Hemopoietic Method Of Benzene And Invigorating The Kidney And Marrow Ctx Induced Aa Rats T - Bet/ifn - Gamma Signaling Pathways Impact Experimental Research

Aplastic Anemia (AA) is a harmful disease with characters including decrease in number and dysfunction of hematopoietic stem cells, damage of hematopoietic microenvironment and decrease of completed blood. Anemia, infection and hemorrhage are the main performance of its clinical syndrome. In recent years, it is found that the disease can be relieved by the mean of Traditional Chinese Medicine, which has drawn great attention of researchers from both China and abroad. When AA happens, the immune system is dysfunctional, which manifests as overactivity of T cells. IFN-y is mainly secreted by Thl cell in CD4+cell and Tcl cells in CD8+cells. During AA, the level of IFN-y is significantly increased, which means secretion function of Thl and Tel are both enhanced.The transcription factors T-bet and GATA-3can adjust the differentiation of ThO cells into Thl and Th2cells, and they are also the key factors for interconversion of Thl/Th2cells. In environment of IL-12secreted by mDCs, T-bet accelerates ThO transformed into Thl by activating factor1(STAT1) with signal transduction and transcription, which results in increase of Type Ⅰ lymphokine such as IFN-y, TNF-a. However, in environment of IL-4, the GATA-3is high expressed by the activating factor6(STAT6). GATA-3accelerates the ThO transformed into Th2that secrete Type II lymphokine such as IL-4. IL-5、IL-13. The IFN-γ genes are activated and endogenous IFN-y production is induced by the ectopic expression of T-bet in AA. IFN-γ can induce CD8+T cells, NK cells and mature B cells to express T-bet that can accelerate differentiated Th2reversely transformed into Thl by means of inhibiting GATA-3expression and blocking the interaction between GATA-3and Th2cytokines. As a consequence. Thl (secrete IFN-γ)/Th2(secrete IL-4) are out of balance and T-bet/GATA-3are abnormally expressed. The functional mechanisms of Bushen Yisui Shengxue Method on IFN-γ/T-bet signal access of rats with AA have been studied by means of flow cytometry method (FCM), radioimmunoassay (RIA) method, immunohistochemistry, Western blot and RT-PCR method on the basis of AA animal models by benzene and cyclophosphamide (CTX). The study provides abundant experimental data for the research on AA’kidney storing essence’theory.Objective:Experiments on animals:the AA animal models were established by benzene and cyclophosphamide (CTX). The pharmacodynamics effects of Bushen Yisui Shengxue Method on rats with AA were studied compared with stanozolol group. CD3, CD4, CD8, CD4/CD8of T lymphatic system were detected with FCM, cell factors including IFN-y, TNF-a, IL-2, IL-4. IL-5and IL-10in serum were detected with radiation immunity method, IFN-γ, T-bet and GATA-3protein in spleen were detected with immunohistochemistry, IFN-γ. T-bet and GATA-3protein expression were detected with Western blot while IFN-γ, T-bet and GATA-3mRNA of bone marrow were detected with RT-PCR.Methods:1.Pharmacodynamics effects of Bushen Yisui Shengxue Method:85clean level SD rats were randomly divided into normal group(10rats) and other group(75rats). Rats in the other group were subcutaneously injected with benzene (1ml/kg) once per two days for7weeks, and with CTX (25mg/kg)3times every2days from sixth Monday for two weeks to establish AA model. Gavage began in the third week. Before that, rats were randomly divided into model group, the stanozolol group, yisui group, wenshen group and zishen group according to weights, each group with16rats. The states and weights were recorded during the experiment. All the rats were killed in the seventh weekend and the blood samples of femoral artery were detected to obtain the level 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. HE was used to observe the pathological changes in spleen.2.FCM and Radiation immunity Method:The level of CD3, CD4, CD8, CD4/CD8of T cell system were detected with the former method while that of cell factors including IFN-γ, TNF-a, IL-2, IL-4, IL-5and IL-10in serum with the latter method.3.Immunohistochemistry:The level of IFN-γ, T-bet and GATA-3protein in spleen of AA rats were detected with this method.4.Western-blot:The level of IFN-γ, T-bet and GATA-3protein in bone marrow of AA rats were detected with this method.5.RT-PCR:The level of IFN-γ, T-bet and GATA-3mRNA in bone marrow of AA rats were detected with this method.6.The experimental data were statistically processed with SPSS17.0.Results:1.The pharmacodynamics effects of Bushen Yisui Shengxue Method on AA rats induced by benzene and CTXAfter the treatment with Bushen Yisui Shengxue Method, all the general state, weight, blood, bone marrow, marrow nucleated cell count and spleen tissue morphology had different levels of improvement.1.1General state:rats were more active, heavier, glossy in hair and stronger in digestion system than rats in the model group with no treatment after treated with Bushen Yisui Shengxue Method.1.2Weight:After2weeks, the weight of the rats of model group decreased obviously. There were statistic differences between normal group and model group (P<0.01). Compared with model group, the weight of the rats in treatment groups increased obviously over time (P<=0.01). Rats in zishen group and yisui group were better than that in wenshen group.1.3Blood:Compared with the normal group, the RBC, WBC, HGB significantly decreased (P<=0.01) in peripheral blood of rats in model group. The RBC, WBC, HGB in each treatment group significantly increased (P<0.05), while the PLT irritability in zishen group significantly increased (P<0.01) compared with the model group.1.4Blood smears:In model group, the erythrocyte were scattered, the number of white blood cells reduced, and the degradation cells were common, while in yisui group, the permeability of the blood cells became better, nucleated cells increased and degradation of the cells decreased.1.5Bone marrow smears:in model group, fat droplets increased significantly, nuclear proliferation reduced, the erythroid, myeloid, megakaryocytic cells reduced, and non-hematopoietic cells appeared while in treatment groups, fat drops in bone marrow cells reduced, nucleated cells increased, and non-hematopoietic cells decreased.1.6The number of bone marrow nucleated cell:compared with model group, they were statistically different in each treatment groups (P<=0.01).1.7HE staining:In model group, lymph nodes shrinked smaller in spleen, the border was unclear between red pulp and white pulp. After treatment, lymph nodes of spleen tended to be normal and the border became clear between red pulp and white pulp.2The effect of Bushen Yisui Shengxue Method on mechanism of immune disorders of AA rats induced by benzene and CTX.2.1FCM:In model group, the number of CD4significantly reduced (P<0.01), CD8significantly increased (P<=0.01), and the ratio of CD4/CD8were significant inverted (P<=0.01) compared with the normal group. The zishen group was significantly better than the yisui group and wenshen group (P<=0.01) compared with the model group.2.2The blood RIA indicators:Compared with the normal group, IFN-γ, IL-2, TNF-α increased obviously (P<=0.01), Compared with the model group, the level of IFN-γ. IL-2and TNF-a in treatment group all decreased obviously (P<=0.01). Compared with the normal group, IL-4, IL-10and IL-5contents decreased obviously (P<0.01) in model group. Compared with the model group, the level of IL-4, IL-5and IL-10in treatment group all increased obviously (P<0.05, P<0.01).3.The effect of Bushen Yisui Shengxue Method on IFN-y, T-bet and GATA-3protein expression in spleen of AA rats induced by benzene and CTX.3.1Results of immunohistochemistry:A small amount of IFN-y and T-bet protein were expressed in spleen cells in normal group; Compared with the normal group, IFN-y and T-bet protein of model group were more strongly expressed (P<0.01); Compared with the model group, the expression of IFN-y and T-bet protein significantly decreased in spleen cells in treatment groups (P<0.01), and zishen group and yisui group were significantly less than the wenshen group (P<0.01). A great amount of GATA-3protein were expressed in spleen cells in normal group; Compared with the normal group, GATA-3protein of model group decreased; Compared with the model group, the expression of I GATA-3protein significantly increased in spleen cells in treatment groups (P<0.01), and zishen group and yisui group significantly increased more than the wenshen group (P<0.01).4.The effect of Bushen Yisui Shengxue Method on IFN-y, T-bet and GATA-3mRNA and protein expression in bone marrow of AA rats induced by benzene and CTX.4.1Results of Western blot:Compared with the normal group, the expression of IFN-y and T-bet protein in model group significantly increased (P<0.01); Compared with the model group, the expression of IFN-y and T-bet protein in treatment groups significantly decreased (P<0.01), while the zishen group was significantly better than the yisui group and wenshen group (P<0.01). Compared with the normal group, the expression of GATA-3protein in model group significantly decreased (P<0.01); and GATA-3protein in treatment group were with a decreased trend.4.2Results of RT-PCR:Compared with the normal group, the expression of IFN-y and T-bet mRNA in model group significantly increased (P<0.01); Compared with the model group, the expression of IFN-y and T-bet mRNA in treatment groups significantly decreased (P<0.01), while the zishen group was significantly better than the yisui group and wenshen group (P<0.01). Compared with the normal group, the expression of GATA-3mRNA in model group significantly decreased (P<0.05); and GATA-3protein in treatment group had a decreased trend.Conclusion:1.BuShen YiSui Shengxue Method play a significant role in the treatment of AA. It can improve general state, gain weight, relieve the pathological changes of spleen tissue and bone marrow, and increase number of peripheral blood cells and nucleated cell of marrow. As concluded in the study, YiSui Shengxue Method is better than Zishen Shengxue Method and Wenshen Shengxue Method on improvement for general state and immune system of AA rats.2. Through the regulation of AA immune function, BuShenYiSui Shengxue Method can reduce CD8+cell, increase CD4+cell, reduce CD4/CD8ratio, and then slow the progression of immune disorders in AA. BuShenYiSui Shengxue Method can reduce the level of negative regulatory factors in Serum, and increase the level of positive regulatory factors in Serum. BuShenYiSui Shengxue Method is able to adjust hematopoiesis and immune system. Zishen Shengxue Method and Yisui Method are better than Wenshen Shengxue Method.3.The BuShenYiSui Shengxue Method can slow down the progression of AA by means of regulating the IFN-γ. T-bet and GATA-3mRNA and protein expression of spleen tissue and bone marrow, as analyzed. It takes effect on the signal transduction access, reduce the secretion of IFN-y and T-bet, make a balance between T-bet and GATA-3, adjust immune system and reduce the immunologic injury, and then slow the progression of AA. These research enrich Chinese medicine of kidney storing the essence theory connotation, and provide new evidence of the clinical treatment for AA.