rets, HGB and HCT were significantly decreased (P<0.01), otherindexes of peripheral blood in Post-T rats were not significantly different (P>0.05).4. Compared with Con rats, the bone marrow tissue in Pre-AA and Post-AA rats displayed a pinelosis, hematopoietic cells decrease, non-hematopoieticcells were significantly increase, and the percentage of hematopeitic cells weresignificantly decreased in Pre-AA and Post-AA rats. The number of bonemarrow capillary significantly reduced and bone marrow mesenchymal blood sinus was dilated in Pre-AA and Post-AA rats. The changes of pathologicmorphology in bone marrow tissue of Pre-T and Post-T rats were significantlyimproved.5. Compared with Con rats, the value of bone marrow mononucleatedcells (BMMNCs) and other bone marrow cells in CIHH, Pre-T and Post-T ratswere not significantly different (P>0.05);The value of BMMNCs andmyeloblasts, segmented granulocytes, basophilic promyelocytes, basophilicerythroblasts, polychromatophilic erythroblasts, normoblasts and megakaryo-cytes in AA rats were significantly decreased (P<0.01), and the percentage ofmyelocytes, metamylocytes, stabform granulocytes, eosinophilic myelocyteslymphocytes and monocytes in Pre-AA and Post-AA rats were significantlyincreased (P<0.01), which the other cells in Pre-AA and Post-AA rats bonemarrow were not significantly different (P>0.05).6. Compared with Con rats, the colonies of CFU-GM, CFU-E, BFU-E andCFU-MK in CIHH and Pre-T rats were not significantly different (P>0.05).The colonies of CFU-GM, CFU-E, BFU-E and CFU-MK in Pre-AA, Post-AAand Post-T rats were significantly decreased (P<0.01).7. Compared with Con rats, the serum concentration of IL-3, EPO, TNF-αand IL-2were not significantly different in CIHH rats (P>0.05); The serumconcentration of IL-3and EPO were significantly decreased, while TNF-α andIL-2were significantly increased in Pre-AA and Post-AA rats (P<0.01). Theserum concentration of IL-3and EPO were significantly increased (P<0.01),while TNF-α and IL-2were not different in Pre-T and Post-T rats (P>0.05).Summary: This study confirmed for the first time that CIHH pre-treatment has preventive effect on incidence of AA, and CIHH post-treatmenthas protective effect on AA, lessening the functional disturbance and improvethe pathological damage of bone marrow tissue in AA rats. The protectiveeffect of CIHH on AA was related to the increase of serum IL-3and EPO, thepositive hematopoiesis regulatory factor, and to the decrease of serum TNF-αand IL-2, the negative hematopoiesis regulatory factor. Part II Effects of chronic intermittent hypobaric hypoxia on T lympho-cytes in aplastic anemia ratsObjective: To investigate the immunological mechanism for protectiveeffect of CIHH against AA through observing the effect of CIHH on Tlymphocyte subpopulations of peripheral blood in AA rats.Methods: Experimental grouping, preparation of AA and CIHH treatmentwere the same as Part. T lymphocytes and subpopulations of CD3~+, CD4~+,CD8~+, CD4~+HLA-DR~+and CD8~+HLA-DR~+in peripheral blood of rats weredetected by flow cytometry (FCM). Serum interferon-gamma (IFN-γ),interleukin-4(IL-4) and interleukin-9(IL-9) in rats were assayed by ELISA.Protein expression of IFN-γ, IL-4and IL-9in bone marrow of rats wasmeasured by immunohistochemistry method.Results:1. Compared with Con rats, the percentage of CD3~+, CD4~+, CD8~+,CD4~+HLA-DR~+and CD8~+HLA-DR~+T lymphocyte subpopulations were notsignificantly different in CIHH and Pre-T rats (P>0.05); The percentages ofCD3~+T lymphocytes, CD8~+and CD8~+HLA-DR~+T lymphocyte subpopulationswere significantly increased and the percentages of CD4~+T lymphocytesubpopulations and the ratio of CD4~+/CD8~+were significantly decreased inPre-AA and Post-AA rats (P<0.01); The percentages of CD3~+, CD8~+and CD4~+HLA-DR~+T lymphocyte subpopulations were not significantly different (P>0.05), while the percentages of CD4~+, CD8~+HLA-DR~+and the ratio of CD4~+/CD8~+were significantly increased in Post-T rats (P<0.05).2. Compared with Con rats, serum IFN-γ, IL-4, IL-9and the ratio ofIFN-γ/IL-4(Th1/Th2) were not significantly different CIHH, Pre-T and Post-Trats (P>0.05); Serum IFN-γ, IL-9and the ratio of IFN-γ/IL-4(Th1/Th2) inPre-AA and Post-AA rats were significantly increased (P<0.01), and serumIL-4were significantly decreased in Pre-AA and Post-AA rats (P<0.05).3. Compared with Con rats, protein expression of IFN-γ, IL-4, IL-9andthe ratio of IFN-γ/IL-4(Th1/Th2) of bone marrow tissue were not significantlydifferent in CIHH, Pre-T and Post-T rats (P>0.05); The protein expression ofIFN-γ, IL-9and the ratio of IFN-γ/IL-4(Th1/Th2) of bone marrow weresignificantly increased (P<0.01), while the protein expression of IL-4of bone marrow were significantly decreased in Pre-AA and Post-AA rats (P<0.01).Summary: Under physiological condition， CIHH treatment had nosignificant effects on T lymphocyte subpopulations and the levels of IFN-γ, IL-9and IL-4in peripheral blood，serum and bone marrow，but effectivelyantagonized the reduction of helper T lymphocytes (CD4~+) and the increase ofcytotoxic T lymphocytes (CD8~+) and activated cytotoxic T lymphocytes (CD8~+HLA-DR~+), keeping the balance of peripheral blood T lymphocyte subsets andthe balance between proinflammatory cytokines and anti-inflammatory cyto-kines. The regulation of CIHH on cellular immunity may be one of themechanisms of anti-AA effect of CIHH.Part III Effects of chronic intermittent hypobaric hypoxia on apoptosis ofbone marrow cells in aplastic anemia ratsObjective: To investigate the anti-apoptosis mechanism for protectiveeffect of CIHH against AA through observing effect of CIHH on bone marrowcells apoptosis in AA rats.Methods: Experimental grouping, preparation of AA and CIHHtreatment were the same as Part. The apoptosis of bone marrow cells wasexamined by FCM and terminal dUTP nick end labeling (TUNEL). Theexpression of apoptosis associated protein Fas, Bax, Bcl-2, Caspase-3andCaspase-8was measured by immunohistochemisty method. The expression ofhypoxia inducible factor-1alpha (HIF-1α) and nuclear factor-kappa B (NF-κB)protein was analyzed by western blot.Results:1. The results of FCM and TUNEL showed that, compared withCon rats, the apoptosis rate of bone marrow cells were not significantlydifferent in CIHH, Pre-T and Post-T rats (P>0.05); The apoptosis rate in thebone marrow cells of Pre-AA and Post-AA rats were significantly increased (P<0.01).2. Compared with Con rats, the protein expressions of Fas, Bax, Bcl-2,Caspase-3, Caspase-8and the radio of Bax/Bcl-2of bone marrow cells werenot significantly different in CIHH, Pre-T and Post-T rats (P>0.05); The proteinexpressions of Fas, Bax, Caspase-3, Caspase-8and the ratio of Bax/Bcl-2were significantly increased (P<0.05-P<0.01), while the protein expression of Bcl-2of bone marrow cells were significantly decreased in Pre-AA and Post-AA rats(P<0.05).3. Compared with Con rats, the protein expressions of HIF-1α and NF-κBof bone marrow cells were not significantly different in CIHH and Pre-T rats (P>0.05); The protein expression of HIF-1α and NF-κB of bone marrow cellswere significantly increased in Pre-AA and Post-AA rats (P<0.05-P<0.01); Theprotein expression of HIF-1α of bone marrow cells were significantly increasedin Post-T rats (P<0.01),and the protein expression of NF-κB of bone marrowcells in the bone marrow cells were not significantly different in Post-T rats (P>0.05).Summary: Under physiological condition, CIHH treatment had nosignificant effect on apoptosis and apoptosis associated protein of bone marrowcells, but effectively antagonized the overexpression of HIF-1α and NF-κBproteins, down-regulated the expression of positive apoptosis proteins Fas, Bax,Caspase-3and Caspase-8, up-regulated the expression of negative apoptosisprotein Bcl-2, resulting in anti-apoptosis eventually. The anti-apoptosis ofCIHH on bone marrow cells might be one of the mechanisms of anti-AA effectof CIHH.Part IV Effects of chronic intermittent hypobaric hypoxia on adhesivenessof bone marrow mesenchymal stem cells in aplastic anemia ratsObjective: To investigate the hematopoietic HIM for protective effect ofCIHH against AA through observing effect of CIHH on BMMSCs and itsadhesiveness.Methods: Experimental grouping, preparation of AA and CIHHtreatment were the same as Part. The morphology of adhension cell andcolony-forming unit-fibroblast (CFU-F) were observed by long-term bonemarrow explant cultures (LTBMC-Ex) dynamically. BMMSCs were culturedand purifucated in vitro by the method of differential velocity adherent cellsculture.Very late antigen-4(VLA-4), vascular cell adhesion molecule-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1), CD162and CD164 of BMMSCs were analyzed by FCM. The protein expression of p38mitogen-activated protein kinase (p38MAPK) was assayed by western blot.Results:1. During LTBMC-Ex, the bone marrow stromal cells in Pre-AAand Post-AA rats were dysplasia, and the most cells were immature precursorcells and fibroblast-like cells. Under the basic physiological condition, CIHHtreatment had no effect on development of bone marrow stromal cells layer, butantagonized effectively the dysplasia of bone marrow stromal cells layer andrenewed a valid layer to support hematopoiesis.2. Compared with Con rats, the CFU-F colonies of bone marrow had nosignificantly changes in CIHH and Pre-T rats (P>0.05); The CFU-F colonies ofbone marrow were significantly decreased in Pre-AA and Post-AA rats (P<0.01); The CFU-F colonies of bone marrow were significantly decreased inPost-T rats (P<0.01).3. Compared with Con rats, the protein expressions of VLA-4, VCAM-1,ICAM-1, CD162and CD164of BMMSCs were not significantly different inCIHH rats (P>0.05). The protein expressions of VLA-4, VCAM-1and ICAM-1of BMMSCs were significantly decreased (P<0.01), while CD162and CD164of BMMSCs were significantly increased in Pre-AA and Post-AA rats (P<0.01). The protein expression of VLA-4, VCAM-1, ICAM-1and CD164ofBMMSCs were not significantly different (P>0.05), but CD162of BMMSCswas significantly increased in Pre-T rats (P<0.01). The protein expressions ofVCAM-1and ICAM-1of BMMSCs were not significantly different in (P>0.05), while VLA-4of BMMSCs was significantly decreased in Post-T rats (P<0.01) and the protein expressions of CD162and CD164of BMMSCs weresignificantly increased in Post-T rats (P<0.01).4. Compared with Con rats, the protein expression of p38MAPK ofBMMSCs was not significantly different in CIHH and Pre-T rats (P>0.05). Theprotein expression of p38MAPK of BMMSCs was significantly increased inPre-AA, Post-AA and Post-T rats (P<0.01).Summary: Under physiological condition,CIHH treatment had nosignificant effect on development of bone marrow stromal cells layer and the adhensiveness of BMMSCs, but effectively antagonized the dysplasia of bonemarrow stromal cells layer to support hematopoietsis and the adhensiveness ofBMMSCs in AA rats. This demonstrates that CIHH treatment improves theadhesion of BMMSCs and enhance nutritional support to HSCs in AA rats,which might be one of mechanism of anti-AA effect of CIHH.Conclusion: CIHH has protective effect agains aplastic anemia of ratsthrough inhibit-ing the overexpression of HIF-1α, NF-κB, p38MAPK anddownstream signal molecules, maintaining homeostasis of hematopoieticregulatory factors, keeping balance of T lymphocyte subpopulations,proinflammatory/anti-inflammatory factors,and adhensive molecules, lesseningthe damage of hematopoietic tissue and inhibition of hematopoietic function inbone marrow, decreasing hematopoietic cell apoptosis, and enhancing theadhesiveness and nutritional support between the bone marrow hematopoieticcells and hematopoietic mic-roenvironment. |
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