λ-卡拉胶引发THP-1细胞免疫响应的结构因素及机制

Structural Factors and Mechanisms of λ-Carrageenan Induce Immune Response in THP-1 Cells

目的:探讨λ-卡拉胶及其降解物引发THP-1单核/巨噬细胞免疫响应的结构因素及其反应机制。方法:THP-1细胞经PMA诱导分化为巨噬细胞;λ-卡拉胶及其不同分子质量的降解产物刺激THP-1细胞,采用ELISA技术检测TNF-α含量,确定引发免疫响应的λ-卡拉胶种类;采用CBA技术检测卡拉胶处理后各类免疫因子的表达;用WesternBlot检测由卡拉胶引起的免疫响应的信号通路。结果:分子质量10～40ku的卡拉胶降解产物(λ-10-40k)最易引起THP-1细胞的免疫响应,用10μg/mLλ-10-40k处理24h,可增加TNF-α的分泌量,为空白对照的2.2倍(P<0.01)。λ-10-40k可通过激活TLR4-NF-κB途径以及MAPK/ERK途径诱导THP-1细胞产生TNF-α,并通过协同效应,增加LPS刺激的TNF-α、IL-1β及IL-6的表达,放大LPS诱导的炎症反应。结论:分子质量范围10～40ku的卡拉胶降解产物易通过TLR4-NF-κB途径及MAPK/ERK途径引发THP-1单核/巨噬细胞免疫响应,并协同增加LPS刺激的炎症反应。

Objective： To explore the structural factors and mechanisms of λ-carrageenan and its degraded forms to induce immune response of THP-1 ceils. Methods： PMA-activated macrophage-like THP-1 cells were treated with 10 μg/mL λ-carrageenan and its degraded forms （dCGN） for 24h. Cell viabihty was determined by MTT colormetric survival assay. The secretion of tumor necrosis factor-α （TNF-α） was measured by enzyme-linked immunosorbent assay（ELISA）. Immune cytokines production was detected by Cytometric Bead Array （CBA）. The expression of immune relative proteins was assessed by Western blot. Results： Low concentration of h-carrageenans（〈20 μg/mL） showed little effect on the via- bility of THP-1 macrophages. The degraded λ-carrageenan with molecular weight of 10-40 ku （λ-10-40k） exhibited the strongest effect to up-regulate the secretion of TNF-α. It could stimulated TNF-α secretion 2.2-fold higher than control （P〈0.01）. TLR4-NF-κB and MAPK/ERK-mediated pathway may involve in λ-10-40k induced TNF-α secretion. In ad- dition, λ-10-40k could coordinated enhance the production of immune cytokines induced by LPS in macrophage-like THP-1 cells. Conclusions： These results indicate that degraded carrageenans with molecular weight of 10-40 ku is more likely to induce immune response through TLR4-NF-κB and MAPK/ERK pathway in THP-1 macrophage cells, λ-10-40k can also synergetically increase the inflammatory effect of LPS.