Expression Of Interleukin-23 In Patients Of Primary Immune Thrombocytopenia And Its Clinical Significance

ObjectiveTo investigate the mRNA and protein expression of interleukin-23(IL-23)in patients with primary immune thrombocytopenia(ITP)and preliminarily elucidate the regulatory role of IL-23 in Thl7 cells from ITP patients.We will also explore whether dexamethasone(DXM)therapy for ITP patients could regulate the expression of IL-23 and the possible underlying mechanisms.MethodThe peripheral blood samples which were anticoagulated by EDTA from ITP patients and healthy control group were collected respectively.The plasmas of both group were collected after centrifugation.Then the peripheral blood mononuclear cells(PBMCs)were separated from the rest cell specimens through Ficoll density gradient centrifugation.The isolated PBMC and plasma were divided into several EP tubes in order to achieve the requirements for different experiments in the future.The total RNA was:.extracted from PBMCs of both group and reverse transcribed into cDNA according to the manufacturer's instructions.The real-time fluorescent quantitative polymerase chain reaction(RT-PCR)using TaqMan-MGB probe method was used to detect the relative mRNA expressions of IL-23p19 subunit,IL-12 p40 subunit,IL-12p35 subunit,IL-23 receptor(IL-23R),IL-12 receptor ?i(IL-12Rp1)and the IL-12 receptor ?2(IL-12Rp2).And the RT-PCR using SYBR Green method was used to analyze the relative mRNA expressions of IL-17A,IL-17 and RAR related orphan receptor C(RORC).The enzyme linked immunosorbent assay(ELISA)method was applied to evaluate the protein level of IL-23 and IL-17.The protein level of IL-23R in both group were obtained using the western blot method.The percentages of Th17 cells were measured by the flow cytometry instrument.The automatic blood cell counter was used to determine the platelet count(PLT)of each subject enrolled in the study.The correlations between the IL-23 level in plasma of ITP patients and the percentage of Thl7 cells,IL-17 protein level in ITP,the PLT in patient's peripheral blood were analyzed using Spearman or Pearson methods.The PBMCs isolated from ITP patients were stimulated with 20ng/ml rhIL-23,10ng/ml rhIL-23 and RPMI-1640 medium respectively,along with 10?g/ml phytohemagglutinin for 24 hours in cell culture incubator.Then the protein levels of IL-6,IL-17,IL-21 in supernatant and the mRNA level of RORC,signal transducers and activators of transcriptions(STATs)were detected by ELISA and RT-PCR methods.In addition,the phosphorylation-STAT3 level was also analyzed.We followed up 15 newly diagnosed ITP patients,to evaluate the protein level changes of IL-23 and IL-17 after taking therapy with 40mg/day DXM for consecutive four days.We used different concentrations of DXM to treat the PBMCs of these ITP patients in vitro aiming to detect the expression changes of IL-23,p38 MAPK and phosphorylation-p38 MAPK.Furthermore,we incubated the PBMCs from ITP patients with anisomycin which is the specific agonist for p38 MAPK and aimed to explore the impact of DXM on IL-23 expression.ResultsCompared to healthy controls,the IL-23p19 subunit,p40 subunit,IL-23R and IL-23R?1 mRNA expressions were elevated in ITP patients significantly.The mRNA expressions of IL-12 p35 subunit and IL-12RP2 were also increased but the differences were not statistically significant.The IL-17A,IL-17F and RORC which is a Thl7 specific regulatory transcription factor were rised in ITP patients.In the plasma of ITP patients,we found that both IL-23 and IL-17 were elevated.When compared with the patients with PLT more than 20�109/L,the increasing of IL-23 and IL-17 in patients with PLT less than 20�109/L was more significant.The results from western blot and flow cytometer indicated that the IL-23R and the percentage of Thl7 cells were expanded.The expression of IL-23 in ITP patients' plasma was positively correlated with the percentage of Th17 cells and IL-17 protein level,meanwhile it was negatively correlated with the PTL in patients.In vitro experiments showed that compared with the RPMI-1640 medium,the 20ng/ml and 10ng/ml rhIL-23 stimulation group had an obvious increase in protein level of IL-6,IL-17,IL-21 and in mRNA level of RORC.In addition,the elevation of IL-17,IL-21 and RORC were more significant in the group stimulated by 20ng/ml rhIL-23.After treated with rhIL-23,the STATs mRNA showed an increasing trend.Among these,the STAT3 had the maximum amplitude increase,meanwhile the p-STAT3 also rised.Through following up 15 newly diagnosed ITP patients,we found the IL-23 and IL-17 in plasma were both decreased significantly after high-does DXM therapy.Using different concentrations of DXM to treat the PBMCs from those patients revealed that the IL-23,p38 MAPK and p-p38 MAPK were declined in an approximate does-dependent manner in vitro.Furthermore,after incubation with anisomycin,the inhibitory effect of DXM on IL-23 expression was significantly weakened.ConclusionThe present study found that the mRNA and protein level of IL-23 were increased in ITP patients,which was associated with disease severity.The results suggested the IL-23 may play a vital role in the pathogenesis and progress of ITP.And the overexpressed IL-23 may mediate the activation of STAT3 expression and the activation of the RORC to stimulate the proliferation and differentiation of Thl7 cells,promoting the development of the disease.The results also suggested that high-dose DXM may inhibit the expression of IL-23 through the p38 MAPK signal pathway and showed an approximate does-dependent manner.