25(OH)D3 and 1.25(OH)2D3 inhibits TNF-α expression in human monocyte derived macrophages

PurposeWe wanted to investigate effects of vitamin D3 (25(OH)D3 and 1.25(OH)2D3) on inflammatory cytokine expression in both activated and non-activated Mφ.Materials and methodsMononuclear cells, isolated from healthy donor buffy coats were cultured for a 6-day differentiation-period. Fully differentiated Mφ were pre-treated with either 25(OH)D3 or 1.25(OH)2D3 for (4, 12 or 24 hours) +/-LPS challenge for 4 hours. Gene expression analyses of VDR, Cyp27b1 and pro-inflammatory markers TNF-α, IL-6, NF-κB, MCP-1, was performed using RT-quantitative PCR. TNF-α protein levels from Mφ culture media were analysed by ELISA.ResultsBoth 25(OH)D3 and 1.25(OH)2D3 significantly inhibited TNF-α expression in both LPS-stimulated and unstimulated Mφ. Also, NF-κB, and to a lesser extend IL-6 and MCP-1 were inhibited. LPS up-regulated Cyp27b1 gene expression which was partly reverted by 1.25(OH)2D3.ConclusionThese data show anti-inflammatory effects of vitamin D3 (25(OH)D3 and 1.25(OH)2D3) in human macrophages, and support, that means for targeting high dose vitamin D3 to the immune system may have beneficial clinical effect in inflammatory conditions.

研究目的：本研究旨在探究维生素D3的两种活性形式——25(OH)D3与1,25(OH)₂D₃，对活化与非活化巨噬细胞（Macrophage，简称Mφ）中炎症细胞因子表达的影响。材料与方法：从健康供体血沉棕黄层（buffy coat）分离得到的单个核细胞，经6天培养诱导分化为成熟巨噬细胞。将完全分化的巨噬细胞分别用25(OH)D3或1,25(OH)₂D₃预处理4、12或24小时，随后予以脂多糖（LPS，Lipopolysaccharide）刺激4小时或不予以刺激。采用逆转录定量PCR（RT-quantitative PCR）检测维生素D受体（VDR，Vitamin D Receptor）、Cyp27b1基因以及促炎标志物肿瘤坏死因子-α（TNF-α，Tumor Necrosis Factor-α）、白细胞介素-6（IL-6，Interleukin-6）、核因子κB（NF-κB，Nuclear Factor κB）、单核细胞趋化蛋白-1（MCP-1，Monocyte Chemoattractant Protein-1）的基因表达水平；通过酶联免疫吸附试验（ELISA，Enzyme-Linked Immunosorbent Assay）检测巨噬细胞培养上清液中TNF-α的蛋白含量。结果：25(OH)D3与1,25(OH)₂D₃均可显著抑制脂多糖刺激及未刺激状态下巨噬细胞的TNF-α表达；此外，NF-κB的活性亦受到抑制，IL-6与MCP-1的表达也受到一定程度的抑制。脂多糖可上调Cyp27b1的基因表达，而1,25(OH)₂D₃可部分逆转该上调效应。结论：本研究数据证实，维生素D3的两种活性形式25(OH)D3与1,25(OH)₂D₃对人源巨噬细胞具有抗炎作用，该结果支持"高剂量维生素D3靶向调节免疫系统，或可在炎症性疾病中产生有益临床疗效"这一观点。