摘要
目的：
利用体外实验研究穿心莲内酯对T细胞行为的影响及其对盲肠结扎穿孔(CLP)诱导的脓毒血症模型的免疫学效应,并探讨其作用机制。
方法：
1.体外实验：研究穿心莲内酯对小鼠T细胞行为的影响,并对其免疫调节作用机制进行初步探讨。分离小鼠淋巴结细胞,以双色荧光抗体染色技术结合流式细胞术检测在刀豆蛋白A (concanavalin, Con A)的刺激下,穿心莲内酯对T细胞早期活化抗原CD69表达的影响；用羧基荧光素乙酰乙酸(carboxyfluorescein diacetate succinimidyl ester, CFDA-SE)染色分析增殖指数；用碘化丙锭(propidium iodide, PI)染色分析细胞周期的分布；用DiOC6(3)单染法测定地塞米松(dexa methason, DEX)诱导的T细胞线粒体膜电势的变化；Griess法检测脂多糖(lipopolysaccharide, LPS)和干扰素-γ(interferon-γ, IFN-γ)诱导的小鼠腹腔巨噬细胞分泌一氧化氮(nitric oxide, NO)的情况,并结合流式细胞术检测无刺激剂作用下巨噬细胞的吞噬情况；
2.体内实验：评价AN对盲肠结扎穿孔(cecal ligation and puncture, CLP)诱导脓毒血症(sepsis)小鼠的影响,并初步研究AN对CLP诱导小鼠脓毒血症T细胞的保护机制。以结扎并穿孔小鼠盲肠末端建立脓毒血症动物模型,观察AN对脓毒血症模型小鼠的胸腺指数、脾脏指数的影响；通过MTT法分析AN对刀豆蛋白A (ConA)刺激下脓毒血症小鼠外周T细胞增殖的影响；通过流式细胞仪检测DEX诱导的胸腺细胞凋亡的影响,并检测中性粒细胞内活性氧自由基的分泌情况。
结果：
1.体外实验结果表明,终浓度为10μmol/L、20μmol/L、30μmol/L、40μmol/L的穿心莲内酯对Con A刺激的早期活化抗原CD69的表达具有明显的抑制作用(P<0.01),且呈剂量依赖性；对Con A刺激的T细胞增殖具有明显的抑制作用(P<0.01)；并明显阻滞T细胞进入S期,且呈剂量依赖性(P<0.01)；并对DEX诱导的细胞线粒体膜电势的变化有明显的调节作用；可促进无刺激剂作用下巨噬细胞的吞噬作用；抑制LPS+IFN-γ诱导的巨噬细胞释放NO,减少炎症的发生。
2.体内实验研究发现,CLP可诱导小鼠胸腺与脾脏出现明显的萎缩现象,而AN药物治疗组小鼠胸腺与脾脏的重量指数明显增加(P<0.05)；体外以刀豆蛋白A (Con A)刺激脓毒血症小鼠外周T细胞的增殖实验结果表明,药物AN治疗组对脓毒血症早期炎症性小鼠淋巴结细胞的增殖具有明显的抑制作用(P<0.01)；并明显抑制胸腺细胞的凋亡(P<0.01)；并抑制中性粒细胞内活性氧自由基的生成。
结论：
AN在一定浓度范围内对T细胞行为和腹腔巨噬细胞的功能均有明显的调节作用,并对小鼠脓毒血症早期炎症具有一定的保护作用。
AIM:
To investigate the effect of Andrographolide on T Lymphocyte behaviors in vitro, and elucidate the immunoregulation effect of AN on sepsis models induced by cecal ligation and puncture, in order to explore the mechanism of AN.
Methods:
1. To investigate the effect of Andrographolide on the behaviors of the mouse lymphocytes in vitro, in order to elucidate the mechanism of the immunosuppressive effect of Andrographolide. Cell suspensions were prepared from mouse lymph nodes. T lymphocytes were treated with different concentrations of Andrographolide, and stimulated with polyclonal activators Con A. Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69. After the staining with CFDA-SE, T lymphocytes were stimulated with polyclonal activators Con A. Flow cytometry (FCM) was used to detect the proliferation of T lymphocytes. The cell-cycle distribution was analyzed by propidium iodide staining. DIOC6(3) staining was used to analyze the apoptosis of lymphocytes induced by dexa methason (DEX). Fluorescence conjugated microbeads and flow cytometry were used to detect phagocytosis. Griess kit was used to detect the production of NO induced by LPS+IFN-y from macrophage. 2. In order to elucidate the effect of Andrographolide on models of CLP and primarily study the protect mechanism on T lymphocytes of sepsis model induced by CLP, the experimental sepsis model was established by CLP. The proliferation related index of mouse lymphocytes was analyzed with MTT. Flow cytometry were used to detect the apoptosis of thymocytes. H2DCFDA staining was used to analyze the changes of reactive oxygen species (ROS) in peripheral blood together with flow cytometry.
Results:
1. The studies in vitro showed that, In a dose-dependent manner, Andrographolide (10μmol/L, 20μmol/L,30μmol/L,40μmol/L) can significantly inhibit T lymphocytes activation index (P<0.01) stimulated by Con A, and inhibit proliferation index (P<0.01) stimulated by Con A, as well as the progression of T lymphocytes in S phase. Andrographolide can significantly reduce the apoptosis of lymphocytes which were induced by DEX. Andrographolide could promote phagocytosis of macrophages, but inhibit the production of NO which was stimulated with LPS+IFN-y.
2. The studies in vivo demonstrated that Andrographolide can significantly inhibit sepsis induced atrophy in spleen and enhance the index of spleen and thymus. The proliferation related index of mouse lymphocytes stimulated by Con A show that Andrographolide can also inhibit the proliferation of T lymphocytes in early state of inflammation of the sepsis, and Andrographolide can also inhibit the apoptosis of thymocytes. Andrographolide can inhibit the production of ROS from neutrophils.
Conclusion:
Andrographolide has significantly effect on behaviors of mouse T lymphocytes and macrophages, and can protect the early state of inflammation of the sepsis model induced by CLP.
引文
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