BAFF调节免疫性血小板减少症模型小鼠的Th17/Treg平衡的研究

Regulation of Th17/Treg Balance by BAFF in a Mouse Model of Immune Thrombocytopenia

目的:探讨B细胞激活因子(BAFF)对免疫性血小板减少症模型小鼠体内辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的调节作用和潜在机制。方法:制备豚鼠抗小鼠血小板抗血清(GP-APS),并将150只无特定病原级别的成年雄性BALB/c小鼠(7~8周龄)随机分为5组,每组30只。分别为对照组(空白对照)和ITP组(GP-APS诱导),ITP+rhBAFF组(ITP组联合静脉注射50μg/kg/50μL重组人BAFF蛋白),并在ITP+rhBAFF组处理的基础上分别联合Notch1的抑制剂(DAPT)或PI3K/Akt的抑制剂Polygalacin D(PGD),设立ITP+rhBAFF+DAPT组和ITP+rhBAFF+PGD组,除对照组和ITP组外,均为静脉注射给药,DAPT注射剂量100μg/kg;PGD注射剂量25μg/kg,静脉注射总体积均为50μL,每日1次。1周后取小鼠1mL外周血并分离血清和单个核细胞。用免疫荧光化学检测单个核细胞中BAFF和Notch1的定位。对外周血中的血小板进行计数。酶联免疫吸附法(ELSIA)检测小鼠外周血血清BAFF的水平。Western blot检测小鼠外周血单个核细胞中PI3K、AKT、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的蛋白表达。流式细胞术检测单个核细胞中Th17/Treg的比例变化。结果:ITP小鼠外周血单个核细胞的BAFF与Notch1共定位在细胞膜。与对照组比较,ITP组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达增加,血小板数目和Treg比例减少,Th17比例增加(P<0.05)。与ITP组比较,ITP+rhBAFF组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达增加,血小板数目和Treg比例减少,Th17比例增加(P<0.05)。与ITP+rhBAFF组比较,ITP+rhBAFF+DAPT组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达降低,血小板数目和Treg比例增加,Th17比例降低(P<0.05)。与ITP+rhBAFF组比较,ITP+rhBAFF+PGD组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达降低,血小板数目和Treg比例增加,Th17比例降低(P<0.05)。结论:BAFF通过激活Notch1/PI3K/Akt信号通路促进免疫性血小板减少症模型小鼠体内Th17比例增加及Treg比例减少。

Objective:To explore the regulatory role of B-cell activating factor(BAFF)on the balance of helper T cell 17(Th17)and regulatory T cell(Treg)in vivo in a mouse model of immune thrombocytopenia(ITP).Methods:Guinea pig anti-mouse platelet serum(GP-APS)was prepared,and 150 adult male BALB/c mice(7-8 weeks old)were randomly divided into 5 groups(n=30 per group):control(blank control),ITP(induced by GP-APS),ITP+rhBAFF(ITP mice treated with intravenous injection of recombinant human BAFF protein at 50μg/kg/50μL),ITP+rhBAFF+DAPT(combined with Notch1 inhibitor DAPT),and ITP+rhBAFF+PGD(combined with PI3K/Akt inhibitor Polygalacin D).DAPT was injected at 100μg/kg,and PGD at 25μg/kg,both with a total volume of 50μL and administered once daily.Peripheral blood(1mL)was collected from mice after 1 week for serum and mononuclear cell isolation.Immunofluorescence staining was used to detect BAFF and Notch1 localization in mononuclear cells.Platelet counts in peripheral blood were determined.Enzyme-linked immunosorbent assay(ELISA)was performed to measure BAFF levels in mouse serum.Western blotting was conducted to assess protein expression of PI3K,AKT,Notch1,p-Akt(Thr308),and p-Akt(Ser473)in mononuclear cells.Flow cytometry was used to analyze changes in Th17/Treg ratio in mononuclear cells.Results:BAFF and Notch1 were co-localized on the cell membrane of mononuclear cells in peripheral blood of ITP mice.Compared with the control group,ITP group showed increased expression of BAFF,Notch1,p-Akt(Thr308),p-Akt(Ser473),increased Th17 proportion,decreased platelet count,and decreased Treg proportion(P<0.05).Compared with the ITP group,ITP+rh-BAFF group exhibited increased BAFF,Notch1,p-Akt(Thr308),p-Akt(Ser473)expression,increased Th17 proportion,decreased platelet count,and decreased Treg proportion(P<0.05).Compared with the ITP+rhBAFF group,both ITP+rhBAFF+DAPT and ITP+rhBAFF+PGD groups showed decreased BAFF,Notch1,p-Akt(Thr308),p-Akt(Ser473)expression,increased platelet count,increased Treg proportion,and decreased Th17 proportion(P<0.05).Conclusion:BAFF promotes an increase in Th17 proportion and decrease in Treg proportion in a mouse model of immune thrombocytopenia by activating the Notch1/PI3K/Akt signaling pathway.