An Experimental Study On The Mechanism Of Yiqi Yangyin Qingre Huayu Recipe To Treat Immune Thrombocytopenia By Regulating Th17/Treg Balance

Objectives: To investigate the effects of Yiqi Yangyin Qingre Huayu Recipe on the expression of Th17 and Treg cells and related cytokines,transcription factors,the thesis establishes the ITP mouse model.The thesis attempts to make a preliminary study on the possible mechanism and the most effective dosage for ITP,so as to provide the theoretical basis for the clinic ITP treatment.Methods: 1.establishing and evaluating the ITP mouse model: 1.Mice were randomly divided into normal group(n = 20)and ITP model group(n = 110).Using passive immunization against platelet surface glycoprotein GPIIb / IIIa establishes the ITP mouse model.Specifically,200 μl of phosphate buffer solution containing 2 μg of rat anti-mouse CD41 monoclonal antibody(MWReg30 monoclonal antibody)is intraperitoneally injected into the model group mice every day,causing a continuous Thrombocytopenia.The normal group is injected with the same amount of saline.The models are evaluated by detecting the peripheral platelet count,the total number of bone marrow megakaryocytes and the number of plate megakaryocytopoiesis on the 1st,3rd,5th and 7th days from modeling.2.Pharmacodynamic research of Yiqi Yangyin Qingre Huayu Recipe on ITP model: On the 8th day,the ITP model group is randomly divided into model group again,TCM(Yiqi Yangyin Qingre Huayu Fang)low,medium and high-dosage-groups,Western medicine control group(prednisone acetate control)(n = 20).Each treatment group is given drug intervention for 2 weeks and the general status of mice in each group is observed,and the changes of peripheral platelet count,the number of megakaryocyte and the number of megakaryocytopoiesis in the plate are detected.All indicators are used to evaluate the efficacy of the recipe.3.Research on the mechanism of Yiqi Yangyin Qingre Huayu Recipe on the ITP model: In order to explore the effect mechanism of Yiqi Yangyin Qingre Huayu recipe,flow cytometry(FCM)is used to detect the changes of the ratio of Th17 、Treg cells and IL-17、IL-21、IL-10 in spleen of mice in each group.q PCR and Western blot are used to detect the expression of RORγt,Foxp3 and STAT3 molecules and protein expression levels.Results: 1.The establishment of the ITP model: Compared with the normal group,the number of peripheral blood platelets in the ITP model group is significantly reduced by half and the total number of the bone marrow megakaryocytes increased,while Plate megakaryocyte production decreased(P <0.05),after injection of the CD41 monoclonal antibody(MWReg30 monoclonal antibody),in line with the platelet characteristics of ITP model mice.2.The change of blood platelet count: Compared with the model group,all blood platelets increased in different amount,of a statistical significance.Among them,the medium dosage group and the blood platelet of Western medicine control group increased most obviously(P<0.01),without statistical significance between the two groups.3.The change of the form of megakaryocyte: Compared with the model group,the number of non-producing megakaryocyte and puerile megakaryocyte of mice in all treatment groups decreased to some extent.And the dosage group boasts a significant effectiveness.4.The change of Th17,Treg cell number: Compared with the normal group,the cell number of Th17 in the model group increased(P<0.05);the cell number of Treg decreased(P<0.05).Compared with the model group,the cell number of Th17 in dosage group and Western medicine control group(P<0.01,P<0.05)decreased while the cell number of Treg increased significantly(P<0.01,P<0.05).5.The changes of Th17,Treg and related factors in spleen: Th17 cells,STAT3,RORγt,IL-17 and IL-21 in ITP model mice are significantly higher than those in normal controls(P <0.01)Compared with the model control group,the expression of Th17 cells and related transcription factors,cytokines(P <0.01)and Th17/Treg ratio increases significantly(P <0.01),and increases the expression of Treg cells and related transcription factors and cytokines(P <0.01).Compared with the model group,the relative expression quantity of RORγt and STAT3 gene and albumen obviously decreased(P <0.01),while the relative expression quantity of Fox P3 gene and albumen increased(P <0.01).6.The change of peripheral platelet count of Th17 and Treg cell factor: Compared with the normal group,the peripheral platelet count of Th17 and the related cell factors of IL-17 and IL-21 increased significantly(P<0.01)and the Treg related cell factors of IL-10 decreased significantly(P<0.01).Compared with the model group,the expression of IL-17 and IL-21 in dosage group and the Western medicine group decreased significantly(P < 0.01),while the expression of IL-10 increased significantly(P<0.01).Conclusion: Yiqi Yangyin qingre huayu Recipe has the effect of increasing platelet and promoting the differentiation of megakaryocytes significantly.Its possible mechanisms include: inhibiting the expression of Th17 transcription factor RORγt and STAT3 in vivo,increasing the expression of Foxp3 in Treg cells,thereby decreasing the production of Th17 and proinflammatory cytokine IL-17,IL-21,increasing the expression of Treg and STAT3 number of inflammatory factors IL-10,adjusting the Th17/Treg ratio,and promoting the recovery of immune homeostasis;to further regulate the maturation of megakaryocytes and a variety of immune cells differentiation and function,and ultimately affect the generation and destruction of platelets,ITP has a positive protective effect.