| 1.ObjectiveImmune thrombotopenic purpura is an autoimmune (ITP) disorder characterized by a low platelet count and mucocutaneous bleeding.A number of features suggest this destruction is immune-mediated,But the exact mechanism of the immune dysfunction, however, is generally not known. More than 70% patients response to treatment with glucocorticoids and splenectomy,but there are about 30% of refractory patients (refractory immune thrombocytopenic purpura, RITP) are defined as those in whom treatment with standard-dose corticosteroids and splenectomy fails and who further therapy because of unsafe platelet counts or clinical bleeding.In this study,ITP mouse model were made by injected exogenous antibodies intraperitoneally guinea pig-antimouse platelet serum (GP-APS) and treated with YiSui granules.Observe the effect of Yisui granules on ITP mouse peripheral blood platelets,bone marrow megakaryocytes,cytokines,spleen and thymus apoptosis index and the apoptotic gene expression,regulatory T cells in the spleen and other factors.Trying to explore Yisui granules effective mechanism on the regulation of immune network in ITP.2.MethodsPreparation of guinea pig anti-Balb/C mice anti-platelet serum (GP-APS) and red blood cell adsorption and reserved.Twenty Balb/c mice were randomly divided into normal group,model group according to the counts of Peripheral platelet.ITP mouse model were made by injected intraperitoneally 1:4 guinea pig-antimouse platelet serum (GP-APS) at a dose of 100μl/20g every other day.Detect the peripheral platelet counts,bone marrow megakaryocyte counts and morphology classification,observe the pathological changes in thymus and spleen to after two weeks in order to judge whether the model was successful or not.After ITP mouse model was verified successful,seventy Balb/C mice were were randomly divided into normal group,model group,Prednisolone acetate group,ShengXue XiaoBan capsule and Yisui granules high,medium and low-dosege groups.The 8th day establishing the model,each group were intragastric administration at a dose of 0.2ml/10g·d-1 volume with normal saline and every treated medicine daily for additional one week respectively.Evaluate the therapeutic effect of YiSui granules according to detecting the peripheral platelet counts,blood coagulation time, bone marrow megakaryocyte counts and morphology classificatio,platelet-associated antibodies,the immune organ index and the pathological changes.After the therapeutic effect of YiSui granule had been determined,the ITP mouse model were established as the methods above-mentioned and treated with high dosage of YiSui granules.Two weeks latter,The levels of immune closely related to Thl/Th2 cytokines (IFN-y and IL-4) and TGF-β1 in peripheral blood serum were detected by ELISA.The spleen and thymus were embedded in paraffin and sliced 4μm,lymphocyte apoptosis index were detected by TUNEL,Bcl-2 and Bax protein expression were detected by immunohistochemistry.The Treg cells were detected by flow cytometry (FCM) in spleen single cell suspension.Explore the possible immune regulation mechanism of Yisui granule on ITP according to detect the factors above-mentioned.3.Results3.1 ITP mouse modelGP-APS is still positive when dilution 1:128,indicating guinea pig has generated a specific antibody in serum, that is guinea pig anti-Balb/C mice platelet antibodies. Compared with the normal group,the peripheral blood platelets counts of ITP mouse model decreased significantly,theer was statistically significant (P<0.05,P<0.01).The peripheral platelet counts was lowest after APS injected 24 hours. The counts of megakaryocytes in bone marrow of model group mice increased significantly,but the megakaryocyte which can produce platelet decreased (P< 0.05,P< 0.01).The blood coagulation time of model group was prolonged than normal group (P< 0.001), spleen index of model group was significantly higher than normal group.But the thymus index has no statistically significant as compared with normal group (P< 0.001).3.2 Experimental study the therapeutic effect of Yisui granuls on ITP mouse modelCompare with model group,the platelet counts of each treatment groups were higher than those of model group,there was statistically significant difference (P<0.01,P<0.001).The counts of platelet in Yisui granules high-dosege group and prednisone group recovered obviously.Compare with model group, immature megakaryocyte proliferation in bone marrow of Yisui granules high-dosage group was inhibited, megakaryocyte which can produce platelet increased,there was statistically significant difference (P<0.01,P< 0.001); The levels of PAIgG of ITP model group were increased significantly than normal group.The levels of PAIgG decreased significantly after treatment with Yisui granules,it was significantly lower than model group (P< 0.001),there was no statistically significant different compared with the normal group.The spleen index of each treatment groups decreased than model group,the Yisui granuls high-dosage group decreased obviously,there was statistically significant difference (P< 0.001)3.3 Experimental study the immunological network regulation mechanism of Yisui granuls on ITP mouse modelCompared with normal group,the levels of IFN-γincreased in model group,the levels IL-4 decreased in model group,the ratio of IFN-γ/IL-4 increased. The balance between Thl and Th2 was brouken out. ITP has cellular immune function dereased and T cell subsets drift, manifestation of a type-1 polarized immune response.The proportion of Treg cells and the levels of TGF-β1 was significantly lower in model group as compared to normal group. Compared to normal group,spleen lymphocyte apoptosis decreased,Bcl-2 protein expression increased,Bax protein expression decreased in model group.But the thymus lymphocyte apoptosis and Bcl-2,Bax protein expression were no statistically significant difference in each group.After treatment,compared with model group,the levels of IFN-γand the ratio of IFN-γ/IL-4 decreased,the levels of IL-4 increased in YiSui Granules group. The proportion of Treg cells and the levels of TGF-β1 was higher in YiSui granules group as compared to model group.However,the proportion of Treg cells was lower in YiSui granules group than that in normal group, there is no significant difference of the levels of TGF-β1 in YiSui granules group as compared to normal group.Compared to model group,spleen lymphocyte apoptosis increased,Bcl-2 protein expression decreased,Bax protein expression increased in YiSui granules group.4.Conclusions4.1 Successfully preparation GP-APS and established ITP mouse models. The peripheral platelet counts decreased in ITP mouse model,and bone marrow megakaryocytes increased and ysmaturity.It's basically consistent with the clinical manifestations of ITP4.2Yisui granules can increase the peripheral blood platelet counts of ITP mice effectively and stimulate the bone marrow megakaryocytes differentiation to maturation.Its curative effect was equal to prednisone4.3 ITP has cellular immune function dereased and T cell subsets drift, manifestation of a type-1 (INF-γ) polarized immune response.The morbidity of ITP may be related to Th1/Th2 cell cytokine imbalance, Treg cells and TGF-β1 was reduced, lymphocyte apoptosis and Bcl-2, Bax protein expression abnormality. Yisui granules may be by regulating the Th1/Th2 cell balance and promote Treg cell proliferation and TGF-β1 secretion, and can regulate lymphocyte apoptosis and apoptosis-regulating genes Bcl-2, Bax abnormal expression, thus regulating the immune abnormalities in ITP,reduceing the platelet-associated antibodies, reducing platelet destruction, increased platelet count, promoting bone marrow megakaryocyte differentiation to mature. surface to achieve therapeutic effectreduce the difference between platelet antibody to reduce platelet destruction, increased platelet count, the promotion of bone marrow megakaryocyte differentiation to mature to achieve therapeutic effect. |
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