Zhou Ru, Wang Jun-xia, Tang Wei, He Pei-lan, Yang Yi-fu, Li Yuan-chao, Li Xiao-yu, Zuo Jian-ping
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Acta Pharmacol Sin. 2006 Dec;27(12):1616-21. doi: 10.1111/j.1745-7254.2006.00457.x.
(5R)-5-hydroxytriptolide (LLDT-8) displayed anti-arthritis and anti-allogenic transplantation rejection activities in our previous studies. Here, we aim to further clarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-gamma.
T cells were activated with anti-CD3 antibody or concanavalin A (ConA). The expression of cell surface molecules was detected with flow cytometry. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to test cell division. IFN-gamma production was determined by enzyme-linked immunosorbent assay. Cell proliferation was evaluated by [3H]-thymidine uptake. Mice were immunized with ovalbumin to assess the in vivo immune response. RT-PCR and Real-time PCR were applied to determine the mRNA expression. The protein phosphorylation levels were detected by Western immunoblot assay.
LLDT-8 at 100 nmol/L did not change the CD25, CD69, and CD154 expressions in anti-CD3-stimulated T cells. LLDT-8 markedly blocked the cell division of CD4 and CD8 T cells after ConA stimulation. LLDT-8 inhibited T cell-derived IFN-gamma production. Moreover, LLDT-8 suppressed the ovalbumin-specific T cell proliferation and IFN-gamma generation. In anti-CD3-activated T cells, LLDT-8 abrogated the mRNA expression of signal transducer and activator of transcription1 (STAT1), T-box transcription factor, IL-12 receptor beta2, STAT4, and interferon regulatory factor 1 in the IFN-gamma expression pathway. Western blot analysis showed that LLDT-8 blocked the phosphorylation levels of extracellular signal-regulated kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28-activated T cells. In addition, LLDT-8 reduced the transcripts of macrophage inflammatory protein (Mip)-1alpha, Mip-1beta, regulated upon activation normally T-cell expressed and secreted, inducible protein-10, IFN-inducible T cell a chemoattractant, and monokine induced by IFN-gamma in IFN-gamma-stimulated murine macrophage cell line Raw 264.7 cells.
LLDT-8 was a potential inhibitor for IFN-gamma-associated signaling.
在我们之前的研究中，(5R)-5-羟基雷公藤内酯醇（LLDT-8）表现出抗关节炎和抗同种异体移植排斥活性。在此，我们旨在进一步阐明LLDT-8对促炎细胞因子γ干扰素（IFN-γ）的作用。
用抗CD3抗体或刀豆蛋白A（ConA）激活T细胞。用流式细胞术检测细胞表面分子的表达。用羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）标记细胞以检测细胞分裂。通过酶联免疫吸附测定法测定IFN-γ的产生。通过[3H]-胸腺嘧啶核苷摄取评估细胞增殖。用卵清蛋白免疫小鼠以评估体内免疫反应。应用逆转录聚合酶链反应（RT-PCR）和实时定量聚合酶链反应（Real-time PCR）测定mRNA表达。通过蛋白质免疫印迹法检测蛋白质磷酸化水平。
100 nmol/L的LLDT-8未改变抗CD3刺激的T细胞中CD25、CD69和CD154的表达。LLDT-8显著阻断了ConA刺激后CD4和CD8 T细胞的细胞分裂。LLDT-8抑制T细胞衍生的IFN-γ产生。此外，LLDT-8抑制卵清蛋白特异性T细胞增殖和IFN-γ生成。在抗CD3激活的T细胞中，LLDT-8消除了IFN-γ表达途径中信号转导和转录激活因子1（STAT1）、T盒转录因子、白细胞介素12受体β2、STAT4和干扰素调节因子1的mRNA表达。蛋白质印迹分析表明，LLDT-8阻断了抗CD3加抗CD28激活的T细胞中细胞外信号调节激酶、应激激活蛋白激酶（SAPK）/c-Jun氨基末端激酶和p38丝裂原活化蛋白激酶的磷酸化水平。此外，LLDT-8降低了γ干扰素刺激的小鼠巨噬细胞系Raw 264.7细胞中巨噬细胞炎性蛋白（Mip）-1α、Mip-1β、正常T细胞激活后表达和分泌的调节因子、诱导蛋白-10、γ干扰素诱导的T细胞趋化因子以及γ干扰素诱导的单核因子的转录本。
LLDT-8是γ干扰素相关信号传导的潜在抑制剂。
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