犀角地黄汤合方含药血清调节ITP患者Teff/Treg分化及功能的研究

Study on the drug serum of Xijiao Dihuang Combined Prescription regulating Teff/Treg differentiation and function in ITP patients

目的探究犀角地黄汤合方含药血清对免疫性血小板减少症(ITP)患者效应性T细胞(Teff)/调节性T细胞(Treg)分化的影响,并探讨磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在其中的作用机制。方法采用随机数字表法将20只大鼠分为含药血清组和空白血清组,每组10只,分别使用犀角地黄汤合方颗粒和蒸馏水连续灌胃3 d,收取2组大鼠全血制备含药血清和空白血清。收集健康志愿者和ITP患者外周血,提取外周血单个核细胞(PBMC),磁珠分选CD4^(+)T细胞,抗CD3/CD28抗体培养体系活化后,分为对照组、模型组和实验组。对照组为健康志愿者CD4^(+)T细胞,模型组及实验组为ITP患者CD4^(+)T细胞,实验组采用含药血清干预72 h,对照组及模型组采用大鼠空白血清干预72 h。流式细胞术检测各组CD4^(+)T细胞中Teff/Treg比例;酶联免疫吸附试验(ELISA)检测各组培养上清液中白细胞介素(IL)-2、干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α、IL-6、IL-17及Treg细胞因子转化生长因子(TGF)-β、IL-10水平;Western blot检测PI3K、Akt、mTOR蛋白及磷酸化蛋白水平。结果CD4阳性率为(99.44±0.01)%。与对照组相比,模型组CD4^(+)T细胞中Teff细胞比例明显升高,Treg比例明显降低,Teff/Treg比值增大,IL-2、IL-6、IL-17、IFN-γ、TNF-α水平以及PI3K、p-PI3K、mTOR、p-mTOR蛋白水平升高,TGF-β、IL-10、p-Akt蛋白水平降低(P<0.05);与模型组相比,实验组CD4^(+)T细胞中Teff细胞比例降低,Treg细胞比例升高,Teff/Treg比值减小,IL-2、IL-6、IL-17、IFN-γ、TNF-α水平以及PI3K、p-PI3K、mTOR、p-mTOR蛋白水平降低,TGF-β、IL-10水平升高(P<0.05)。结论犀角地黄汤合方含药血清能够调节ITP患者Teff/Treg的分化及细胞因子的分泌,可能与抑制PI3K/Akt/mTOR信号通路PI3K、mTOR蛋白表达及其磷酸化水平有关。

Objective To investigate the effect of drug serum of Xijiao Dihuang Combined Prescription(XJDH)on the differentiation of effector T cell(Teff)/regulatory T cell(Treg)in patients with immune thrombocytopenia(ITP),and to investigate the mechanism of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Twenty rats were divided into the drug-containing serum group and the blank serum group by random number table method,with 10 rats in each group.The rats of the two groups were given intragastric administration of XJDH granules and distilled water for 3 consecutive days,respectively.The whole blood samples were collected in the two groups,and drug-containing serum and blank serum were prepared.The peripheral blood samples were collected in healthy volunteers and ITP patients.The peripheral blood mononuclear cells(PBMC)were extracted,and CD4^(+)T cells were sorted by magnetic beads.After the activation of anti-CD3/CD28 antibody culture system,the cells were divided into the control group,the model group and the experimental group.The control group consisted of CD4^(+)T cells of healthy volunteers.The model group and the experimental group were CD4^(+)T cells of ITP patients.The experimental group was treated with drug-containing serum for 72 h,and the control and the model groups were treated with blank serum for 72 h.The proportion of Teff/Treg in CD4^(+)T cells was detected by flow cytometry in each group.The levels of interleukin(IL-2),interferon(IFN)-γ,tumor necrosis factor(TNF)-α,IL-6,IL-17,Treg cytokine transforming growth factor(TGF)-βand IL-10 in supernatant were detected by enzyme-linked immunosorbent assay(ELISA).The expressions of PI3K,Akt,mTOR and phosphorylated proteins were detected by Western blot assay.Results CD4 positive rate was(99.44±0.01)%.Compared with the control group,the proportion of Teff cells in CD4^(+)T cells was significantly increased in the model group.The proportion of Treg was significantly decreased,and the ratio of Teff/Treg was increased(P<0.05).The levels of IL-2,IL-6,IL-17,IFN-γand TNF-αwere increased in the model group,while the levels of TGF-βand IL-10 were decreased(P<0.05),the protein levels of PI3K,p-PI3K,mTOR and p-mTOR were increased in the model group,the level of p-Akt protein was decreased(P<0.05).Compared with the model group,the proportion of Teff cells in CD4^(+)T cells was decreased in the experimental group,the proportion of Treg cells was increased,and the Teff/Treg ratio was decreased(P<0.05).The levels of IL-2,IFN-γ,TNF-α,IL-6 and IL-17 were decreased in the experimental group,while the levels of TGF-βand IL-10 were increased(P<0.05).Compared with the model group,the protein levels of PI3K,p-PI3K,mTOR and p-mTOR were decreased in the experimental group(P<0.05).Conclusion The drug serum with XJDH can regulate the differentiation of Teff/Treg and secretion of cytokines in ITP patients,which may be related to the inhibition of expression levels of PI3K,mTOR proteins and their phosphorylated proteins on the PI3K/Akt/mTOR signaling pathway.