| BackgroundPrimary immune thrombocytopenia (ITP) is an acquired autoimmune disease, which is related with platelet destruction and/or platelet reduction caused by the autoreactive T cell and B cell activated by platelet antigen. At present, glucocorticoids is the main treatment drug for adult ITP with70%-80%effective rate. Indoleamine2,3-dioxygenase, IDO is the limiting velocity enzymes of tryptophan along the kynurenine pathway. Tryptophan is the essential amino acid for cell proliferation and survival. T cells are sensitive to tryptophan depletion. T cells proliferation is prevented under the low tryptophan concentration conditon. Tryptophanyl-tRNA synthease, TTS can make the tryptophan bind to its specific tRNA to form the Trp-tRNA complex, which constitutes the tryptophan library of protein synthesis and annotates the inhibition of the IDO to T cell proliferation. In this research, we predicte the sensitivity of glucocorticoid therapy in ITP patients by detecting the change of tryptophan metabolic pathway before and after glucocorticoid treating and comparing the change rule of the valid and invalid treatment groups.Objective1. Observation of the clinical efficacy of high-dose dexamethasone treatment in ITP patients.2. Discussion of the role of the IDO mediated tryptophan metabolic pathways in ITP patients.3. To analyze change rules of valid and invalid treatment groups according to the expression and activity of IDO and TTS, tryptophan and kynurenine concentration before and after the treatment.Methods 1. Selection of study samples:Twenty ITP patients were investigated, all patients met the diagnosis criteria of ITP. Diabetes, hypertension, peptic ulcer, hepatitis, tuberculosis and other acute or chronic infectious disease were excluded. All selected patients without glucocorticoids or immunosuppressive treatment at least one month before exsanguinate. Twenty healthy people were recruited as control group.2. Isolation of peripheral blood mononuclear cells (PBMCs):The venous blood is drawed from healthy people and ITP patients into EDTA anticoagulant tubes, PBMCs were isolated by Ficoll-Hypaque density centrifugation and washed twice under sterile condition.3. Detection of T lymphocyte proliferation function:Lymphocytes were adjusted to5×105/ml concentration with cell culture medium, and then were cultured in96-well plates. Blank group, negative control group and experimental group were set up and three wells were repeated each group.1-methy-tryptophan (1-MT)was added to each experimental group, and not added to blank group and negative control group. Cells were cultured for48hours, then CCK-8was added to each well and continued to culture for4hours. Finally, the OD value of each well was read with a microplate reader, and the stimulation index was calculated.4. Measurement of plasma tryptophan and kynurenine concentration:Plasma samples were separated by centrifugation from the ITP patients and health control. Tryptophan and kynurenine concentration were measured by Liquid chromatography tandem mass spectrometry (LC-MS/MS).5. IDO and TTS expression in CD4+、CD8+T cells were measured by flow cytometry, and IDO and TTS mRNA expression were measured by RT-PCR.Result1. The effective rate of ITP patients treated by high dose dexamethasone was80%.2. Plasma tryptophan and kynurenine level in ITP patients:Before treatment, the level of plasma tryptophan and kynurenine in ITP patients were both higher than that in healthy controls[(8.776±1.398vs.4.456±0.747umol/L, P<0.05),(0.132±0.034vs.0.106±0.039umol/L, P<0.05)]. The kynurenine to tryptophan (Kyn/Trp) ratio(0.018±0.001vs.0.013±0.001umol/L, P<0.05) was significantly higher in the ITP patients than that in the healthy controls; after high-dose dexamethasone treatment, effective treatment group tryptophan concentration(3.047±0.209um01/L vs.8.776±1.398umol/L, P<0.05)was significantly lower than before treatment, kynurenine concentration(0.175±0.008umol/L vs.0.132±0.034umol/L,P<0.05)was significantly higher than before treatment,Kyn/Trp ratio was significantly higher(0.025±0.003um01/L vs.0.018±0.00lumol/L,P<0.05):ineffective group,the concentrat ion of tryptophan(7.736±1.630um01/L vs.8.380±0.841umol/L,P)O.05)and kynurenine(0.091±0.042umol/L vs.0.113±0.073umol/L, P>0.05)there were no significant difference compared with before treatment, Kyn/Try(0.016±0.006umol/L vs.0.017±0.007umol/L,P<0.05)had no significant di fference.3.Inhibition ratio of T cell proliferation:After treatment,T cell proliferation was significantly lower than that before treatment(0.7430±0.0076vs.1.3803±0.0257,P<0.05).High dose dexamethasone can induce high expression of IDO,which inhibited T cell proliferation.T cell proliferation was significantly promoted by IDO inhibitor1-MT(1.2827±0.0168vs.0.7430±0.0076,P<0.05).4.MFI of IDO(456.67±51.59vs220.33±51.79,P<0.01)and TTS(1430.34±113.29vs774.67±67.17P<0.05)were significantly increased in PBMCs in ITP patients compared with healthy people.But MFI of IDO was significantly decreased in CD4+T cells(183.35±39.01vs508.67±47.01P<0.01)and CD8’T cells(141.00±16.09vs241.00±57.86P<0.05)in ITP patients,MFI of TTS was significantly increased in CD4+T cells(4319.37±120.25vs1812.44±93.73) and CD8’T cells(5267.67±436.51vs1308.00±118.51,P<0.01)in ITP patients compared.with healthy people:In effective treatment group after high-dose dexamethasone treatment, MFI of IDO was significantly increased in PBMCs(517.00±69.78vs456.67±51.59),CD4+T cells(935.34±61.87vs183.35±39.01)and CD8+T cells(828.12±63.15vs141.00±16.09)in ITP patients compared with before treatment group,P<0.05:MFI of TTS was significantly decreased in PBMCs(830.51±79.38vs1430.34±113.29),CD4+T cells(1127.46±188.32vs4319.37±120.25)and CD8+T cells(1710.33±191.92vs5267.67±436.51)in ITP patients compared with before treatment group,P<0.05.In ineffective group,there were no significant difference between after and before treatment for MFI of IDO and TTS in PBMCs[(393.37±57.25vs363.45±42.78),(1255.75±112.29vs1323.25±108.34)P<0.05],CD4+T cells[(152.07±23.45vs172.12±20.33).(4309±167.28vs4211±87.64),P<0.05]and CD8+T cells[(132.35±11.57vs126.41±19.60),(5249.00±422.95vs4987.75±344.84),P>0.05].5. Before treatment, the relative amount of IDO mRNA (0.457±0.003vs0.040±0.001) and TTS mRNA (0.624±0.052vs0.040±0.001) in ITP patients are significantly higher than that in healthy people, P<0.05. In effective treatment group with high-dose dexamethasone, the relative amount of IDO mRNAhas no differences between before and after treatment (0.423±0.004vs0.457±0.003). The relative amount of TTS mRNA was significantly decreased compared with before treatment group (0.0235±0.002vs0.624±0.052), P<0.05.ConclusionHigh-dose dexamethasone can significantly increase the platelet count for ITP patients in short-term. In ITP patients, the expression of IDO and TTS are abnormal, IDO/TTS-mediated tryptophan metabolism pathway play a critical role in the pathogenesis of ITP. High-dose dexamethasone treatment in ITP patients can induce high expression of IDO, downregulate the expression of TTS, inhibit the proliferation of T cells. Fruthermore, the IDO and TTS expression levels analysis can forecast the sensitivity to hormone therapy in ITP patients. Finally, we hope to find a new therapy of ITP via the IDO and TTS expression mechanism study on T cells... |
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