摘要
To investigate the anti-inflammatory mechanism of curcumin in THP-1 cells induced by LPS, the effect of curcumin on inflammatory cytokine mRNA expression was assessed by Real-time PCR. Covalent post-translational modification profiles were determined by a PathScan? Kit. Western blotting analysis verified the PathScan results and the effects of curcumin on nucleus-related protein changes. Annexin V-FITC and PI staining was used to detect early cellular apoptosis by flow cytometry. Anti-inflammatory effects of curcumin were mediated by reducing the levels of IL-1β, IL-6, TNF-α and ICAM-1 mRNA. However, PathScan and Western blotting results showed that curcumin inhibited inflammatory cytokine mRNA expressions by preventing p65 nuclear translocation without interfering with p38 and JNK/MAPK phosphorylation or IκBα degradation. Moreover, curcumin promoted PARP-1 cleavage and inhibited intranuclear lamina protein Lamin B1 expression. Flow cytometry results showed that curcumin enhanced Annexin V-FITC positive cells when LPS induced inflammation. This study provided evidence that curcumin inhibited p65 nuclear translocation and cytokine transcription by regulating nuclear protein structure and function, suggesting that early apoptosis involved the suppression of p65 nuclear translocation.
为进一步研究姜黄素抑制LPS诱导的THP-1细胞炎症机制,采用Real-time PCR的方法检测姜黄素对细胞炎症因子mRNA表达的影响。PathScan?试剂盒检测细胞翻译后共价修饰蛋白的变化,并用western blot确认PathScan的结果以及检测细胞核相关蛋白表达的变化。THP-1细胞经AV-PI染色后流式细胞术检测细胞凋亡情况。结果显示姜黄素可明显降低炎症因子IL-1β, IL-6, TNF-α和ICAM-1 mRNA的表达水平。PathScan和western blot结果显示姜黄素抑制了p65转录因子的核转移,而对p38和JNK/MAPK磷酸化、IκBα降解没有抑制作用;姜黄素促进了PARP-1蛋白的剪切、抑制核纤层LaminB1蛋白的表达。流式细胞术显示姜黄素增加了AV-PI阳性细胞比例。本研究表明,姜黄素抑制p65核转移以及炎症因子的转录,是由于对核蛋白结构和功能的调节导致的,凋亡通路参与了姜黄素对p65核转移的抑制作用。
基金
National Natural Science Foundation of China(Grant No.81202543)
