| ObjectiveImmune Thrombocytopenic Purpura (ITP) is an autounmune disease characterized by a low platelet count and mucocutaneous bleeding, and is a common hemorrhagic disease of blood system in clinics, it be long to "blood syndrome',"muscle apostaxis',"macula syndrome" etc, in Traditional Chinese Medicine (TCM). Its definite pathogenic mechanism is unknown, And with the development to the depth, except for humoral immunity, cellular immunity may be one of the pathogenesises of ITP, it is one of the research hotspot about ITP in the world at present. Imbalances of positive and negative costimuLatory signals in immunocytes may leads to autoimmune diseases.High excepression of Positive costimulatory molecules may lead to overactivation of T lymphocytes and abnormal cytokine secretion. samely, overactivation of B lymphocytes may bring more antiplatelet antibodies, which mainly destroy platelet. While negative costimulatory molecules may regulate function of T lymphocytes such as the activation, proliferation and secreting cytokines. Thl and Th2lymphocytes mediated cellular and humoral immunity respectively, they can stimulate themself Proliferation by autocrine cytokines Down-Regulate cytokines Secretion each other and mutual conversion in some condition, so as to maintain the normal immune function. The abnormal secretion of cytokine can cause many immune disease.In this study we try to investigate the specific mechanism of Positive and negative costimulatory signals cause ITP through clinical and animal researches. In order to study the function of T cells changed by costimulatory signals (CD80,CD86,PD-1) and Thl and Th2lymphocytes cytokines (IL-4,IFN-γ) which cause ITP, treatment with ShuFeng LiangXue BuShen therapy. techniques of monoclonal antibody, Flow cytometry (FCM)and enzyme linked immunosorbent assay (ELISA) be used in the experiments.on this basis, we will research the effects of serum containing ShuFeng LiangXue BuShen Therapy on costimulatory molecules and Th1and Th2lymphocytes-secreting cytokines (IL-4,IFN-γ) from ITP patients and animal model. Try to evaluate the effect of ShuFeng LiangXue BuShen Therapy, Evaluate the effect of ShuFeng LiangXue BuShen Therapy,and explore the possible immune regulation mechanism of ShuFeng LiangXue BuShen Therapy on CITP. The study may provides the new theory basis and practice for treating CITP through chinese medical science.Methods1Animal Experiment StudyTwenty BALB/C mices were randolnly divided into normal group, model group according to the counts of peripheral platelet.ITP mouse model were made by injected intraperitoneally1:4guinea pig-antimouse platelet serum(GP-APS)at a dose of100μL/20g at the first, third, fifth, seventh, ninth, eleventh, thirteenth day, totally7days. Detect the peripheral platelet counts, bone marrow megakaryocyte counts and morphology classiflcation, observe the pathological changes in thymus and spleen to in order to judge whether the model was successful or not to be established after two weeks. In the animal experiment,85BALB/C mice with SPF, ordered were randomly divided into six groups (10mice for normalg roup;15mice per group respectively for Blank group, prednisone group and high-dosage e/midst-dosage/low-dosage groups of ShuFeng LiangXue BuShen Therapy). The latter five groups were improved with yang yufei methods and were injected intraperitoneally APS (Dilution with1:4proportion)100μ L Per time at every other day in13days and the CITP mice models were made. From the beginning of sixth day, these groups were administrated intragastrieally with a single dose Per day of experiment substance (administration quantity0.2mL/10g,10times for clinical dosage) in the next nine days, which were gavaged respectively with saline prednisone (0.5mg/mL, decuple of clinical dose),high-dosage (12.75g/Kg-1. d-1), midst-dosage(8.5g/Kg-1. d-1),low-dosage (5.68g/Kg-1. d-1) of the ShuFeng LiangXue BuShen Therapy. At the same time, the general state of health, the adverse reaction and the death amount were observed. Afterwards all mice were sacrificed and the needed specimens were taken in order to observe the change of Platelet counts,CD80,CD86,PD-1,IFN-γ and IL-4. 2Clinical Study39CITP (chronic immune thrombocytopenic purpura, CITP) Patients were randomly divided to three groups (13Patients administrated with the ShuFeng LiangXue BuShen Therapy as A group;13Patients with the same decoction And Prednisone as B group;13Patients with Prednisone as C group. To more explain, B group administration way referred to A and C group.C group was constituted of Prednisone. The Prednisone administration quantity was given lmg·kg·d-1Per day. The abovet reatment was4weeks as one time of therapy. After3times of therapy, a series of observation were made as before.Results1Animal Experiment Study results1.1The experiment successfully made the CITP mouse model according to injecting intraperitoneally the APS(dilution proportion1:4)with100μL Per time resectively at the first, third, fifth, seventh, ninth, eleventh, thirteenth day.The duration time of the CITP model was72hours and the result was the same as that of correlative data.1.2With regard to Platelet counts of the CITP mice, the high-dosage and midst-dosage could all improve Platelet counts and was considered significant compared with the control group (P<0.05).However, there was no significant difference among the high-dosage and midst-dosage decoction. there was no significant difference the high-dosage,Prednisone groups and model group (P>0.05)1.3With regard to the expression of PD-1of the CITP mice, the amount of the high-dosage,midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered and was considered significant compared with the control group (P<0.05), There was no significant difference among the four groups (P>0.05)1.4With regard to the expression of CD80,CD86of the CITP mice, the amount of the high-dosage,midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered (P<0.05), There was no significant difference among the four groups (P>0.05)1.5With regard to the expression of IL-4of the CITP mice, the amount of the high-dosage,midst-dosage and low-dosage decoction groups and Prednisone groups could all be lowered and IFN-γ be improved (P<0.05). IL-4was lowered more significantly in the high-dosage group and the Prednisone group(P<0.01). 2Clinical Experiment Study results2.1With regard to the therapeutic effect, there was no significant difference among the three groups (P>0.05), But the ShuFeng LiangXue BuShen Therapy group and the ShuFeng LiangXue BuShen Therapy combined with Prednisone group have a effective trend than Prednisone group. With regard to the markedly effective, the ShuFeng LiangXue BuShen Therapy combined with Prednisone group is superior to the Prednisone group.2.2With regard to the integration of hemorrhage symptoms, Three groups were improved significantly after therapy(P<0.05), the ShuFeng LiangXue BuShen Therapy group and the ShuFeng LiangXue BuShen Therapy combined with Prednisone group are better than the Prednisone group(P<0.05).2.3With regard to the dosage of prednisone, after three period, the ShuFeng LiangXue BuShen Therapy combined with Prednisone group and the Prednisone group significantly reduced (P<0.01), the ShuFeng LiangXue BuShen Therapy combined with Prednisone group more significantly reduced than the Prednisone group (p<0.01)2.4With regard to Platelet counts, that of the ShuFeng LiangXue BuShen Therapy group was raised obviously after therapy and there was significant difference compared with that before therapy(P<0.05). But the result of the ShuFeng LiangXue BuShen Therapy group was similar to that of the ShuFeng LiangXue BuShen Therapy combined with Prednisone group.2.5Aspects of the expression of CD80,CD86of the CITP patients, three groups could all be lowered and was considered significant compared with the control group(P<0.05). There was no significant difference among the three groups (P>0.05)2.6The ShuFeng LiangXue BuShen Therapy group could lowered the expression of PD-1(P<0.05), There was no significant difference among the three groups.2.7Post-therapy, the Prednisone group and The ShuFeng LiangXue BuShen Therapy group significantly higher than those in control group(P<0.05), the ShuFeng LiangXue BuShen Therapy combined with the Prednisone group was significantly lower than the Prednisone group and the ShuFeng LiangXue BuShen Therapy group(P<0.05).2.8The ShuFeng LiangXue BuShen Therapy group could improved the expression of INF-γ (P<0.01), There was no significant difference among the three groups. Pre-therapy, the three groups significantly lower than those in control group. Post-therapy, the Prednisone group higher than those in control group (P<0.05).ConclusionThe result showed that the disturbance of costimulatory molecules (pathway) and T lymphocytes subsets imbalance exist in CITP, the function of Thl cell is weak, but the function of Th2cell prevail in immune dysfunction of CITP. All the analysis shows that over-expression of costimulating molecules CD80, CD86and PD-1, and abnormal expression of related cytokines IL-4,IFN-γ be one of the pathogenesises of CITP. The ShuFeng LiangXue BuShen Therapy has good clinical effect on CITP. one of its mechanism is that it can adjust the imbalance of costimulatory molecules (pathway), upregulate the expression of negative costimulatory molecules (pathway) and inhibit the over-expression of positive costimulatory molecules, down regulate immune responses mediated by T lymphocytes; The other mechanism is regulating the imbalance of Thl/Th2cells, decrease the expression level of IL-4and increase the level of IFN-γ, inhibit over-autoimmune reaction, Reduce the destruction of platelets, a good clinical effect was achieved. Therefore, by correcting the abnormal expression of CD80, CD86,PD-1and Th1/Th2to regulate immune balance is one of the treatment strategy, which has important clinical significance, It worth further research.
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