| Background:Primary immune thrombocytopenia(ITP)is an acquired autoimmune disease characterized by autoantibody-mediated platelet destruction and/or impaired platelets production.The clinical manifestations are mainly reduced platelet counts to varying degrees,and the number of megakaryocytes in the bone marrow of most ITP patients is normal or increased.Current treatment options include glucocorticoids,intravenous immunoglobulins,splenectomy,thrombopoietin receptor agonists,anti-CD20 monoclonal antibodies,and other immunosuppressive drugs.The long-term remission rate of ITP is only one-third,and the immunosuppressive effect of non-specific treatment is equivalent to the incidence of bleeding.Therefore,it is significant to explore immunomodulatory therapies that enable patients to achieve long-term remission,rebuild immune tolerance,and avoid serious complications.The pathogenesis of ITP is complex,and the mechanism that causes platelet autoantigen immune intolerance is still unclear.Many immune cells participate in the process of ITP immune tolerance imbalance,involving the interaction between antigen presenting cells,T cells and B cells.Regulatory T cells(Tregs),as a subset of immunosuppressive T cells,play a key role in inducing and maintaining immune tolerance.Many autoimmune diseases are associated with reduced numbers and impaired function of peripheral blood Tregs.The damage of Tregs in the peripheral blood may lead to excessive proliferation and unrestricted function of autoreactive effector T cells.Many studies have shown that deficiency of Tregs leads to autoimmune intolerance in ITP patients.In addition,previous work in our laboratory has shown that monocytes/macrophages recognize platelets sensitized by autoantibodies through Fcy receptors on the membrane surface,and then mediate platelet phagocytosis and release inflammatory factors,causing inflammatory responses.Compared to healthy controls.monocytes in peripheral blood from newly diagnosed ITP patients showed stronger Fcy receptor-mediated phagocytosis.Clinical and experimental studies have shown that treatments such as dexamethasone can exert a therapeutic effect in part by regulating the activation of Tregs and reducing the phagocytosis of monocytes/macrophages.18?-glycyrrhetinic acid(18?-GA)is the main active ingredient of glycyrrhizic acid,which can achieve most of the pharmacological properties of glycyrrhizic acid by regulating the steady state of T subsets.18?-GA has a variety of biochemical and pharmacological activities,including immunomodulatory and anti-inflammatory activities.In addition,18?-GA is also a new HMGB1 pharmacological inhibitor,which directly inhibits its cytokine activity by binding to HMGB1.At present,there are various dosage forms containing 18?-GA on the market,and they are widely used in clinical practice.High mobility group box protein B1(High mobility group box 1,HMGB1)is a non-histone nuclear protein that not only functions as a DNA chaperone in cells,but also outside of the cell as the damage associated molecular pattern(DAMP)molecule.HMGB1 was first identified as an advanced inflammatory mediator,and its elevation was correlated with disease progression.It was later discovered that it could mediate the development of autoimmune diseases,act on cell surface receptors and trigger intracellular signaling.In autoimmune diseases such as hepatitis B,inflammatory bowel disease,systemic lupus erythematosus,and rheumatoid arthritis,the expression of HMGB1 in serum and joint synovium is increased,accompanied by increased pro-inflammatory factors,such as IL-6 and IL-17,and this affects the balance of Thl/Th2 and Th17/Tregs.HMGB1 stimulation can significantly reduce the expression of Foxp3 in mice and humans,and reduce the level of IL-10 secreted by Tregs.Studies have shown that HMGB 1 levels in spleen and serum are higher in ITP patients compared to healthy controls.18?-GA is an inhibitor that can directly bind to HMGB 1.It is not clear whether 18?-GA has the potential to reinstate immune tolerance in ITP.The role of HMGB1 and 18?-GA in ITP needs further study.Objective:This study aimed to assess whether 18?-GA can reduce symptoms and restore immune tolerance in ITP patients and mice.Through in vivo and in vitro experiments,the role of 18?-GA in regulating Tregs and monocytes/macrophages and their underlying molecular mechanisms were investigated.A prospective cohort study was performed to evaluate the efficacy and safety of 18?-GA in ITP patients.Our aim is to provide new ideas for ITP treatment.Methods:1.An active murine model of ITPPlatelets were collected from wild-type(WT)C57/BL6 mice,and 108/100 ?l were infused into C57/BL6 CD61-KO mice once a week for four consecutive weeks,and then the mice were euthanized to prepare a spleen single cell suspension.Severe combined immunodeficient(SCID)mice of the same background were irradiated with 180cGy throughout the body,and within 3 hours after irradiation,the immune spleen cell preparation(5×104 cells/mouse)was injected intraperitoneally into SCID mice to establish an active ITP murine model.The mice in the 18?-GA group were injected intraperitoneally with 18?-GA(30mg/kg,3%DMSO),and the control group received the same volume of 3%DMSO once every other day.After 5 weeks,SCID mice were euthanized.Get the peripheral blood,the spleen,the thymus,groin lymph nodes and the liver.1.1 Platelet count(after 1/10 dilution)and body weight were monitored weekly using a whole blood cytometer.1.2 After 5 weeks,in vivo imaging:DIR(1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)labeled platelets from WT mice were injected into two groups of ITP mice,and the mice were euthanized after 30 minutes.We removed their spleens and livers.In vivo imaging systems were used to measure fluorescence intensity to assess platelet retention in organs.1.3 After 5 weeks,the ratios of CD4+CD25+Foxp3+Tregs,CD11b+CD115+Ly6c+monocytes,and Ml type F4/80+CD80+CD86+macrophages were detected by flow cytometry.2.Evaluation of prospective clinical cohort studiesFrom June 2019 to September 2019,34 patients with primary ITP who were not treated before were recruited and screened.16 patients were treated with 18?-GA(75mg,3 times a day×7 days)and dexamethasone(DXM;40mg,once a day×4 days);18 patients were treated with dexamethasone alone(DXM;40mg,once a day×4 days).The primary endpoints were complete response(CR),response(R),and overall response(OR).Secondary endpoints were response time and adverse events.After the intervention,the treatment response was assessed weekly.The study was registered on ClinicalTrials.gov(#NCT03998982).3.Patients and samplesFrom December 2017 to March 2019,this experiment included 40 ITP patients,and 20 healthy volunteers were matched by age.5 mL of heparinized peripheral blood was extracted,and peripheral blood mononuclear cells(PBMCs)were isolated.CD4+T cells,naive CD4+T cells and CD 14+monocytes were separated using antibody-coated magnetic beads and MS columns.3.1 In vitro cell experiments:culture mediums of PBMCs and CD4+T cells were supplemented with recombinant human IL-2(10ng/mL),anti-human CD3 antibody(1?g/mL),and anti-human CD28 antibody(1?g/mL).The culture medium of naive CD4+T cells needed to be supplemented with anti-TGF?1 antibody(5ng/mL).Cells were cultured with a series of concentrations(12.5?M/25?M/50?M/100?M)of 18?-GA or recombinant human HMGB1 protein(rhHMGB1,100ng/mL),and 0.1%DMSO was used as a control.After several hours of incubation,the cells were collected for flow cytometry,immunofluorescence,RT-PCR and western blot detection.3.1.1 Flow cytometry and immunofluorescence to detect phagocytosis of macrophages:monocytes were sorted with anti-CD 14 magnetic beads,and stimulated with 18?-GA/DMSO and PMA overnight.Macrophages and anti-CD41 antibody-coated platelets labeled by CMFDA(1:10)were co-incubated for 1 hour.Flow cytometry and immunofluorescence were used to determine the average fluorescence intensity of platelets phagocytosed by macrophages.3.1.2 Detect the apoptosis level of PBMCs before and after the treatment of 18?-GA by flow cytometry with Annexin V and PI staining.3.1.3 Flow cytometry detection of the ratio of CD4+CD25+Foxp3+Tregs in PBMCs/CD4+T cells/naive CD4+T cells,before and after treatment with 18?-GA/rhHMGB1.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of HMGB1 in the cell supernatant.The expression of Foxp3 was detected by PCR.3.1.4 Tregs inhibitory function test:CD4+CD25-effector T cells(Teffs)were isolated by magnetic beads,stained with CFSE,and seeded in round bottom 96-well plates at 2×105/well.CD4+CD25+Foxp3+Tregs were isolated by magnetic beads and added to round bottom 96-well plates for co-culture at a ratio of 1:4.After 6 days,the proliferation of CD4+Teffs was measured with or without the treatment of 18?-GA by flow cytometry.3.2 Compare the level of HMGB1 between ITP patients and healthy controls,and evaluate the clinical diagnostic value of HMGB1.3.2.1 ELISA to detect the expression level of plasma HMGB1 in ITP patients and healthy controls;to analyze the correlation between platelet count in patients' blood and plasma HMGB1 level;to assess the specificity and sensitivity of HMGB1 as an auxiliary diagnostic indicator of ITP by the area under the ROC curve(AUC).3.2.2 ELISA and western blot were used to detect the expression of HMGB1 in PBMCs/CD4+T cells from ITP patients and healthy controls.Results:1.18?-GA relieved the symptoms of immune thrombocytopenia in an active murine model.1.1 Platelet count and body weight:On the 14th day after the model was established,the platelet count decreased to the lowest point.On the 28th day,a significantly higher platelet count was observed in the 18?-GA treatment group compared with the control group(**P28=0.0017,**P35=0.0012).18?-GA had no significant effect on the body weight of ITP mice.1.2 Platelet retention in liver and spleen:After 35 days,ITP mice were injected with DIR-labeled platelets in the tail vein,and euthanized after 30 minutes.Liver and spleen were removed,and platelet residues were assessed using an in vivo imaging system.It was concluded that platelet phagocytosis by the liver and spleen was weaker after 18?-GA treatment(**Pliver=0.005 1,Pspleen=0.1212).1.3 The ratio of Tregs/monocytes/macrophages in each organ:After 35 days,the flow cytometry results showed that compared with the control group,18?-GA treatment can increase CD4+CD25+Foxp3+Tregs in spleen,inguinal lymph nodes and thymus of mice,and it can reduce the percentage of CD 11 b+CD 115+ Ly6c+monocytes in peripheral blood and spleen;and reduce F4/80+CD80+CD86+M1 type macrophages in spleen and liver.2.In a prospective cohort analysis,the clinical response of DXM combined with 18?-GA in ITP patients was superior to that of DXM alone.2.1 The OR(CR/R)ratio of the DXM group(n=18)was 50%(9/18),and that of the DXM+18?-GA group(n=16)was 62.5%(10/16),and no significant adverse events.2.2 In both groups of patients who achieved CR/R,platelet counts increased significantly and the symptoms of thrombocytopenia were relieved after treatment;and platelet counts also increased significantly in both groups of NR patients.2.3 ?platelets was defined as changes in platelet counts of one patient before and after treatment.Compared with DXM alone,?platelets increased significantly after treatment with DXM+18?-GA.That was 19(-1,211)× 109/L in the DXM group,and 71(7,268)×109/L in the DXM+18?-GA group.(*P=0.0433).3.18?-GA attenuated platelet phagocytosis of macrophages in ITP patients.3.1 Flow cytometry results showed that compared with the 0.1%DMSO group,macrophages had less platelet phagocytosis in the 18?-GA group.(**P=0.0067).3.2 Immunofluorescence images showed that fewer platelets were phagocytosed by macrophages in 18?-GA group than in DMSO group.4.18?-GA increased the ratio of Tregs in ITP patients and healthy controls in a concentration-dependent manner,and did not induce apoptosis significantly at 25?M.Flow cytometry and PCR results showed that when the dose was 25 ?M and 50 ?M,18?-GA increased the number of Tregs,and the percentage of CD4+T cells in PBMCs did not change at 25?M;Foxp3 mRNA expression in the 18?-GA group was higher than control group;PMBCs can be induced to apoptosis in ITP patients at high doses of 50?M and 100?M.Therefore,25?M was chosen as the optimal dose of 18?-GA.5.18?-GA in creased the production of Tregs from naive CD4+T cells and enhanced its inhibitory function.5.1 We sorted CD4+T cells by magnetic beads in PBMCs of ITP patients.Flow cytometry showed that 25?M 18?-GA can increase the number of Tregs.We sorted naive CD4+T cells,and repeated the experiment.The results showed that 18?-GA can stimulate differentiation and activation of Tregs from naive CD4+T cells.5.2 18?-GA enhances the inhibitory function of Tregs:We co-cultured CFSE-labeled effector T cells(CD4+CD25-Teffs)and CD4+CD25+Foxp3+Tregs at a ratio of 4:1 for 5 days.Flow cytometry results showed that Tregs significantly inhibited the proliferation of effector T cells in the 18?-GA group and the DMSO group.Compared with DMSO,18?-GA can significantly enhance the inhibitory function of Tregs.18?-GA itself has no obvious effect on the proliferation of effector T cells,indicating that 18?-GA can enhance the function of Tregs to inhibit the proliferation of effector T cells.6.18?-GA directly acted on CD4+T cells and increased the proportion of Tregs by inhibiting the function of HMGB1.6.1 ELISA and RT-PCR results showed that the levels of HMGB1 in the plasma of active ITP patients and the mRNA expression of HMGB1 in PBMCs were higher than those in healthy controls6.2 Blood examinations of ITP patients revealed a negative correlation between plasma HMGB1 levels and platelet counts(r=-0.7602,R2=0.5778,*P=0.0174).HMGB1 is helpful for ITP diagnosis and can be used to determine disease severity.6.3 The area under the ROC curve(AUC)analysis was used to determine the diagnostic value of plasma HMGB1 for ITP:the AUC of HMGB 1 was 0.751(0.5843-0.9176,*P=0.0194),and its best sensitivity and specificity were 90.91%and 69.57%,respectively.6.4 ELISA and western blot results showed that CD4+T cells can directly produce HMGB1,and the expression of HMGB1 in CD4*T cells was higher in ITP patients than in healthy controls.6.5 Rescue experiment by flow cytometry results proved that the role of 18?-GA in ITP is related to the inhibition of HMGB1 function:In ITP patients,compared with DMSO group,18?-GA increased the production of Tregs in CD4+T cells(18?-GA vs.DMSO,***P<0.0001).With the addition of 100ng/ml rhHMGB1,this change was significantly reduced(18?-GA vs.rHMGB1+18?-GA,**P=0.0028).HMGB1 itself directly reduces the number of Tregs(DMSO vs.rHMGB1,****P<0.0001),and this inhibition is weakened when 18?-GA is added(rHMGB1 vs.rHMGB 1+18?-GA,****P<0.001).Conclusion:In this study,we evaluated whether 18?-GA can restore immune tolerance in ITP through in vivo and in vitro experiments.Vivo experiments have shown that 18P-GA can increase platelet numbers in ITP mice.Vitro experiments have shown that 18?-GA stimulates the production of Tregs,and enhances the inhibitory effect of Tregs by regulating the function of HMGB1.18?-GA can reduce the platelet phagocytosis of the mononuclear phagocytic system in vitro and in vivo.A cohort study showed that 18?-GA combined with DXM is safe and effective in ITP patients.Therefore,we suggest that 18?-GA can be a potential drug for treating ITP patients. |
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