| Idiopathic thrombocytopenic purpura (ITP) is an acquired organ specific autoimmune disease. The mechanisms and etiology of ITP are recognized to be an autoimmune condition, involving platelet destruction induced by autoantibody and reduced platelet production caused by over phagocytose and destruction of mononuclear phagocytic system. This is the etiopathogenisis of humoral-mediated immunity. However, the pathogenesis of ITP can not be explained completely only by this theory. Recently, many studies suggested that not only humoral immunity but also cell-mediated immunity were included in the pathogenesis of ITP. Previous studies indicated that function disorder of T help1(Thl) cell will cause severe inflammation reaction and multi autoimmune disorders. Thl cell mediates cytoimmunity by secreting interferon-γ (INF-y). However, autoimmune diseases were also found in interferon-y (INF-y) or interferon-γ receptor (INF-γR) defect mice in some researches, which indicated that there are some other Th subgroups participating in the development of autoimmune disorders besides Th1. Th17is a neotype of responsiveness CD4+T cell subgroup, which takes his specificity effect by secreting cytokines such as interleukin-17(IL-17) etc. Nowadays, more and more evidence indicated that Th17cell plays important role in the pathogenesis of many autoimmune diseases.Sarcandra glabra is a kind of plant belongs to Chloranthaceae. It functions as promoting blood flow, heat-clearing, detoxicatingim and immunoregulation. It has been reported recently that Sarcandra glabra has immunosuppressive role on macrophage, T and B lymphocytes. Therefore, Sarcandra glabra has therapeutic effect on some autoimmune diseases, such as rheumatoid arthritis, psoriasis, ITP and chronic non-specificity ulcerative colitis etc. However, the mechanism of Sarcandra glabra in the treatment of ITP and the molecular target still need further investigation.The study was divided into two parts. In the first part, Th17cell population and the expressions of related cytokines in peripheral blood were detected to study the variation and the role of Thl7in the development of ITP. In the second part, we investigated the contribution of Sarcandra glabra to Th17cell population and the expressions of related cell differentiation, regulation and transcription factor in vivo by constructed ITP mouse model.Objective:To investigate the role and the significance of Th17in the pathogenesis of ITP, we examined Th17cell proportion, the expression of related cytokines, regulation and transcription factor in peripheral blood from the patients with ITP. Furthermore, to explore the mechanism of Sarcandra glabra in the treatment of ITP, we investigated the contribution of Sarcandra glabra to Th17cell population and the expressions of related cell differentiation, regulation and transcription factor in vivo by constructed ITP mouse model. This study will illuminate the mechanism of Sarcandra glabra in the treatment of ITP and other autoimmune diseases, and will provide new rational target molecule treatment for ITP and other autoimmune diseases.Method:1.30cases of ITP patients with a low platelet count (among5×109/L-78×109/L, median:30×109/L)and10healthy people (platelet count among143×109/L-243×109/L, median:174×109/L) as normal controls were selected for investigation. ITP patients were eliminated of possible secondary causes, for example:infection, virus hepatitis, diabetes, cardiovascular disease, fetation and connective tissue disease according to FTP diagnostic criteria.Peripheral blood samples from ITP patients and normal controls were examined for Thl7cell proportion by flow cytometry (FCM). The expressions of IL-17, IL-23,IL-21, IL-6and TGF-β1in hematoplasma were detected by ELISA; The mRNA expression levels of IL-17and ROR γ t in peripheral blood mononuclear cell (PBMCs) from patients with ITP and normal countrols were measured by RT-PCR technique, and the expressions of pSTAT3and ROR γ t proteins by Western-blot.2:Randomly,60BALB/c mouse were divided into normal control, ITP, positive control group treated with prednisone acetate and low, medium and high dosage Sarcandra giabra treated groups. The ITP mouse model was constructed by injection guinea pig-antimouse platelet serum(GP-APS) into abdominal cavity of mouse. Then the role of Sarcandra glabra on platelet, leukocyte and erythrocyte in preperipheral blood of ITP mice were observed. Furthermore, the Th17cell proportion in peripheral blood, the expressions of related cytokines such as IL-17and IL-23in serum, the mRNA expression levels of IL-17and ROR Y t, and the expressions of pSTAT3and ROR γ t protein in spleen tissues were measured by FCM, ELISA, RT-PCR and Western-blot techniques separately to investigate the role of Sarcandra glabra on ITP mouse model.Results:First part:①The Th17cell proportion (0.60±0.93%) in peripheral blood from patients with ITP was greatly increased when compared with normal control group (0.11±0.07%, P<0.05).②The expressions of IL-17(28.44±26.90pg/ml), IL-23(0.35±0.18ng/ml), IL-6(190.9±134.5pg/ml) and TGF-01(1.19±0.81ng/ml) in hematoplasma in ITP patients were all significantly higher than that of the normal control group (7.61±9.38pg/ml,0.19±0.06ng/ml,86.3±22.6pg/ml,0.65±0.32ng/ml, all P<0.01); There were no significant differences between ITP (0.92±0.43ng/ml) and control group (0.84±0.40ng/ml) as to the expression of IL-21(P>0.05).③The mRNA expression levels of IL-17and ROR Y t in PBMCs from patients with ITP were much higher than that of normal controls (P＜0.05).④The protein expressions of pSTAT3(0.30±0.07) and ROR γ t (1.04±0.31) in PBMCs of ITP patients were greatly increased when compared with control (0.17±0.05,0.45±0.17, P<0.05). Second part:①The platelet count in preperipheral blood of ITP mice were decreased dramatically when compared with normal control (P<0.01). However, the platelet count of the Sarcandra glabra treated groups were increased greatly when compared with ITP group (P＜0.01), especially in high dosage group.②The populations of Thl7cells in spleen tissues of ITP mouse were increased obviously than normal control group (P＜0.01) while the populations decreased greatly in medium and high dosage Sarcandra glabra treated groups when compared with ITP group (P<0.05).③The expressions of IL-17and IL-23in hematoplasma of ITP mouse were significantly higher than that of the normal control groups (IL-17:P<0.05, IL-23:P<0.01); The expression of IL-17decreased greatly in medium and high dosage Sarcandra glabra treated groups when compared with ITP group (P<0.05). Nevertheless, there were no significant difference between all treated groups and control group as to the expression of IL-23(P>0.05).④The mRNA expression levels of IL-17and ROR γ t in spleen tissues of ITP mouse were greatly increased when compared with normal control groups (P<0.01); When compared with ITP group, the mRNA expression levels of IL-17decreased greatly in medium and high dosage Sarcandra glabra treated groups (P<0.01) while ROR Y t mRNA level decreased dramatically in all Sarcandra glabra treated groups (all P＜0.01).⑤The protein expressions of pSTAT3and ROR γ t in spleen tissues of TIP mouse were greatly increased when compared with normal control group (all P＜0.01). However, the expressions were greatly decreased when the mouse were treated with Sarcandra glabra of medium and high dosage (P<0.01, P＜0.05).Conclusion:In summary, we found:①The Th17cell subgroup may play some role in the occurrence and development of ITP, which may participate in the pathogenesis of ITP by increasing Th17cell proportion and altering the expression levels of Th17related cytokines, regulation and transcription factors.②the GP-APS was prepared and ITP mouse model was established successfully.③Sarcandra glabra can improve the condition of mouse with ITP. The herb increased the food-intake and activities of IPT mouse, decreased bleed and increased the platelet count in preperipheral blood of ITP mice, which indicated that the herb may have treatment role on ITP since it can suppress differentiation and proliferate of Thl7cell, down regulating the expression of IL-17by blocking the expression of Th17cell related differentiation, regulation and transcription factor.④To regulate Th17cells of patients with ITP may be a new strategy for treatment of ITP. |
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