| BackgroundImmune thrombocytopenia (ITP) is an autoimmune disorder in which autoreactive T and B cells are activated by platelet autoantigens, resulting in immune-mediated platelets destruction or suppression of platelets production. Clinical features of ITP patients include subcutaneous mucosal bleeding, increase of megakaryocyte and visceral hemorrhage. Tripterygium wilfordii (TW) is a common medicine of traditional Chinese medicine. It has immunosuppressive activity and has been used as an anti-inflammatory agent in traditional Chinese medicine to treat the immunological disease. Indoleamine 2,3-dioxygenase (IDO) is an initial and rate-limiting enzyme which uses tryptophan degradation via the kynurenine pathway to induce or maintain peripheral immune tolerance. Tryptophanyl-tRNA synthetase (TTS) can bind tryptophan with its specific tRNA to form a reservoir of tryptophan for protein synthesis, which can antagonize IDO-mediated immunosuppression by depriving the available tryptophan. In this study, we will explore the influence of TW to the expression of IDO and TTS on T cells subset in ITP patients through observing the clinical efficacy in patients who were treated by TW, and the expression of IDO and TTS on T cells before and after using TW.Objective1.To observe the clinical efficacy of TW treatment in ITP patients.2. To explore the influence of TW in the IDO/TTS mediated tryptophan metabolic pathways through the expression of IDO and TTS on T cells in ITP patients which before and after treated with TW.3.To detect the effect of TW or dexamethasone on tryptophan metabolism pathway through TW or dexamethasone stimulates PBMCs cells of ITP patients.Methods1. Selection of study samples:25 newly diagnosed or relapse ITP patients were recruited and treated with TW with the daily does:1-1.5mg/per·kg.25 healthy subjects were selected as the control group. The heparin anticoagulant blood samples were obtained before treatment and the 5th day after treatment.The patients with diabetes, tuberculosis, hepatits, pepticulcer or hypertension were excluded. All subjects without glucocorticoids or immunosuppressive treatment at least one month before exsanguinate.2. Isolation and cultivation of PBMCs:The heparin anticoagulant venous blood was drawed from healthy people and ITP patients, and PBMCs were isolated by 1.077g/ml Ficoll-Hypaque gradient centrifugation and washed twice under sterile condition. The numbers of cells were adjusted as 5×106/ml and cultivated in 48-well microtiter plates with indicated concentration of TW(2nM) or dexamethasone (luM) for 12 hours.3. The necrosis ratio of PBMCs cells:Cells concentration were adjusted to 5×105ml.5ul PI flourescent dye were added to each tube. The necrosis ratio were tested by flow cytometry after incubation 15min in dark at room temperature.4.Expression of IDO and TTS:105/ml PBMCs were resuspended and incubated with CD4, CD8, IDO and TTS. The expression of IDO and TTS on CD4+ and CD8+ cells were detected with flow cytometry. Message RNA of IDO and TTS on CD4+ and CD8+ cells in PBMCs were measured by RT-PCR method.Result1. The clinical efficiency ratio of ITP patients treated by TW was 80%.2. The expression of IDO on CD4+ and CD8+ cells were lower than healthy control groupe, but the expression of TTS on these two cells were significantly higher than healthy control group (P<0.05).After treatment, the expression of IDO on CD4+and CD8+ in effective group were significantly higher than before treatment, the expression of TTS on CD4+ and CD8+ were significantly lower than before treatment (P<0.05). In ineffective group, the expression of IDO and TTS on CD4+ and CD8+ were no significantly difference between before and after treatment (P>0.05).3. In effective group, compared to control group, the expression of IDO mRNA was decreased, the expression of TTS mRNA was increased (P<0.05). After treatment, the expression of IDO mRNA was higher than before treatment, the expression of TTS mRNA was lower than before treatment (P<0.05)4. The necrosis ratios of control group, patients blank group, solvent group, TW-stimulating group and dexamethasone-stimulating group were 0.70%,0.46%,0.70%,0.94% and 0.79%, respectively. All necrosis ratios were below 1.0%.5. The expression of IDO on CD4+ and CD8+ in patients blank group were lower than those of control group, but the expression of TTS were significantly higher than those of control group (P<0.05).After cocultured with TW, the expression of IDO on CD4+and CD8+ were significantly higher than those of patients blank group, however, the expression of TTS on CD4+ and CD8+ were significantly lower than patients blank control group (P<0.05). Compared to patients blank control, the expression of IDO on CD4+and CD8+ were significantly increased in group of cells were cocultured with dexamethasone, the expression of TTS on CD4+ and CD8+ were significantly decrease (P<0.05).There were no significantly difference between patients blank group and solvent group. The same result were observed between PBMCs cocultured with TW and PBMCs cocultured with dexamethasone (P>0.05)6. Compared to healthy blank control group, the expression of IDO mRNA decreased in patients blank control group, the expression of TTS mRNA increased (P<0.05). After cocultured with TW, the expression of IDO mRNA was higher than patients blank control group, contrastly, the expression of TTS mRNA was lower than patients blank control group (P<0.05). Compared to patients blank control, the expression of IDO mRNA increased after cocultured with dexamethasone, the expression of TTS mRNA had significantly decrease (P< 0.05).There were no significant difference between patients blank group and solvent group. The same result were observed between PBMCs cocultured with TW and PBMCs cocultured with dexamethasone (P>0.05).ConclusionTW can effectively increase the numbers of platelets of the ITP patients. It played a role in treatment of ITP patients via the IDO/TTS-mediated tryptophan metabolic pathway, similar with dexamethasone. Hopefully glucocorticoid can be replaced by TW to treat the ITP patients, avoiding the adverse reactions happened whenever the ITP patients use the glucocorticoid. |
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