为了探究阿胶不同分子质量组分免疫活性的强弱,通过Superdex 75pg葡聚糖凝胶柱将阿胶分为分子质量<10 kDa、10~30 kDa、>30 kDa(F1、F2和F3)的3个组分,并比较它们刺激RAW264.7小鼠巨噬细胞释放NO和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的能力,然后以免疫活性最强组分为研究对象,研究其对巨噬细胞的激活作用及其机制。结果表明,在同等浓度下,F2组分比F1和F3组分能刺激巨噬细胞释放更多的NO和TNF-α,且其可以激活RAW264.7细胞,促进细胞释放活性氧(reactive oxygen species,ROS)和白细胞介素-6(interleukin-6,IL-6),说明阿胶中10~30 kDa的组分(F2)具有相对较强的免疫活性。此外,实时荧光定量PCR(quantitative real-time PCR,q-PCR)结果表明F2可以促进细胞TNF-α、IL-6、诱导型一氧化氮合酶( inducible nitric oxide synthase ,iNOS)和 Toll 样受体 4(Toll-like receptor 4,TLR4)基因的表达,增加其在细胞内的mRNA含量,说明F2组分能够与TLR4受体结合,并且能够促进细胞中C-Jun 氨基末端蛋白激酶(C-Jun N-terminal protein kainse,JNK)、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)和P38蛋白的磷酸化,表明阿胶中F2组分可以通过丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路发挥免疫调节作用。以上研究结果将为优化阿胶的生产加工条件以制造出高免疫活性阿胶产品提供理论指导。
To identify the immune activity of different molecular weight components of Colla corii asini, it was separated into three components: <10 (F1), 10-30 (F2), and >30 kDa (F3) by Superdex 75pg dextran gel column, and their abilities to stimulate cells to release NO and TNF-α were compared. Then the fraction with the strongest immunomodulatory ability was used to study the activation effect on macrophages and its mechanism. Results showed that F2 could stimulate macrophages to release more NO and TNF-α than F1 and F3 at the same concentration. In addition, it could activate RAW264.7 cells and promote the release of ROS and IL-6. The results of q-PCR showed that F2 could promote the expression of TNF-α, IL-6, iNOS and TLR4 genes in cells and bind to TLR4 receptors. The phosphorylation of JNK, ERK and P38 proteins was promoted, which indicated that F2 played an immunomodulatory role through the MAPK signaling pathway. This research will provide theoretical guidance for optimizing the production and processing conditions of Colla corii asini to produce high-quality product.
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