Department of Medicine, Division of Rheumatology, MetroHealth Medical Center/Case Western Reserve University, 2500 Metro Health Drive, Cleveland, OH 44109, USA.
Arthritis Res Ther. 2010;12(5):R195. doi: 10.1186/ar3166. Epub 2010 Oct 18.
Pomegranate has been revered throughout history for its medicinal properties. p38-MAPK is a major signal-transducing pathway in osteoarthritis (OA) and its activation by interleukin-1β (IL-1β) plays a critical role in the expression and production of several mediators of cartilage catabolism in OA. In this study we determined the effect of a standardized pomegranate extract (PE) on the IL-1β-induced activation of MKK3/6, p38-MAPK isoforms and the activation of transcription factor RUNX-2 in primary human OA chondrocytes.
Human chondrocytes were derived from OA cartilage by enzymatic digestion, treated with PE and then stimulated with IL-1β. Gene expression of p38-MAPK isoforms was measured by RT-PCR. Western immunoblotting was used to analyze the activation of MAPKs. Immunoprecipitation was used to determine the activation of p38-MAPK isoforms. DNA binding activity of RUNX-2 was determined using a highly sensitive and specific ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with siRNAs.
Human OA chondrocytes expressed p38-MAPK isoforms p38α, -γ and -δ, but not p38β. IL-1β enhances the phosphorylation of the p38α-MAPK and p38γ-MAPK isoforms but not of p38δ-MAPK isoform in human OA chondrocytes. Activation of p38-MAPK in human OA chondrocytes was preferentially mediated via activation of MKK3. In addition, we also demonstrate that polyphenol rich PE inhibited the IL-1β-induced activation of MKK3, p38α-MAPK isoform and DNA binding activity of the transcription factor RUNX-2.
Our results provide an important insight into the molecular basis of the reported cartilage protective and arthritis inhibitory effects of pomegranate extract. These novel pharmacological actions of PE on IL-1β stimulated human OA chondrocytes impart a new suggestion that PE or PE-derived compounds may be developed as MKK and p38-MAPK inhibitors for the treatment of OA and other degenerative/inflammatory diseases.
石榴在历史上因其药用特性而备受推崇。p38-MAPK 是骨关节炎 (OA) 中的主要信号转导途径，其被白细胞介素-1β (IL-1β) 激活在 OA 中几种软骨分解代谢介质的表达和产生中起着关键作用。在这项研究中，我们确定了标准化石榴提取物 (PE) 对 IL-1β 诱导的 MKK3/6、p38-MAPK 同工型和转录因子 RUNX-2 在原代人 OA 软骨细胞中的激活的影响。
通过酶消化从 OA 软骨中获得人软骨细胞，用 PE 处理，然后用 IL-1β 刺激。通过 RT-PCR 测量 p38-MAPK 同工型的基因表达。Western 免疫印迹用于分析 MAPK 的激活。免疫沉淀用于确定 p38-MAPK 同工型的激活。使用高度敏感和特异的 ELISA 测定 RUNX-2 的 DNA 结合活性。使用 siRNA 转染进行阐明涉及途径的药理研究。
人 OA 软骨细胞表达 p38-MAPK 同工型 p38α、-γ 和 -δ，但不表达 p38β。IL-1β增强人 OA 软骨细胞中 p38α-MAPK 和 p38γ-MAPK 同工型的磷酸化，但不增强 p38δ-MAPK 同工型的磷酸化。人 OA 软骨细胞中 p38-MAPK 的激活主要通过 MKK3 的激活介导。此外，我们还证明富含多酚的 PE 抑制了 IL-1β 诱导的 MKK3、p38α-MAPK 同工型和转录因子 RUNX-2 的 DNA 结合活性的激活。
我们的结果为报道的石榴提取物对软骨的保护作用和关节炎抑制作用提供了重要的分子基础。PE 对 IL-1β 刺激的人 OA 软骨细胞的这些新的药理作用提示，PE 或 PE 衍生的化合物可能被开发为 MKK 和 p38-MAPK 抑制剂，用于治疗 OA 和其他退行性/炎症性疾病。
