绿原酸对人外周血T淋巴细胞体外增殖及免疫调节功能的影响

Effect of chlorogenic acid on proliferation and immunomodulation of human peripheral blood T lymphocytes in vitro

目的绿原酸对T淋巴细胞的影响尚不清楚。文中旨在探讨绿原酸对植物凝集素(PHA)刺激人外周血T淋巴细胞体外增殖及免疫调节功能的影响。方法采用Ficoll离心法分离人外周血单个核细胞,分为对照组、PHA组、PHA+绿原酸50μg/mL组、PHA+绿原酸100μg/mL组。CCK-8法检测细胞增殖,瑞氏-吉姆萨染色观察细胞形态,实时荧光定量PCR检测T细胞增殖标志物Ki-67,Th1细胞因子IFN-γ、TNF-α,转录因子T-bet和Th2细胞因子IL-4、IL-13及转录因子GATA-3的mRNA表达水平;活性氧试剂盒检测细胞内ROS变化。结果与对照组比,PHA组刺激后T细胞明显增殖并转化为淋巴母细胞,增殖标志物Ki-67和Th1相关因子IFN-γ、TNF-α、T-bet mRNA表达水平明显升高(P<0.05),Th2相关因子IL-4、IL-13的表达上升(P>0.05),GATA-3 mRNA表达明显下降(P<0.01),胞内ROS水平明显增加(P<0.01);与PHA组比较,PHA+绿原酸50μg/mL组、PHA+绿原酸100μg/mL组处理后T细胞增殖、转化率和Ki-67、IFN-γ和T-bet mRNA表达水平显著降低(P<0.05),IL-4、IL-13、GATA-3 mRNA水平显著增加(P<0.05),胞内ROS水平明显降低(P<0.01),且呈剂量依赖性。结论绿原酸可抑制人外周血T淋巴细胞体外增殖,并可能通过下调ROS水平、抑制Th1因子的表达促进Th2分化发挥免疫调节功能。

Objective Chlorogenic acid has a various of biological effects such as anti-inflammatory and antioxidant.T cells are an important part of the immune system,and the effect of chlorogenic acid on T lymphocytes is unknown.This paper aims to study the effect of chlorogenic acid on the proliferation and immunomodulatory function of human peripheral blood T lymphocytes stimulated with phytohaemagglutinin(PHA)in vitro.Methods Human peripheral blood mononuclear cells(PBMCs)were isolated by Ficoll centrifugation,and divided into Control group,PHA group,the group co-treatment with PHA and 50μg/mL,100μg/mL chlorogenic acid.CCK-8 assay was used to detect cell proliferation.The morphology of the lymphocytes was observed by Wright's-Giemsa staining.The mRNA expression of T cell proliferation markers Ki-67,Th1 cytokines interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),and transcription factor T-bet,Th2 cytokines Interleukin 4(IL-4),Interleukin 13(IL-13),and transcription factor GATA-3 were detected by quantitative real-time PCR.Reactive oxygen species kits were used to detect intracellular ROS levels.Results Compared with Control group,T cells significantly proliferated and transformed into lymphoblastoid cells in PHA group.Following PHA treatment,the mRNA expression levels of T cells proliferation markers Ki-67,and Th1 related factors IFN-γ,TNF-α,and T-bet were significantly increased(P<0.05),and Th2 related factors IL-4 and IL-13 were increased(P>0.05).GATA-3 mRNA was significantly decreased(P<0.05),and the level of intracellular ROS was significantly increased with PHA stimulation(P<0.01).After co-treatment with PHA and 50μg/mL,100μg/mL chlorogenic acid,compared with PHA group,the proliferation and transformation of T lymphocytes and the mRNA expression of Ki-67,IFN-γ,and T-bet were significantly decreased(P<0.05).IL-4,IL-13,GATA-3 mRNA level significantly increased(P<0.05),and dose-dependently reduced intracellular ROS levels(P<0.01).Conclusion Chlorogenic acid inhibits the proliferation of human peripheral blood T-lymphocyte in vitro.Chlorogenic acid may exert immunomodulatory functions by downregulating ROS levels and inhibiting Th1 factor expression to promote Th2 differentiation.