Department of Medicine/Rheumatology, MetroHealth Medical Centre, Case Western Reserve University, 2500 MetroHealth Drive, Cleveland, OH 44109, USA.
Arthritis Res Ther. 2011 Jun 17;13(3):R93. doi: 10.1186/ar3368.
Epigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes.
Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).
Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.
Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.
表没食子儿茶素没食子酸酯（EGCG）是绿茶中的一种生物活性多酚，通过抑制信号事件和基因表达发挥强大的抗炎作用。白细胞介素-1β（IL-1β）是与骨关节炎（OA）软骨降解相关的主要细胞因子。本研究的目的是评估 EGCG 对人软骨细胞中与 OA 发病机制相关的 IL-1β诱导的蛋白质表达的整体影响。
用 EGCG（10 至 100 μM）预处理原代 OA 软骨细胞，然后用 IL-1β（5ng/ml）刺激 24 小时。将细胞培养上清液与细胞因子抗体阵列孵育，并通过增强化学发光法观察免疫反应性蛋白（80 种蛋白）。通过实时 PCR 验证 EGCG 对 18 种选定基因的 IL-1β诱导表达的影响，并使用特定的 ELISA 测定 IL-6、IL-8 和肿瘤坏死因子-α（TNF-α）的产生。用 Western 免疫印迹法分析 EGCG 对 IL-1β刺激的软骨细胞中白细胞介素-1受体相关激酶 1（IRAK-1）和肿瘤坏死因子受体相关因子 6（TRAF-6）蛋白的影响。通过使用核因子 kappa-B（NF-κB）和丝裂原激活蛋白激酶（MAPKs）的特异性抑制剂（NF-κB 的 MG132 和 MAPKs 的 p38-MAPK、SB202190；JNK-MAPK、SP600125、ERK-MAPK、PD98059），确定选定基因的调节机制和 EGCG 介导的这些基因调节的机制。
在 80 种存在于阵列上的蛋白质中，有 14%的蛋白质的组成型表达被 EGCG 处理改变。未观察到对与软骨合成反应相关的蛋白质有明显的刺激作用。IL-1β刺激增强了 29 种蛋白质的表达。用 IL-1β 上调的所有 29 种蛋白质的表达均被 EGCG 抑制。EGCG 还抑制了信号中间体 TRAF-6 在 50 和 100 μM 浓度下的表达（P <0.05）。我们的结果确定了 EGCG 的几个新靶点，包括上皮中性粒细胞激活肽-78（ENA-78）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）、生长相关癌基因（GRO）、GRO-α、IL-6、IL-8、单核细胞趋化蛋白-1（MCP-1）、MCP-3、巨噬细胞炎症蛋白-1β（MIP-1β）、粒细胞趋化蛋白-2（GCP-2）、MIP-3α、干扰素-γ诱导蛋白-10（IP-10）、核小体组装蛋白-2（NAP-2）和白血病抑制因子（LIF）。EGCG 的抑制作用主要是通过抑制人软骨细胞中 NF-κB 和 c-Jun N 端激酶（JNK）-MAPK 的激活来介导的。
我们的结果表明，EGCG 在 OA 治疗/预防中的潜力可能与其在人软骨细胞中全局抑制炎症反应的能力有关。这些结果确定了 EGCG 的其他新靶点，并主张 EGCG 可能是 OA 的一种有效的软骨保护剂。
