黄芪多糖诱导SOCS3表达对鸡巨噬细胞炎症反应的抑制作用

doi:10.16431/j.cnki.1671-7236.2021.11.038

摘要/Abstract

摘要： 试验旨在探究黄芪多糖（Astragalus polysaccharide，APS）对LPS诱导的鸡巨噬细胞（HD11）炎症模型的抗炎效果和作用机制。用不同浓度的LPS刺激鸡巨噬细胞（HD11），通过CCK8法检测细胞活力，实时荧光定量PCR检测白细胞介素-1β（IL-1β） mRNA表达的变化，以确定构建细胞炎症模型的LPS最适添加浓度。将HD11分为对照组（C）、脂多糖（LPS）组（L）、黄芪多糖（APS）组（A）和黄芪多糖抑制脂多糖组（A+L），在LPS刺激后的2、6、12、24、48 h用实时荧光定量PCR法检测IL-1β、肿瘤坏死因子-α（TNF-α）、核转录因子（NF-κBp65）、p38丝裂原活化蛋白激酶（p38MAPK）和细胞因子信号转导抑制因子3（SOCS3） mRNA表达的变化，Western blotting法检测NF-κBp65、p38MAPK和SOCS3蛋白含量变化，ELISA检测IL-1β和TNF-α蛋白含量变化。细胞活力和IL-1β检测结果表明，构建细胞炎症模型的LPS最适添加浓度为0.5 μg/mL。实时荧光定量PCR结果表明，与对照组相比，L和A组IL-1β、TNF-α、NF-κBp65、p38MAPK和SOCS3 mRNA表达均显著升高（P<0.05）；与L组相比，A+L组在LPS刺激后的IL-1β、TNF-α、NF-κBp65和p38MAPK mRNA表达含量均显著降低（P<0.05），SOCS3 mRNA表达显著增加（P<0.05）。Western blotting结果表明，与对照组相比，A组P-NF-κBp65/NF-κBp65、P-p38MAPK/p38MAPK和SOCS3/α-tubulin比值均显著增加（P<0.05）；与L组相比，A+L组P-NF-κBp65/NF-κBp65和P-p38MAPK/p38MAPK比值均显著降低（P<0.05）；A+L组SOCS3/α-tubulin比值显著升高（P<0.05）。ELISA结果表明，与对照组相比，L组IL-1β和TNF-α蛋白含量在2~48 h均显著增加（P<0.05）；与L组相比，A+L组IL-1β和TNF-α的蛋白含量在LPS刺激后12~48 h均显著降低（P<0.05）。以上结果表明，在LPS诱导的HD11细胞炎症模型中，APS通过促进SOCS3的表达抑制NF-κBp65和p38MAPK信号通路的过度活化，从而发挥抗炎作用。

关键词: 黄芪多糖; 抗炎; 细胞因子信号转导抑制因子3(SOCS3)

Abstract: The aim of this study was to investigate the anti-inflammatory effect and action mechanism of Astragalus polysaccharide (APS) on LPS-induced chicken macrophage (HD11) inflammation model. HD11 cells were stimulated with different concentrations of LPS. Cell viability was detected by CCK8 assay and interleukin-1β(IL-1β) mRNA expression was detected by Real-time quantitative PCR in order to determine the optimal concentration of LPS for the construction of cellular inflammation model. HD11 cells were divided into control group (C), lipopolysaccharide (LPS) group (L), APS group (A) and APS inhibited LPS group (A+L). The mRNA expressions of IL-1β, tumor necrosis factor-α (TNF-α), NF-κBp65, p38MAPK and suppresser of cytokine signaling 3 (SOCS3) were detected by Real-time quantitative PCR, the protein contents of NF-κBp65, p38MAPK and SOCS3 were detected by Western blotting, and the protein contents of IL-1β and TNF-α were detected by ELISA at 2, 6, 12, 24 and 48 h after LPS stimulation. The results of cell viability and IL-1β detection showed that the optimal addition concentration of LPS for construction of cellular inflammation model was 0.5 μ g/mL LPS. Real-time quantitative PCR results showed that compared with control group, the mRNA expressions of IL-1β, TNF-α, NF-κBp65, p38MAPK and SOCS3 in L and A groups were significantly increased (P<0.05). Compared with L group, the mRNA expression levels of IL-1β, TNF-α, NF-κBp65 and p38MAPK in A+L group were significantly decreased (P<0.05), and the mRNA expression level of SOCS3 was significantly increased (P<0.05). Western blotting results showed that compared with control group, the ratios of P-NF-κBp65/NF-κBp65, P-p38MAPK/p38MAPK and SOCS3/α-tubulin in A group were significantly increased (P<0.05). Compared with L group, the ratios of P-NF-κBp65/NF-κBp65 and P-p38MAPK/p38MAPK in A+L group were significantly decreased (P<0.05), and SOCS3/α-tubulin ratio in A+L group was significantly increased (P<0.05). ELISA results showed that compared with control group, the protein contents of IL-1β and TNF-α in L group were significantly increased within 2-48 h (P<0.05), the protein contents of IL-1β and TNF-α in A+L group were significantly decreased within 12-48 h after LPS stimulation when compared with L group (P<0.05). The results suggested that in the LPS-induced HD11 cell inflammation model, APS could inhibit the overactivation of NF-κBp65 and p38MAPK signaling pathways by promoting SOCS3 expression, and thus played an anti-inflammatory role.

Key words: Astragalus polysaccharide; anti-inflammatory; suppresser of cytokine signaling 3 (SOCS3)

中图分类号:

S831.7

陶新磊, 刘丹华, 田旭, 郑世民, 高雪丽, 吕晓萍, 刘超男. 黄芪多糖诱导SOCS3表达对鸡巨噬细胞炎症反应的抑制作用[J]. 中国畜牧兽医, 2021, 48(11): 4284-4291.

TAO Xinlei, LIU Danhua, TIAN Xu, ZHENG Shimin, GAO Xueli, LYU Xiaoping, LIU Chaonan. Inhibitory Effect of Astragalus Polysaccharide on Inflammatory Response of Chicken Macrophages by Inducing SOCS3 Expression[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4284-4291.

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