The Mechanism Of High Mobility Group Protein 1(HMGB1)in Children With Chronic Immune Thrombocytopenia

Immune thrombocytopenia(ITP)is one of the most common hemorrhagic diseases of children;immune mediated damage of the platelets and megakaryocytes leads to a significant decrease in the number of peripheral blood platelets and results in related clinical manifestations.As a special group,the children have low immunity and are susceptible to infection with bacteria and viruses,so that 60%to 70%of those with immune thrombocytopenia are acute,and the remission rate is high.But many children who suffer from the ITP gradually turn into chronic,and requir routine blood test intermittently and take hormones and so on continuously.However,the mechanism underline chronic ITP remains unclear.The high mobility group box-1 protein(HMGB1),a non-histone nucleoprotein,can be used as a damage-associated molecular pattern(PAMP)or alarmins to stimulate the innate immune system itself,or as part of the cytokine complex.After the activation of HMGB1,NLRP3 can be triggered to bind to the corresponding receptor(Tolls,RAGE,etc),then its molecular conformation changes,inducing the oligomerization of NLRP3 molecules whichbinds to ASC protein.The procaspase-1,Caspase-1 cleave IL-1? and 1L-18 precursors into active IL-1?and 1L-18 that will modulate various immune and inflammatory responses.The interaction between HMGB1 and NLRP3 inflammasom in autoimmune diseases and some chronic diseases has been reported.In the research,we examinated the level of HMGB1 and cytokines IL-17?IL-10 in peripheral blood between children with chronic ITP and normal control groups,and the correlation between the HMGB1,IL-17,IL-10 and clinical indicators was explored.Then the expressions of TLR2 and TLR4 in peripheral blood CD 14+CD16+monocyte subsets and CD1c dendritic cells of children with chronic ITP and of normal control groups,and the expressions of NRLP3 and cytokines Il-1? and il-18 in the CD 14+monocytes and CDlc dendritic cells were examined.Then we cultured of macrophage from adult peripheral blood,and examined interaction between HMGB1 and NRLP3 in macropahges.We found the proper young mice model of ITP.Last the therapeutic potential of HMGB1 inhibitors were assessed in rat model of chronic ITP.This experiment is divided into four chapter,the research contents are as follows:PART ?:Correlation study of plasma HMGB1 level elevation and clinical indices in children with chronic immune thrombocytopeniaObjective:Understanding the expression of HMGB1,Cytokines IL-17,IL-10 in serum of chronic ITP of children and the relationship between HMG1 and the clinical data of chronic ITP of children.Method:1.Collected and analyzed clinical data of chronic ITP of children and normal control groups.2.Detecting the expression of peripheral blood HMGB1,IL-17,IL-10 of normal children and the chronic ITP of children by ELISA.3.Analyzed the correlation between HMGB1,IL-17,IL-10 and clinical data.Result:1.The age of first diagnosis of chronic ITP of children was obviously younger than that of the normal control group(P<0.05).The mucocutaneous bleeding symptoms in chronic ITP of children were more serious than those in normal control group.(P<0.05).For chronic ITP of children,the platelet values before treatment and after treatment were significantly lower than those of normal control group.Children with chronic ITP expressed high percentage of Th cells than normal control group and their Ts cells significantly reduced than those of normal control group.At the same time,Th/Ts cells of chronic ITP of children compared with normal control group were significantly reduced,the difference was statistically significant(P<0.05).2.The Expression of HMGB1 and IL-17 in chronic ITP of children increased,while IL-10 in plasma of chronic ITP of children decreased,(P<0.05).3.HMGB1 was positively correlated with IL-17,and negatively correlated with IL-10.Plasma HMGB1 was negatively correlated with IL-10 in normal control group(P<0.05).4.In this study,the serum HMGB1 protein in chronic ITP of children was positively correlated with platelet values before treatment,there were no significant correlation for the remaining results.PART ?:The role of inflammatory NLRP3 and pathway-related components in children with chronic immune thrombocytopeniaObjective:Comparing the expression of TLR2 and TLR4 in CD 14+CD16+monocyte subsets and CD1c dendritic cells of chronic ITP of children and that of normal control group,and the expression of NRLP3 and cell factor L-1pand IL-18 after CD14+monocytes,CD1c+dendritic cells were separated.Exploring the effect of HMGB1 on expression of NLRP?L-1 and IL-18 human in PBMC derived macrophages.Method:1.Using flow cytometry antibody to mark chronic ITP of children and normal control group peripheral blood,and detecting TLR2 and TLR4 expression in CD 14+CD16+monocyte subsets and CD1c+dendritic cells.2.Sorting CD 14+monocytes and CD1c+dendritic cells with flow cytometry,and then using micro-RNA kit,the expression of NRLP3 and cytokine L-1?and IL-18 in monocytes and dendritic cells were detected by RT-PCR.3.Ex vivo cultured PBMC derived macrophage will be stimulated with various doses of HMGB1(0ng/mL?50ng/mL?100ng/mL?200ng/mL)and ATP in vitro,then the level of IL-1?and IL-18 in supernatant will be assessed by ELISA and expression of NRLP3 in macrophage will be assessed by Western blotting.Result:1.For chronic ITP of children,the positive rate of CD 14++CD 16-monocyte decreased and the positive rate of CD14+CD16++monocyte increased,there were significant differences(P<0.05),the positive rate of TLR2 on the surface of CD14++CD16+CD 16+monocyte decreased significantly comparing with the normal control group,the positive rate of TLR2 on the surface of CD14++CD16+monocyte increased significantly comparing with the control group,and the difference was statistically significant(P<0.05).However,the positive rate of TLR4 in the monocyte subgroup significantly increased comparing with the normal control group,and the difference was significant(P<0.05).The expression of CD1c+dendritic cells in the two groups showed no obvious abnormality.The expression of TLR2 on the surface of CD1c+ dendritic cells in the chronic ITP group decreased,while the expression rate of TLR4 increased,and the difference was significant(P<0.05).2.For chronic ITP of children,the relative expression levels of NLRP3,IL-1?and IL-18 in CD 14+monocytes and CD1c+dendritic cells all significantly increased,the differences were statistically significant(P<0.05).3.In the presence of different concentrations of ATP and rHMGB1 stimulation,as the concentration increases,the expression of NLRP3,IL-1?and IL-18 increased gradually,but the expressions of protein increased in different degree.PART ?:Comparison of different chronic young mice model of ITP constructed with different antiplatelet antibodiesObjective:Comparing the effects of two different antiplatelet antibodies in the construction of chronic young mice model of ITP and select the appropriate oung mice model of ITP.Method:1.building two chronic young mice model of ITP with two different antibodies:guinea pigs anti-platelet antibody and rats Anti-CD41 antibody.2.Comparing the general conditions,platelets,biochemical and hepatosplenic indices of the two models.Result:1.The general situation of animal models of normal control group were better than that of animal model group.2.The platelet values of two ITP animal model groups were significantly lower than that of respective control groups,but the subsequent increase of platelets in the antiplatelet antibody group of guinea pigs was significant.There was no significant difference between the two models in biochemical and hepatic splenic index.PART ?:The effect of HMGB1 and its inhibitor on chronic young mice model of ITPObjective:In order to validate the in vivo experiment;Studying the effects of HMGB1 and its inhibitor in animal modelsMethod:1.The animal models were divided into control group,ITP group,Anti-HMGB1 group and Liquiritigenin group.The expressions of platelet,HMGB1,IL-10 and IL-17 were detected in peripheral blood of chronic young mice model of ITP.2.The mononuclear macrophages and dendritic cells in the spleen cells were selected by magnetic beads.the expressions of mononuclear macrophages,dendritic cells TLR2 and TLR4 in each spleen group were detected,the gene expressions of NLRP3,IL-1?,IL-18 were detected.3.With WB and ELISA,the expressions of NLRP3,IL-1?and IL-18 of bone marrow macrophages in each young mice group were examined.Result:1.Anti-HMGB1 neutralization antibody group,prednisone acetate group and liquiritigenin group could significantly improve the platelet count in the young mouse model,with significant difference(P<0.05).The expression of HMGB1 protein in the ITP model group,the prednisone acetate group and liquiritigenin group increased comparing with that of the normal control group.The ITP model group,Anti-HMGB1 neutralizing antibody group and liquiritigenin group,all of them could reduce the expression of HMGB1 protein in the young mice model of ITP,and the differences were significant(P<0.05).The expression levels of IL-17 in each model group and the normal control group increased,and the difference was significant(P<0.05).Anti-HMGB1 neutralization antibody group,prednisone acetate group and liquiritigenin groupc all could decrease the expression of i1-17 protein in the young mice model of ITP,and the differences were significant(P<0.05).The expression levels of IL-10 protein in each model group decreased significantly compared with that in the normal control group,with significant difference(P<0.05).The expression of IL-10 protein in the ITP model could be increased in the anti-hmgbl neutralizing antibody group,prednisone acetate group and liquiritigenin group.The difference was significant(P<0.05).It was also found that HMGB1 was positively correlated with IL-17 in the ITP model group.2.The expression of TLR2 and TLR4 on the surface of CD11b+mononuclear macrophages in each model group was higher than that in the control group.The expression of TLR2 in the ITP model group was significantly higher than that in the Anti-HMGB1 neutralized antibody group,the prednisone acetate group and liquiritigenin group,and the difference was statistically significant(P<0.05).The TLR2 and TLR4 expression levels of CD11c+dendritic cell surface in each model group were higher than that in the control group,The TLR2 expression in ITP model group was reduced by Anti-HMGB1 neutralization antibody group,prednisone acetate group and liquiritigenin group,the difference was significant(P<0.05).There was significant difference in TLR2 expression between the prednisone acetate group and the anti-hmgbl neutralization group(P<0.05).Anti-HMGB1 neutralization antibody group and liquiritigenin group could reduce the expression of TLR4 in ITP model group,with significant difference(P<0.05).There was significant difference between the prednisolone acetate group and the anti-hmgbl neutralizing antibody group(P<0.05).The relative expression of NLRP3 and IL-1?in each model group was significantly higher than that in the control group,with significant difference(P<0.05).Anti-HMGB 1 neutralization antibody group,prednisone acetate group and liquiritigenin group could significantly reduce the relative expression of NLRP3,IL-1 ?and IL-18 in ITP model,and the difference was significant(P<0.01).The relative expression of IL-18 in ITP model group,Anti-HMGB 1 neutralizing antibody group and liquiritigenin group increased obviously comparing with normal control group,there were significant differences(P<0.05);The relative expression of IL-18 in the anti-hmgbl neutralized antibody group and the prednisone acetate group was lower than that of liquiritigenin group,and the difference was significant(P<0.05).3.The expressions of NLRP3,IL-1? and IL-18 in each model group increased with the increase of rHMGB1 concentration,but the rise level was different.The expression of NLRP3,IL-1? and IL-18 in the ITP model reduced in different degree in Anti-HMGB 1 neutralizing antibody group,prednisone acetate group and liquiritigenin group.Conclusion:1.The expression of HMGB1 increased in chronic ITP of children,and related to the expression of cytokine IL-17,IL-10,HMGB1 may be used as an early warning alarm of childhood chronic ITP,and invoved in its disease development.2.The expression of TLR2 and TLR4 in monocytes and dendritic cells in children with chronic ITP were changed,and the expression of NLRP3,IL-1? and IL-18 in macrophages induced by monocytes,dendritic cells and peripheral blood was increased too,HMGB1 may affect the occurrence and development of chronic childhood ITP through NLRP3,TLR2/TLR4 pathway.3.The establishment of the model of chronic ITP by using Anti-CD41 antibody could maintain low platelet and ensure the construction of ITP model of chronic young mouses.4.It may be that anti-HMGB1 neutralizing antibodies,prednisone acetate and glycyrrhizin can improve the condition of the animal model of chronic ITP and protect the body function.The protective effect of HMGB1 antagonist on chronic ITP in children provides basic research for clinical trials,and HMGB1 may be a therapeutic target for chronic ITP of children.