血必净促进内毒素／脂多糖刺激调节性T淋巴细胞凋亡并介导辅助性T淋巴细胞漂移的作用

The enhancing effect of ＂Xuebijing injection＂ on lipopolysaccharide-induced apoptosis of regulatory T cells and mediation of polarization of helper T cells

目的了解血必净注射液促进LPS刺激CD4^＋CD25^＋调节性T淋巴细胞（Treg细胞）凋亡过程及介导辅助性T淋巴细胞（Th）漂移的调节作用。方法免疫磁珠法分选获得大鼠脾脏CD4^＋CD25^＋Treg细胞，分为常规培养对照组、抗CD3／CD28组、抗CD3／CD28＋LPS组、抗CD3／CD28^＋血必净组和抗CD3／CD28＋LPS＋血必净组，培养3d后应用流式细胞术检测Treg细胞凋亡率及叉头翼状螺旋转录因子3（Foxp3）表达。将CD4^＋CD25^＋Treg细胞与CD4^＋CD25^-T淋巴细胞1：1培养，伴刀豆球蛋白A刺激68h，检测上清液中Th1分泌的γ干扰素（IFN-γ）、Th2分泌的IL-4、Th17分泌的IL-17水平。结果抗CD3／CD28＋LPS＋血必净组Treg细胞凋亡率为（45．14±2．7）％，明显高于抗CD3／CD28＋LPS组[（29．4±1．6）％，P〈0．01]；2组Foxp3平均荧光强度分别为95±9、140±18，差异有统计学意义（P〈0．01）。同时，抗CD3／CD28＋LPS＋血必净组IFN-γ分泌水平显著高于抗CD3／CD28＋LPS组（P〈0．01），IL4则呈相反变化（P〈0．05），抗CD3／CD28＋LPS＋血必净组IFN-γ／IL-4较对照组升高（P〈0．01）；抗CD3／CD28＋血必净组IL-17分泌水平较抗CD3／CD28组明显下降（P〈0．05）。结论CD4^＋CD25^＋Treg细胞活化介导了Th1向Th2功能性极化；血必净对LPS诱导的T淋巴细胞免疫功能有重要调节作用，可促进CD4^＋CD25^＋Treg细胞凋亡并介导Th2向Th1漂移，从而缓解细胞免疫抑制状态。

Objective To investigate the enhancing effect of Chinese medieine-Xuebijing injection on fipopolysaceharide （LPS）-induced apoptosis of CD4^＋ CD25^＋ regulatory T cells （Tregs） and polarization of helper T cells （Th）. Methods CD4^＋ CD25^＋ Tregs collected from rat spleen in vitro by immunomagnetie beads assay were divided into the control group, anti-CD3/CD28 group, anti-CD3/CD28 ＋ LPS group, anti-CD3/CD28 ＋ ＂ Xuebijing injection＂ group and anti-CD3/CD28 ＋ LPS ＋ ＂ Xuebijing injection＂ group. Tregs apoptosis rate and expression of winged helix transcription factor （Foxp3） in Tregs were detected by flow cytometry on 3rd post culture day. CD4^＋ CD25^- T cells were co-cultured with CD4^＋ CD25 ^-Tregs （1：1） for 68 hours with canavalin A stimulation. Interferon γ （γ-IFN）, interleukin （IL）-4 and IL-17 in su- pernatants, which respectively was secreted by Thl, Th2 and Thl7, were measured by ELISA. Results Tregs apoptosis rate of anti-CD3/CD28 ＋ LPS ＋ ＂ Xuebijing injection＂ group （45. 1 ± 2.7% ） was significantly higher than that of anti-CD3/CD28 ＋ LPS group （29.4 ±1.6% , P 〈 0.01 ）. Meanwhile, Foxp3 expressions in Tregs in above 2 groups were 95 ± 9 and 140 ± 18 respectively, showing statistically significant difference between them（ P 〈 0.01 ）. γ-IFN levels secreted in anti-CD3/CD28 ＋ LPS ＋ ＂ Xuebijing injec- tion＂ group were significantly higher than those in anti-CD3/CD28 ＋ LPS group （ P 〈 0. 01 ）, while IL-4 levels had an opposite tendency compared with γ-IFN （ P 〈 0.05） , resulting in a marked increase in the ratio of 7-IFN /IL-4 in anti-CD3/CD28 ＋ LPS ＋ ＂ Xuebijing injection＂ group （ P 〈 0. 01 ）. In anti-CD3/ CD28 ＋ ＂ Xuebijing injection＂ group, IL-17 secretion levels were significantly decreased compared with anti- CD3/CD28 group （ P 〈 0.05）. Conclusions Activation of CD4^＋ CD25^＋ Tregs induced by LPS may mediate Thl shift to Th2 response. ＂ Xuebijing injection＂ can effectively regulate immune function of T cells, increase the LPS-indueed apoptosis of CD4^＋ CD25^＋ Tregs as well as enhance the polarization of Th2 to Thl, thereby abating the suppressive state of cell-mediated immunity.