Ranjan Dinesh, Chen Changguo, Johnston Thomas D, Jeon Hoonbae, Nagabhushan Moolky
Transplant Section, Department of Surgery, University of Kentucky, College of Medicine, Lexington, Kentucky 40536, USA.
J Surg Res. 2004 Oct;121(2):171-7. doi: 10.1016/j.jss.2004.04.004.
T cell mediated acute rejection of transplanted organ continues to be a noticeable problem in solid organ transplantation. We showed that Curcumin is a potent inhibitor of Cyclosporin A resistant T cell CD28 co-stimulation pathway. Here we report the inhibitory effects of Curcumin on mitogen-stimulated lymphocyte proliferation, IL-2 synthesis/signaling, and NFkappaB (transcription factor of IL-2 promoter) activation.
Human lymphocytes were isolated from fresh human spleen (SP-L). Mitogens [final concentrations of 2 microg/ml concanavalin A (Con A), 5 microg/ml phytohemagglutinin (PHA), and 20 ng/ml of phorbol-12-myristate-13-acetate (PMA)] were added to the designated wells in a 96-well plate with 0.2 million SP-L and cultured for 48 h and then assayed for IL-2 synthesis by ELISA and 3H-thymidine uptake. In another parallel experiment we added IL-2 (0.5 nM) to stimulate the cells to check if Curcumin's inhibition of IL-2 synthesis is the sole reason for inhibition of cell proliferation. Electrophoretic mobility shift assay (EMSA) was performed in PMA (20 ng/ml, 1 h) stimulated cells with or without Curcumin to assay NFkappaB activation.
Curcumin at 2.5 microg/ml inhibited Con A, PHA, and PMA stimulated SP-L proliferation at 77, 23, and 48%, respectively, over controls and Curcumin at 5 microg/ml completely (nearly 100%) inhibited the mitogen stimulated proliferation. Curcumin inhibited IL-2 synthesis in Con A, PHA, and PMA stimulated SP-L in a concentration-dependent manner with an ED50 (concentration required for 50% inhibition) measured at 3.5 microg/ml. Exogenous IL-2 stimulated SP-L proliferation was also inhibited by Curcumin in a concentration-dependent manner with an ED50 of 2 microg/ml. EMSA assay indicated that PMA at 20 ng/ml stimulated NFkappaB activation 253% over control, which was inhibited by 24, 38, and 73%, respectively, with Curcumin at final concentrations of 2.5, 5, and 10 microg/ml, respectively.
Curcumin has profound immunosuppressive effects mediated via inhibition of IL-2 synthesis, mitogen, and IL-2 induced activation of human lymphocytes. This effect may be mediated via NFkappaB inhibition.
在实体器官移植中，T细胞介导的移植器官急性排斥反应仍然是一个显著问题。我们发现姜黄素是环孢菌素A耐药T细胞CD28共刺激途径的有效抑制剂。在此，我们报告姜黄素对丝裂原刺激的淋巴细胞增殖、白细胞介素-2（IL-2）合成/信号传导以及核因子κB（IL-2启动子的转录因子）激活的抑制作用。
从新鲜人脾脏中分离出人淋巴细胞（脾淋巴细胞，SP-L）。将丝裂原[最终浓度为2微克/毫升的刀豆球蛋白A（Con A）、5微克/毫升的植物血凝素（PHA）和20纳克/毫升的佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）]加入到含有20万SP-L的96孔板的指定孔中，培养48小时，然后通过酶联免疫吸附测定法（ELISA）和3H-胸腺嘧啶核苷摄取来检测IL-2的合成。在另一个平行实验中，加入IL-2（0.5纳摩尔）刺激细胞，以检查姜黄素对IL-2合成的抑制是否是其抑制细胞增殖的唯一原因。对用或不用姜黄素处理的PMA（20纳克/毫升，1小时）刺激的细胞进行电泳迁移率变动分析（EMSA），以检测核因子κB的激活情况。
2.5微克/毫升的姜黄素分别使Con A、PHA和PMA刺激的SP-L增殖比对照组降低了77%、23%和48%，而5微克/毫升的姜黄素完全（接近100%）抑制了丝裂原刺激的增殖。姜黄素以浓度依赖的方式抑制Con A、PHA和PMA刺激的SP-L中IL-2的合成，半数有效浓度（ED50，即50%抑制所需的浓度）为3.5微克/毫升。外源性IL-2刺激的SP-L增殖也被姜黄素以浓度依赖的方式抑制，ED50为2微克/毫升。EMSA分析表明，20纳克/毫升的PMA刺激核因子κB激活比对照组高253%；最终浓度分别为2.5、5和10微克/毫升的姜黄素分别使其抑制了24%、38%和73%。
姜黄素通过抑制IL-2合成及丝裂原以及IL-2诱导的人淋巴细胞激活而具有显著的免疫抑制作用。这种作用可能是通过抑制核因子κB介导的。
