Experimental Study On The Protective Effect Of Zishen Yisui Shengxue Recipe On The Bone Marrow Protection Of AA Mice And The Influence Of T-bet/GATA-3

Aplastic Anemia(AA)is a bone marrow failure disease caused by genetic,physical,chemical,biological and various unknown causes.Red blood cells,white blood cells and platelets in AA patients are reduced,and severe anemia,bleeding,and infection may occur.The occurrence of AA is closely related to abnormal immune function.T lymphocytes are divided into two subgroups of CD8+T that mainly mediate cytotoxic immunity and CD4+T cells that regulate immunity,according to the different molecular antigens on the membrane surface.CD4+T/CD8+T ratio imbalance is closely related to immune destruction of AA bone marrow hematopoietic tissue.The original CD4+T cells are also called Th0 cells.Th1 and Th2 are differentiated by Th0 through STAT1 and STAT6 pathways respectively.IL-12 secreted by dendritic cell cells can promote the activation of Th1 cells and secrete them.Type ? cytokines that induce type ? immune response mainly include IFN-y,IL-2,TNF-?,etc,activated Th2 cells mainly secrete type ? cytokines,mainly including IL-10,IL-5,IL-4.With IL-13,the immune pathogenesis of acquired AA is related to the polarization of Th1 cells and the imbalance of Th1/Th2.GATA-3 is a specific transcription factor that can regulate the differentiation of CD4+T cells into Th2 cells.It can specifically initiate the expression of Th2 cytokines;the specific transcription factor T-bet can initiate the polarization process of Th0 cells to Th1 cells,and also Induce T cells and NK cells to start the expression of T-bet transcription factor through IFN-y,and block the polarization of Th2 cells.At present,the immunosuppressant cyclophosphamide is mainly used in clinical treatment,but there are adverse effects such as large side effects,and clinical application of traditional Chinese medicine can often achieve good results.Zishen Zishen Yisui Shengxue Fang is a traditional Chinese medicine compound based on the traditional Chinese medicine theory of "kidney essence,essence filling,marrow shengxue".Our previous animal experiments found that the Zishen Yisui Shengxue method induced AA rats induced by benzene and cyclophosphamide The improvement of bone marrow failure is better than that of warming kidney and marrow,and nourishing kidney and marrow.Therefore,in this study,60Co-? radiation combiued with immune cell injection was used to construct an immune-induced AA mouse model,and the Swiss-Giemsa staining,flow cytometry,HE staining,enzyme-linked immunoassay,immunohistochemistry,Western Blot and RT-PCR method to study the effect of Zishen Yisui Shengxue Fang on the transcription factors T-bet and GATA-3 in AA mice,and provide experimental basis for the theory of "Kidney accumulates essence,essence produces marrow"in Chinese medicine.Objective:60Co-? radiation combined with immune cell injection was used to construct an immune-induced AA mouse model.Stanozolol was used as a control drug to explore the pharmacodynamic effects of Zishen Zishen Yisui Shengxue Fang on immune-induced AA mice.Flow cytometry Detect the changes of CD3,CD4,CD8 and CD4/CD8 in T lymphocytes of immune-induced AA mice.Swiss-Gimsa staining was used to observe the morphological changes of bone marrow cells in immune-induced AA mice.IFN-?,IL-2,TNF-?,IL-5,IL-13,IL-4,IL-10 and other 7 cytokines;immunohistochemical study of immune-induced spleen tissue of AA mice T-bet and GATA-3 protein expression;Western Blot detection of immune-induced changes in T-bet and GATA-3 protein expression in bone marrow of AA mice.Methods:1.Pharmacodynamic experiment:55 clean-grade male inbred BALB/c mice were randomly divided into 10 normal groups.The remaining 45 mice were irradiated with 60Co-? for 5.5 Gy modeling.After the irradiation,DBA/2 mice were quickly completed through the tail vein.Immune cell suspension injection,with an injection volume of 1�106 immune cells each,induces an AA mouse model;mice that survived at week 2 were randomly divided into a model group,Kang Li Long group and Zishen Yisui Shengxue group,administer daily and observe the general state of the mice in each group,weigh and record the changes in body weight every week;at the end of 4 weeks of intragastric administration,remove the eyeballs and take anticoagulant to detect HGB in the peripheral blood of mice in each group,RBC,WBC,PLT changes,take mouse femur to prepare bone marrow suspension,observe and count the number of mouse bone marrow nucleated cells with a hemocytometer under an inverted microscope;prepare blood smear,observe the immune induction by Gimsa staining Blood cell changes in AA mice;bone marrow smears were made and Gimsa staining was used to observe the morphological changes of bone marrow cells in AA mice;spleen tissue sections of AA mice were made and their pathological morphological changes were observed by HE staining.2.Flow cytometry:To detect the changes of CD3,CD4,CD8 and CD4/CD8 in peripheral immune cells of AA mice.3.Enzyme-Iinked immunoassay:To detect the changes of IFN-?,IL-2,TNF-?,IL-5,IL-13,IL-4,IL-10 cytokines in the serum of AA mice.4.Immunohistochemistry:To detect the changes of T-bet and GATA-3 protein expression in the spleen tissue of AA mice.5.Western Blot:To detect the changes of T-bet and GATA-3 protein expression in bone marrow tissues of AA mice.6.RT-PCR:To detect the changes of T-bet mRNA and GATA-3 mRNA expression in bone marrow tissue of AA mice.Results:1.The effect of Zishen Yisui Shengxue Fang on the pharmacodynamics of 60Co-? combined immune induction AA mice:1.1 General state:The treatment groups such as Zishen Yisui Shengxue Fang can significantly improve the general state of AA mice,increase activity and diet,and maintain the body mass of AA mice.1.2 Body weight:Compared with the normal group of mice,the body weight of AA mice in the model group was lower(P<0.01);while the body weight of the Zishen Yisui Shengxue Fang group was significantly higher than that of the model group(P<0.01).1.3 Peripheral blood:Compared with the normal group,the WBC and HGB values of the AA mice in the model group were significantly reduced(P<0.01);the number of RBCs was reduced(P<0.05),and the PLT changes were not statistically significant(P>0.05);compared with the model Compared with the two groups,the HGB values of the Zishen Yisui Shengxue Prescription group were significantly increased(P<0.01),and the WBC and RBC of the Stanozolol group were significantly increased(P<0.01,P<0.05).1.4 Blood smear:Compared with the normal group of mice,the AA mice in the model group had fewer blood cells and more red blood cells with abnormal shapes.The Zishen Yisui Shengxue Fang group had a certain improvement.1.5 Bone marrow smear:The bone marrow of the AA mice in the model group showed more large fat droplets and fibers and other non-hematopoietic tissues,and the number of immature cells in each line of the bone marrow decreased;the non-hematopoietic cells in the bone marrow of the AA mice in the Zishen Yisui Shengxue Fang group were significantly reduced,and the bone marrow declined significantly Get better.1.6 The number of nucleated cells of bone marrow:Compared with the normal group,the number of bone marrow nucleated cells in the model group of AA mice was significantly reduced,and the number of bone marrow nucleated cells in the mice of the Stanozolol and Zishen Yisui Shengxue Fang group increased(P<0.01,P<0.05).1.7 Spleen HE staining:model group immunization induced AA mice with irregular spleen lymph node morphology,red pulp and white pulp borders are unclear,white pulp borders are irregular,and splenic blood sinus and splenic cord are difficult to distinguish clearly;In the treatment group,the lymph nodes of the spleen tended to be normal,the border of the red pulp and white pulp became clear,the blood sinus and the splenic cord were more obvious,and the red and white pulp were more regular than the model group.2.Effect of Zishen Yisui Shengxue Fang on 60Co-y ray combined with immune cell injection to induce immune function in AA mice:2.1 Changes in serum factors:The levels of IFN-?,TNF-?,IL-2,IL-5,and IL-13 in AA mice in the model group were significantly increased,and IL-4 and IL-10 were significantly decreased(P<0.01).Compared with the model group,except for the IL-5 in the Stanozolol group,the levels of IFN-?,TNF-?,IL-2,IL-5,and IL-13 in the Stanozolol group and the Zishen Yisui Shengxue group were reduced(P<0.01,P<0.05);while the levels of IL-4 and IL-10 increased(P<0.01,P<0.05).2.2 Flow cytometry detection:the number of CD8,CD3 and CD4 immune cells of model-type immune AA mice is at a low level(P<0.01),the ratio of CD4/CD8 is reduced(P<0.01);and the model group immunization Compared with type AA mice,CD4 and CD8 were increased in the other three treatment groups(P<0.01,P<0.05),and CD3 was increased in Zishen group and Huangqi group(P<0.05).3.The effect of Zishen Yisui Shengxue Fang on TCO and GATA-3 protein in spleen tissue of AA mice induced by 60Co-y combined immunization:Immunohistochemical results showed that:Compared with the GATA-3 protein expression in the normal group,the GATA-3 protein expression in the spleen tissue of the AA mice in the model group was reduced(P<0.01).Compared with the model group,the GATA-3 protein in the spleen tissue of the mice in each treatment group The expression increased(P<0.01);there was no difference in GATA-3 protein expression between the Stanozolol group and the Zishen Yisui Shengxue group(P>0.05).Compared with the normal group of mice,the expression of T-bet protein in the cytoplasm of the spleen tissue cells of the model group AA mice was strongly positive(P<0.01).Compared with the model group,the T-bet protein expression of each treatment group was significantly reduced(P<0.01);there was no difference in T-bet protein expression between Stanozolol group and Zishen Yisui Shengxue group(P>0.05).4.Effect of Zishen Yisui Shengxue Fang on T-bet,GATA-3 protein and mRNA in bone marrow of AA mice induced by 60Co-? combined immunization:4.1 Western Blot test results show:The expression of GATA-3 protein in bone marrow tissue of AA mice in the model group was lower than that in the normal group(P<0.01).There was no statistically significant change in GATA-3 expression in bone marrow of mice in the Zishen Yisui Shengxue group and Stanozolol group(P>0.05).The expression of T-bet in the bone marrow of AA mice in the model group was higher than that in the normal group(P<0.05),while the expression of T-bet in the bone marrow of AA mice in the Stanozolol group and Zishen Yisuishengxue group was lower than that in the model group(P<0.01,P<0.05).4.2 RT-PCR test results show:The expression of T-bet mRNA in the bone marrow of AA mice in the model group and each treatment group was higher than that in the normal group(P<0.01),and the expression of T-bet mRNA in the bone marrow of AA mice in each treatment group was lower than that in the model group(P<0.01).Compared with the Stanozolol group,the expression of T-bet mRNA in bone marrow of AA mice in the Zishen Yisui Shengxue group decreased(P<0.01);the expression of GATA-3 mRNA in the bone marrow of AA mice in the model group was significantly lower than normal Group(P<0.01).Compared with the model group,the expression of GATA-3 mRNA in the Stanozolol group and the Zishen Yisui Shengxue group increased(P<0.01).Conclusion:1.Zishen Yisui Shengxue Fang can alleviate the general state of AA mice,improve the body weight of mice,increase the number of RBC,WBC,and HGB in the peripheral blood of AA mice,increase the number of bone marrow nucleated cells,and improve AA small Pathological damage of mouse spleen reduces excessive immune damage and protects bone marrow hematopoietic function.2.Zishen Yisui Shengxue Fang can increase the number of CD3 and CD4 cells in the peripheral blood of AA mice,increase the ratio of CD4/CD8,and reduce the IFN-?,IL-2,TNF-?,IL-5 in the peripheral serum of AA mice,IL-13 and other cytokines,increase IL-4,IL-10 and other cytokines,thereby reducing the immune damage caused by abnormal immune activation and maintaining the immune balance and steady state of AA mice.3.Zishen Yisui Shengxue Fang can effectively inhibit the expression of T-bet protein and mRNA in the spleen and bone marrow tissues of AA mice,and increase the expression of GATA-3 protein and mRNA;Zishen Yisui Shengxue Decoction may regulate T-bet/GATA-3 related signal pathways regulate the body's immune balance and delay the process of bone marrow failure in AA mice.