Research On The Therapy Of Shenguiyiqiwenyang Decoction And The Correlated Immune And Transcriptional Factor Level In CITP Patients

Thrombocytopenic purpura was named Idiopathic thrombocytopenic purpura(ITP) at the beginning. In 2007 the International Working Group(IWG) recommended to change its name to Immune thrombocytopenia(ITP). The word "Immune", was to emphasize that the cause of the disease was immune, In addition, some patients have no or have light hemorrhage, so the word "purpura" should be abandoned, named "immune thrombocytopenia". However, its abbreviation retains "ITP".Based on his many years temporary experience, professor Ma Rou, my tutor, proposed a series of therapeutic regimen that committed not only to the disease but also to the patients symptoms. Professor Ma Rou subtyped CITP into two groups: spleen-kidney yang deficiency and dual deficiency of the lung-spleen. He used the method of tonifying qi and warming yang, and set up Shenguiyiqiwenyang decoction to therapy CITP and obtained good effects.The Objective of this study was to discuss tutor's therapeutic ways for CITP and verify its clinical effect, and to discuss the immune disorderly conditions of CITP's patients without glucocorticoid interfering. Together with T-bet/GATA-3,Foxp3 mRNA transcribe molecular biology study, further expounded the therapeutic mechanisms of Shenguiyiqiwenyang decoction.The results was that in treatment group(28 cases),4 cases(14.29%) were excellence, 3 cases(10.74%) were good,5 cases(17.86%) were improvement,16cases(57.14%) were invalid. That is,12cases were effective and total effective rate was 42.86%.The platelet counts was 23.02±13.69 before treatment in treatment group, and after treatment was 46.18±50.47.And there was significant difference(P<0.05)between before and after treatment. It proveed that Shenguiyiqiwenyang formula had significant curative effect for ITP.We used flow cytometry of BECKMAN COULTER to detect CD3+T lymphocyte, CD3+CD4+T lymphocyte, CD3+CD8+T lymphocyte, CD4+/CD8+proportion, CD3+CD25+ active T lymphocyte, CD3+DR+ activate T lymphocyte, CD4+CD25+ Treg, CD3+CD16CD56+ NKT cell, CD3-CD16CD56+NK cell, CD19+B lymphocyte, CD19+CD5+ B lymphocyte and the change of Thl/Th2/ThO between before and after treatment.Compared to the healthy group, they increased significantly(P<0.05) with CD3+CD19- Total T lymphocyte, CD3+CD8+T lymphocyte, NKT cell, CD3DR+ active T lymphocyte cell counts, and decreased significantly(P<0.05) with CD4+/CD8+ proportion, CD19+CD5+/CD19+ cell proportion, CD4+CD25+Treg cell counts of before treatment in treatment group. And after treatment,they decreased significantly(P<0.05) with CD3+CD19- Total T lymphocyte, NKT cell, CD3DR+ active T lymphocyte cell counts, and increased very significantly(P<0.01) with CD19+CD5+/CD19+ cell proportion, CD4+CD25+Treg cell counts. There was no significant difference of CD3+CD8+T lymphocyte,CD4+/CD8+ proportion and Th1,Th2,Th0 between before and after treatment in treatment group.In effect group, the result showed that:CD4+CD25+Treg cell proportion increased significantly(P<0.05) after treatment. In inefficacy group, the study showed that: after treatment, CD3+CD19- Total T lymphocyte proportion decreased significantly(P<0.05); CD4+CD25+Treg cell counts CD19+CD5+/CD19+ cell proportion increased significantly(P<0.05). Compared to before treatment there was no significant difference with CD3+CD8+ CTL cell and CD3DR+ cell after treatment, but compared to healthy control group, there was significant difference(P<0.05).In all, in CITP patient organism, we can deduce that the low CD4+CD25+Treg cell and CD19+CD5+B cell expressed low level and had immunologic function defect and CD3+CD19- Total T lymphocyte CD3+CD8+ CTL cell NKT cell and CD3DR+ cell had hyperfunction, All that were important link in pathogenic mechanism. The target improved after treatment, which evident further that factor had important effect in pathogenesy. It's worth noting that in inefficacy group CD3+CD8+ CTL and CD3DR+ cell expressed high level, and had not descend with therapy interfering in. What demonstrateed:CTL cell and CD3DR+ active cell were important factor in CITP disease course.By multivariantly gradual regressive analysis, there were no linear correlations among every T,B lymphocyte subset and peripheral blood cells.Using Real-Time-RT-PCR, we detected transcription factor T-bet, GATA-3, Foxp3 mRNA transcriptional level in before treatment and after treatment, and discussed associativity among transcription factor's transcriptional level, Th1, Th2, CD4+CD25+ cell and peripheral blood cells.The results showed that:in the treatment group, GATA-3mRNA transcriptional level was notably higher than in healthy control group(P<0.01). After treatment, the transcriptional level decreased significantly(P<0.01). There had no statistical significant difference comparing after treatment in treatment group with healthy control group. T-bet mRNA transcriptional level had no statistical significance comparing before and after treatment in treatment group with healthy control group. The transcriptional level of Foxp3mRNA had no statistical significance comparing before treatment in treatment group with healthy control group, but it decreased in after treatment. After treatment, the transcriptional level of Foxp3mRNA was lower than healthy control group(P<0.05).Before treatment, GATA-3mRNA transcriptional level was strengthened, which concerned with autoregulation reaction in body's gene deck. The high expressing of GATA-3mRNA made Th balance drift toward Th2 superiority, that profited to immune tolerance and patient's condition relieving. With therapeutic reaction and Treg accommodate cell's raising, GATA-3 mRNA transcriptional level descended. It consistent with the disease variation processed. Foxp3mRNA transcriptional level descended after treatment, this indicated that peripheral Treg accommodate cell population increased and it's function strengthened with the disease remission and then adjusted genetic transcription level by negative feedback, making it's level express down. It proved that although Foxp3 was the Treg's specific regulating factor, which accepted Treg's negative feedback regulation.Using the SPSS 17.0, we analyzed the double variable relations among the 3 factors of transcription factors of T-bet,GATA-3,Foxp3 of before and after treatment. Before treatment, T-bet and GATA-3 was negative correlation ship(P<0.05), but dependability was not very intimate(coefficient correlation<0.5); with Foxp3 was in obviously negative correlation(P<0.01, coefficient correlation>0.5). After treatment, Foxp3 and GATA-3 was in positive coefficient correlation(P<0.05), coefficient correlation was 0.427.It proved that GATA-3 feedback high expression will restrain relatively T-bet transcription in CITP chronic phase; Foxp3 transcription in high level will restrain T-bet function too, which was in line with the morbid state of CITP and in line with functions of every transcription factor. In paracmasis, the transcription of Foxp3 and GATA-3 degrade concomitantly, which proved that along with the regaining of the periphery blood Treg adjusting cell and the improving of the immune transient status, the level of transcription about Foxp3 and GATA-3 was be degraded by degenerative feedback mechanism.By multiple linear regression analysis, we further investigated the correlation among transcription factor T-bet,GATA-3,Foxp3 expression and peripheral blood cells. The result showed, after treatment GATA-3 and PBC was negative correlation(P<0.05), and the equation was PLT=90.299-64.086gata. It descripted that the ascensus of platelet in peripheral blood had dependability with the descending of GATA-3 transcriptional level.To summarize above all, through the study, we can further verified that Shenguishenqiwenyang decoction is effective and safe for CITP patiets, and its prostecdtive efficacy (further observing 6 months after platelet ascensus) is stable. The research of mechanism detected that the proportion of CD4+CD25+Treg cells increased notably(P<0.05) in utility groups(excellence+good). In ineffective group, CD3+CD19-Total T lymphocyte proportion decreased after treatment, CD4+CD25+ Treg cell counts CD19+CD5+/CD19+ cell proportion increased, and there was significant difference(P<0.05); CD3+CD8+ CTL cell and CD3DR+ cell keep still high expression, they descend rarely after treatment(P<0.05). The study indicated that it was the possible remission mechanism that CD4+CD25+Treg cell and CD19+CD5+/CD19+ cell proportion increased or their function strengthened, Meanwhile,CD3+CD8+ CTL cell NKT cell and CD3DR+ active T cell functions restrained. It's worth noting that in ineffective group CD3+CD8+ CTL and CD3DR+ cell expressed with high level, and they did not descend with therapy, so this demonstrate that CTL cells and CD3DR+ active cells were the important factor in CITP disease course.