Study On Autoimmune Deficiency Mechanism And Drug Intervention Mechanism Of Bone Marrow Mesenchymal Stem Cells In Patients With Idiopathic Thrombocytopenic Purpura

Background and purposeImmune thrombocytopenia(ITP)is a common acquired autoimmune hemorrhagic disease in clinic.The typical clinical manifestations of ITP are decreased peripheral blood platelet count,bone marrow megakaryocyte maturation disorder and clinical bleeding symptoms.Th17 and regulatory T cells(Treg)are two important subsets of helper T(Th)cells.The imbalance of Th17/Treg is involved in the occurrence of various infectious diseases,metabolic diseases and autoimmune diseases.Previous studies have shown that the plasma of ITP patients exhibits an increase of Th17/Tregs ratio.The down-regulation of miR-146 a,miR-326 and miR-142-5p in immune-related miRNAs in peripheral blood mononuclear cells(PBMC)of patients may be related to ITP.Mi R-142-5p and miR-146 a are negatively correlated with the levels of Th17 cells,and miR-146 a is positively correlated with the levels of Tregs and platelet counts.Therefore,immune-related miRNAs may be inherently related to Th17/Treg balance in ITP.Bone marrow mesenchymal stem cells(BMSCs)exert an immunomodulatory function in autoimmune tolerance.After transplantation of BMSCs into ITP mice,the platelet production,the number of Tregs,and the levels of IL-10 and TGF-β are significantly increased.In addition,mesenchymal stem cells(MSCs)act as paracrine,such as exosomes.Exosomes are membrane sacs secreted by cells through exocytosis.Exosomes contain proteins,lipids,transcription factors,DNA,m RNA,and micro RNA(miRNA),which act on target cells and perform their functions by binding to target-tissue receptors or fusing with plasma membranes.The up-regulation of miR-146 a in exosomes of bone marrow mesenchymal stem cells(BMSCs-Exo)suggests that BMSCs-Exo miR-146 a may regulate Th17/Treg imbalance in ITP.Predictive analysis of the target genes of miR146 a revealed that miR-146 a interacted with IL-1R-associated kinase-1(IRAK1).IRAK1 has been confirmed to be involved in the regulation of Th17/Treg differentiation and balance.Therefore,we speculated that extraneous BMSCs may secrete exosome miR-146 a to CD4+ T cells,and regulate the imbalance of Th17/Treg by targeting IRAK1 in ITP.Chidamide is a kind of anticancer drug,which has broad-spectrum anti-tumor effect.Its anticancer mechanism mainly includes activating NK cells,inhibiting MDSC/Treg cells,playing an immunomodulatory role;restoring drug sensitivity of tumor cells through epigenetic regulation mechanism;inducing tumor cell apoptosis and inhibiting cell cycle.Chidamide is seldom studied in ITP.It has been proved that low-dose Chidamide can not only improve thrombocytopenia in ITP model mice,but also weaken the function of antibodies swallowed by platelet macrophages,stimulate the production of natural Tregs,promote the peripheral transformation of T cells to Treg,and restore Treg inhibition in vivo and in vitro.BMSCs itself can trigger the production of Treg,while Treg can negatively regulate the immune response.BMSCs in patients with ITP has its own defects,such as increased rate of apoptotic cells,impaired ability of proliferation,lack of immunosuppressive ability and Treg induction ability.Human platelet-derived growth factor-BB(PDGF-BB)can inhibit the senescence and apoptosis of BMSCs in patients with ITP by inhibiting p53/p21 pathway.Therefore,we scientifically hypothesized that low-dose cedarbamine can correct the immune deficiency of BMSCs in patients with ITP and restore the proliferation of BMSCs in patients with ITP.Method(1)The effect of exosome of bone marrow mesenchymal stem cells on Th17/Treg imbalance in patients with ITP in vitro1、In this study,20 patients with ITP and 20 healthy controls were recruited to isolate and identify human BMSCs.Human BMSCs were cultured in vitro,and the culture medium was collected.BMSC exosomes(BMSCs-Exo)were isolated by Exo Quick kit.Transmission electron microscope,particle size analysis and marker westernblot(WB)were used to identify the exosomes.2、Peripheral blood samples were collected from patients with ITP and healthy controls.The proportion of Th17/Treg cells in peripheral blood of patients with ITP and healthy controls was measured by flow cytometry,and the levels of inflammatory cytokines IL-17,IL-10 and TGF-β were detected by ELISA.3、CD4+T cells were isolated from peripheral blood of ITP patients and healthy controls.Then CD4+T cells were co-incubated with BMSCs-Exosome or PBS,respectively,and the proportion of Th17 and Treg cells in CD4+T was determined by flow cytometry.The levels of inflammatory cytokines IL-17,IL-10 and TGF-β were detected by ELISA.(2)To explore the effect of BMSC-EXO on Th17/Treg imbalance through the delivery of miR-146a1、The expression of miR-146a-5p in BMSC and BMSC-Exo was detected by q RT-PCR,and the same method was used to detect the expression of miR-146a-5p in CD4+ T cells of ITP patients incubated with PBS and BMSCs-Exo.Exosomes were extracted after BMSCs was transfected into miR-146a-5p inhibitor,the expression of miR-146a-5p in BMSCs-Exo was detected by q RT-PCR.Then,CD4+T cells were incubated with exosomes of BMSCs silenced by miR-146a-5p(miR-146a-5p I-Exo),and the expression of miR146a-5p in CD4+T cells was detected by q RT-PCR.2、Mi R-146a-5p inhibitor or inhibitor NC was transfected into BMSCs,and the exosomes were extracted as miR-146a-5p I-Exo and NCI-Exo,respectively.Mi R-146a-5p I-Exo and NCI-Exo were co-incubated with CD4+T cells.The proportion of Th17 cells and Tregs in CD4+T cells was detected by flow cytometry,and the levels of IL-17,IL-10 and TGF-β were detected by ELISA.(3)To explore the molecular mechanism of the imbalance of Th17/Treg regulated by BMSC-Exo transduction miR-146a-5p.1、 Luciferase activity assay identified the binding of miR-146a-5p and IRAK13’UTR.RIP and RNA pull-down experiments verified the role of miR-146a-5p in regulating IRAK1 in CD4+T cells.After CD4+T cells of ITP patients were transfected with miR-146a-5p mimic or inhibitor,q RT-PCR and western blot respectively to detect the expression level of IRAK1 in CD4+ T cells.Exosomes were extracted after BMSCs transfection into miR-146a-5p inhibitor or miR-146a-5p inhibitor NC.CD4+ T cells were incubated with miR-146a-5p I-Exo,NCI-Exo,normal BMSCs-Exo or PBS.The gene and protein expression of IRAK1 in CD4+ T was detected by q RT-PCR and western blot.2、Mi R-146a-5p mimic and IRAK1 overexpression vectors were co-transfected into CD4+ T cells of ITP patients.The proportion of Th17 and Treg cells in CD4+ T cells was detected by flow cytometry,and the levels of IL-17,IL-10 and TGF-β in culture medium were detected by ELISA.3 、 IRAK1 overexpression vector was transfected into CD4+ T cells of ITP patients and incubated with BMSC-Exo.The proportion of Th17 and Treg cells was detected by flow cytometry,the expression of ROR γ t and Foxp3 was detected by q RT-PCR,and the levels of IL-17,IL-10 and TGF-β in culture medium were detected by ELISA.(4)Low dose Chidamide can correct the defect of BMSCs in ITP patients,restore BMSCs immunity in ITP patients,and protect BMSCs from apoptosis.1.The third generation BMSCs was collected,the proliferation ability of BMSCs in ITP patients was detected by CCK8.Then the cell cycle and apoptosis experiments were used to further verify the effect of Chidamide(CHD)on the cycle and apoptosis of BMSCs.2.The mechanism of protecting ITP-BMSCs from apoptosis.the experiment was divided into three groups: HC-BMSCs,ITP-BMSCs and ITP-BMSCs+2u CHD.The site changes of mitochondrial membrane were detected,and the levels of PI3 K,Akt,p-PI3 K,P-AKT,p53,P21 Bax,Bcl-2,caspase-9 and caspase-3 were detected by western blot.3.Detection of the ability of BMSCs to inhibit T cell proliferation,induce Tregs and inhibit anti-GPIIb-IIIa antibody in ITP patients: the experiment was divided into three groups: T 、 T+PHA 、 T+PHA+MSC-control,T+PHA+MSCT-ITP,T+PHA+MSC-control+CHD、T+PHA+MSCs-ITP+CHD.CD4+T cells were isolated from healthy human peripheral blood and co-cultured with BMSCs in each group for5 days.The proliferation of T cells and the proportion of Tregs cells were detected by flow cytometry.After PBMC cells and BMSCs were co-cultured for 10 day,the level of anti-GPIIb-IIIa antibody in the undiluted culture supernatant was detected by ELISA.Result(1)BMSCs-exosomes repressed the proportions of Th17 cells and enhanced the proportions of Tregs in ITPThe Th17/Treg ratio of ITP patients was notably higher than that of healthy control,and the IL-17 level of ITP patients was higher than that of healthy control.However,there was no significant difference in IL-10 levels between ITP patients and healthy control.The expression of TGF-β in ITP patients was lower than that in healthy subjects.When CD4+T cells were incubated with BMSCs-Exo,the ratio of Th17/Treg in CD4+T cells decreased significantly in the presence of BMSCs-Exo.BMSCs-Exo significantly inhibited the level of IL-17 in CD4+T cells,while BMSCs-Exo significantly increased the levels of IL-10 and TGF-β in CD4+T cells.The data of flow cytometry and ELISA showed that BMSCs-Exo had no effect on the imbalance of Th17/Treg in CD4+T cells of healthy control group.(2)BMSCs-derived exosomal miR?146a?5p regulated the imbalance of Th17 cells and Tregs in the CD4+ T cellsThe results of q RT-PCR showed that miR-146a-5p was highly expressed in BMSCs exosomes compared with BMSCs.When CD4+T cells were treated with BMSCs-Exo,miR-146a-5p was up-regulated in CD4+T cells.In addition,we silenced the expression of miR-146a-5p,miR-146a-5p in BMSCs and decreased in BMSCs-Exo.After CD4+T cells were co-incubated with exosome(miR-146a-5p I-Exo)of miR-146a-5p-silenced BMSCs,the expression of miR-146a-5p in CD4+T cells decreased compared with NCI-Exo group.In addition,the ratio of Th17/Treg in CD4+ T cells treated with miR-146a-5p I-Exo was significantly higher than that in NCI-Exo group.Compared with NCI-Exo group,the level of IL-17 in miR-146a-5p I-Exo group increased significantly,while the levels of IL-10 and TGF-β in CD4+ T cells decreased.(3)BMSCs-exosomal miR-146a-5p repressed Th17/Treg ratio in CD4+T cells by repressing IRAK1 expressionWe performed luciferase reporter assay to verify the relationship between miR-146a-5p and IRAK1,showing that miR-146a-5p interacted with IRAK1.RIP and RNA pull-down assays also demonstrated that miR-146a-5p directly targeted IRAK1 in CD4+T cells.Over-expression of miR-146a-5p down-regulated the level of IRAK1 in CD4+T cells.After transfection with miR-146a-5p inhibitor,the expression of IRAK1 was enhanced.After BMSCs-Exo treatment,the expression of IRAK1 gene and protein in CD4+T cells decreased significantly.However,the expression of IRAK1 gene and protein was significantly increased when miR-146a-5p I-Exo in CD4+T cells.Co-transfected with miR-146a-5p mimic and pc DNA3.1IRAK1 in CD4+T cells,over-expression of miR-146a-5p reduced the proportion of Th17/Treg cells in CD4+T cells,while the proportion of Th17/Treg cells was up-regulated by IRAK1.Meanwhile,the overexpression of miR-146a-5p inhibited the expression of IL-17,but increased the levels of IL-10 and TGF-β.The effect of overexpression of IRAK1 was eliminated by overexpression of miR-146a-5p.over-expressing IRAK1 of CD4+T cells were co-incubated with BMSCs-Exo.BMSCs-Exo can reduce Th17/Treg ratio in CD4+T cells.Over-expression of IRAK1 leads to an increase in Th17/Treg ratio in CD4+T cells,and the effect of IRAK1 up-regulation can be corrected by BMSCs-Exo.BMSCs-Exo decreased the level of IL-17 in CD4+T cells and increased the level of IL-10 and TGF-β.The up-regulation of IRAK1 promoted the level of IL-17 in CD4+T cells and inhibited the levels of IL-10 and TGF-β,which could be partially eliminated by BMSCs-Exo.(4)Chidamide can restore the immunomodulatory effect of MSCs-ITP and protect MSCs-ITP from apoptosisCCK8 experiment showed that the proliferation level of bone marrow mesenchymal stem cells in ITP patients(ITP-BMSCs)was higher than that in other drug groups after adding 2umol/L Chidamide.The results of apoptosis showed that adding 2umol/L Chidamide could improve the nuclear morphology,down-regulate the apoptosis rate of ITP-BMSCs,and reverse the decrease of G0/G1 cells in ITP-BMSCs group.The mitochondrial membrane potential in ITP+CHD group was significantly higher than that in ITP-BMSCs group.At the protein level,compared with the HC-BMSCs group,the Bcl-2/Bax ratio and caspase-9 and caspase-3activities were decreased in the ITP-BMSCs group,and this effect could be corrected by Chidamide.Chidamide significantly decreased the expression of p53 and p21 and upregulated the expression of PI3 K,AKT and Survivin at the protein level.Chidamide also partially reversed the abnormal subcellular localization of p53 and p21 in ITP-BMSCs.When ITP-BMSCs treated with Chidamide was co-cultured with CD4+T cells,the proliferation of T cells induced by PHA decreased significantly and the number of Tregs increased.The ability of ITP-BMSCs treated with Chidamide to inhibit the production of anti-GPIIb-IIIa antibody was restored.Conclusion1 、 These findings demonstrate that BMSC-derived exosomal miR-146a-5p regulates Th17/Treg imbalance in ITP by repressing IRAK1 expression.Thus,this work suggests that BMSCs-exosomal miR-146a-5p may be a potential therapeutic target for ITP.2、Low dose Chidamide can correct the functional defect of ITP-MSCs,restore the immunity of ITP-BMSCs and protect ITP-BMSCs from apoptosis through P53/P21 and PI3K/AKT pathway.Therefore,low-dose Chidamide has a potential therapeutic effect on ITP-BMSCs.