| Aplastic Anemia (AA) is a disease of the hematopoietic system caused by a variety of factors, and the pathogenesis of aplastic anemia is that excessive hematopoietic negative regulatory factors secreted by abnormal activation of T cells induced the apoptosis of the hematopoietic stell cells by some mechanisms, thus resulting in bone marrow failure. The whole process which starts from the hematopoietic stem cells to the mature cells is regulated by a complex regulation of the cell signal transduction. The transcription factor GATA-1, PU.1regulate hematopoietic stem/progenitor cells into erythroid megakaryocyte and granulocyte monocyte differentiation. Whole blood cells in the patients with aplastic anemia are reduced, with the reduction most apparent in erythroid dysplasia, EPO-mediated JAK2/STAT5pathways and specific transcription factor GATA-1are essential in erythroid development. Guided by the theory of "kidney storing essence", the functional mechanisms of Bushen Yisui Shengxue method on hematopoietic stem/progenitor cells into granulocyte proliferation and specific transcription factor PU.1, and hematopoietic stem/progenitor cells into erythroid regulated by transcription factor GATA-1and JAK2/STAT5signaling pathway of rats with AA have been studied by flow cytometry method (FCM), radioimmunoassay (RIA) method, Western blot and RT-PCR method on the basis of AA animal models by60Co-y rays and cyclophosphamide (CTX). The study provides abundant experimental data for the AA "kidney storing essence" theory.Objective:Experiments on animals:the AA animal models were established by60Co-y rays and cyclophosphamide. The pharmacodynamics and immunity effects of Bushen Yisui Shengxue Method on rats with AA were studied.Experiment on cells:hematopoietic stem/progenitor cells were cultured in methyl cellulose semi-solid medium, and the effect of Bushen Yisui Shengxue Method medicated serum of rats with aplastic anemia on the number of hematopoietic progenitor cells and the effect of Bushen Yisui Shengxue Method medicated on the expression of JAK2, STAT5, GATA-1protein and mRNA in erythroid cells and PU.1mRNA in myeloid cells from molecular biology were evaluated using Western blot, RT-PCR methods.Methods:1. Pharmacodynamics effects of Bushen Yisui Shengxue Method:110SD rats were randomly divided into the normal group (10rats) and the other groups (100rats). Rats in the other group were irradiated disposable body by60Co-y ray at the dose of4.0Gy. Intraperitoneal injection of cyclophosphamide (25mg/kg) for3times every days after the forth day of exposure. Rats were randomly divided into model group, the stanozolol group, yisui group, wenshen group and zishen group according to their weights. All the rats were sacrificed after being gavaged for14days, and the blood samples of femoral artery were detected to measure the levels of RBC, WBC, HGB and PLT. Blood smears were treated by Wright stain to observe blood cell morphology. The bone marrow smears were treated by Wright stain to observe marrow cell morphology. The number of bone marrow cells was obtained by preparing the bone marrow suspension. Serum was separated for cell culture at late stage.2. FCM:The expressions of peripheral blood T lymphocyte subsets CD3, CD4, CD8were detected.3. Radiation immunity Method:cell factors including Fas, FasL and TGF-β1in serum were detected.4. Hematopoietic progenitor cell culture and colony count:normal rat bone marrow mononuclear cells were separated, hematopoietic stem/progenitor cells were cultured in semi-solid methylcellulose medium with serum prepared in the first experiment, then the colonies at the corresponding time were counted.5. Western blot:The levels of JAK2, STAT5, GATA-1protein in erythroid cells were detected using this method.6. RT-PCR:The levels of JAK2, STAT5, GATA-1and GATA-lmRNA in erythroid cells and PU.1mRNA in myeloid cells were detected by this method.7. The experimental data were processed with SPSS17.0.Results:1. The pharmacodynamics effects of Bushen Yisui Shengxue Method on AA rats induced by60Co-γ rays and cyclophosphamide.1.1General state:After the treatment with Bushen Yisui Shengxue Method, rats were more active, glossier in hair and showed better appetite, Conspicuously, they manifested improvement of anemia symptoms than rats in the model group. The improvement of the general state in zishen group was the most evident.1.2Blood:Compared with the normal group, the RBC, WBC, HGB and PLT significantly decreased (P<0.01) in peripheral blood of rats in the model group. The RBC, WBC, HGB and PLT in each treatment group significantly increased (P<0.05, P<0.01) when compared with the model group; Compared with the yisui group and the wenshen group, RBC, WBC, and PLT in zishen group significantly increased (P<0.05, P<0.01).1.3Blood smears:In the model group, the erythrocyte were scattered, and the number of white blood cells reduced, and the degradation cells were common, while in the yisui group, the permeability of the blood cells improved, the number of the nucleated cells increased while the-degradation of the cells decreased.1.4Bone marrow smears:in the model group, fat droplets increased significantly. The nuclear proliferation as well as the number of erythroid, myeloid, megakaryocytic cells reduced, and non-hematopoietic cells appeared while in the treatment groups, fat drops in the number of bone marrow cells and non-hematopoietic cells reduced, while that of nucleated cells increased1.5The number of bone marrow nucleated cell:compared with the model group, they were statistically significant in each treatment group (P<0.01). Compared with the yisui group and the wenshen group,_they significantly increased (P<0.05, P<0.01) in zishen group.2. The effect of Bushen Yisui Shengxue Method on mechanism of immune disorders of AA rats induced by60Co-y rays and cyclophosphamide.2.1The changes of T cell subsets:Compared with the normal group, CD4significantly decreased (P<0.01), CD8significantly increased (P<0.05) and CD4/CD8significantly decreased (P<0.01) in model group. CD4and CD4/CD8significantly increased (P<0.05, P<0.01), CD8significantly decreased (P<0.05) in each treatment group comparing with the model group.2.2Changes of cytokines in serum:Compared with the normal group, the level of Fas, FasL in serum significantly reduced (P<0.01), TGF-β1significantly increased (P<0.01) in model group. The level of Fas and FasL significantly increased (P<0.05, P<0.01), TGF-β1significantly reduced (P<.01) in each treatment group comparing with the model group.3The effect of Bushen Yisui Shengxue Method on myeloid hematopoietic progenitor cell proliferation and differentiation of rat and PU.1expression3.1The number of CFU-GM:Compared with normal control group, the number of CFU-GM in model group significantly decreased (P<0.01); Compared with the model group, the number of CFU-GM in the each treatment group increased significantly (P<0.01).3.2The expression of PU.1mRNA:Compared with normal control group, the expression of PU.1mRNA in model group significantly decreased (P<0.01); Compared with the model group, the expression of PU.1mRNA in each treatment group significantly increased (P<0.01).4The effect of Bushen Yisui Shengxue Method on erythroid hematopoietic progenitor cell proliferation and differentiation and JAK2/STAT5signaling pathway.4.1The number of CFU-E, BFU-E:Compared with normal control group, the number of CFU-E, BFU-E in model group significantly decreased (P<0.01); Compared with the model group, the number of CFU-E, BFU-E in the each treatment group significantly increased (P<0.01), and zishen group significantly increased more than yisui group and the wenshen group (P<0.01)).4.2The expression of JAK2, STAT5, GATA-1protein and mRNA:The expression of JAK2, STAT5, GATA-1protein and mRNA in model group significantly decreased (P<.01); Compared with the model group, the expression of JAK2, STAT5, GATA-1protein and mRNA in each treatment group significantly increased (P<0.05, P<0.01), and zishen group increased more than yisui group and wenshen group (P<0.05, P<0.01).Conclusion:1. Bushen Yisui Shengxue Method plays a significant role in the treatment of AA. It can improve rats’general state, increase the number of peripheral blood cells and nucleated cell of marrow, rectify the abnormal morphology of blood cells and pathological changes of bone marrow. YiSui Shengxue Method is better than Zishen Shengxue Method and Wenshen Shengxue Method on the improvementof hematopoietic functions.2. CD4, CD4/CD8levels were upregulated, and CD8level was downregulated using Bushen Yisui Shengxue Method. Meanwhile, Fas, Fasl, the two anti-apoptotic factors, were increased while TGF-β1, a negative regulator, was decreased. Therefore, we conclude that Bushen Yisui Shengxue Method has a regulatory role in immunological functions in rats. Compared with Zishen Shengxue Method and Wenshen Shengxue Method, YiSui Shengxue Method is apparently superior.3. Bushen Yisui Shengxue Method can increase the number of CFU-GM, promote PU.1mRNA expressions, and then promote hematopoietic progenitor cell into myeloid; Bushen Yisui Shengxue Method can also increase the number of CFU-E, BFU-E, improve the expressions of JAK2, STAT5, GATA-1protein and mRNA, and then promote hematopoietic progenitor cell into erythroid; Zishen Shengxue Method are superior to Yisui Shengxue Method and Wenshen Shengxue Method in terms of promoting blood cell levels.4. Bushen Yisui Shengxue Method has a positive effect on the AA rats as it can improve the hematopoietic and immune function. It can also promote the proliferation and differentiation of hematopoietic progenitor cells. It can also accelerate the erythroid differentiation and maturation by regulating JAK2/STA5signaling pathways. Thus, this study provides evidence for the theory of "kidney storing essence". |
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