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红景天苷对脂多糖诱导小鼠巨噬细胞炎性介质分泌的影响

黄倩, 胡晓兰^*

浙江大学医学院生理学系，杭州 310058

摘要

本文旨在探讨红景天苷(salidroside, Sal)对脂多糖(lipopolysaccharide, LPS)诱导小鼠巨噬细胞系J774.1炎性活化的影响及其可能机制。J774.1细胞分为PBS对照组、LPS (0.5 &micro;g/mL)刺激组和不同剂量(5、25、125 &micro;g/mL)Sal预处理+LPS组。CCK-8比色法检测细胞活性，ELISA测定培养上清中TNF-α、MCP-1和MIP-2含量，硝酸还原酶法测定上清中NO含量，RT-PCR检测细胞iNOS mRNA表达，Western blot检测胞浆iNOS蛋白和胞浆与胞核NF-κB/p65蛋白表达，TransAMTM NF-κB/p65活性检测试剂盒测定NF-κB/p65 DNA结合活性。结果显示，0.5 &micro;g/mL LPS以及不同剂量(5、25、125 &micro;g/mL) Sal处理细胞12 h对J774.1细胞活力无影响；与LPS刺激组比较，LPS刺激前Sal预处理J774.1细胞，培养上清中TNF-α、MCP-1、MIP-2和NO含量呈剂量依赖性降低(P < 0.05)，细胞iNOS mRNA和蛋白表达水平下调(P < 0.05)，胞核NF-κB/p65蛋白表达降低(P < 0.05)而胞浆NF-κB/p65蛋白相应增加(P < 0.05)，且NF-κB/p65 DNA结合活性呈剂量依赖性降低(P < 0.05)。以上结果提示，Sal预处理能够降低LPS诱导的巨噬细胞炎性活化，其机制可能通过干扰LPS/TLR4/NF-κB信号通路，从而降低炎性介质及细胞因子的过度表达和分泌。

关键词： 红景天苷; 脂多糖 ; 巨噬细胞 ; 炎症介质

分类号：Q291

Effects of salidroside on the secretion of inflammatory mediators induced by lipopolysaccharide in murine macrophage cell line J774.1.

HUANG Qian, HU Xiao-Lan^*

Department of Physiology, Zhejiang University School of Medicine, Hangzhou 310058, China

Abstract

To investigate the effect of salidroside (Sal) on the inflammatory activation of lipopolysaccharide (LPS)-induced murine macrophage cell line J774.1 and its possible mechanism, the cells were treated with PBS, LPS (0.5 &micro;g/mL) or different doses of Sal (5, 25, 125 &micro;g/mL) + LPS (0.5 &micro;g/mL). CCK-8 colorimetric method was used to detect the cell activity. The enzyme-linked immunosorbent assay (ELISA) was used to detect the contents of TNF-α, MCP-1 and MIP-2 in the supernatant, and the content of NO in the supernatant was determined by nitrate reductase method. The expression levels of iNOS mRNA was detected by RT-PCR. Western blot was used to detect the expression levels of iNOS protein in cytoplasm and NF-kappaB/p65 (NF-κB/p65) protein in both cytoplasm and nucleus, and DNA binding activity of NF-κB/p65 was detected by using TransAMTM NF-κB/p65 activity assay kit. The results showed that the treatment with 0.5 &micro;g/mL LPS and different doses of Sal (5, 25, 125 &micro;g/mL) for 12 h had no effect on cell viability. Compared with LPS stimulation group, pretreatment with Sal significantly reduced the contents of TNF-α, MCP-1, MIP-2 and NO in culture supernatant induced by LPS in a dose dependent manner (P < 0.05), downregulated the expression levels of iNOS mRNA and protein (P < 0.05), decreased the expression level of NF-κB/p65 protein in nucleus (P < 0.05) while accordingly increased that in cytoplasm (P < 0.05), and decreased DNA binding activity of NF-κB/p65 in a dose dependent manner (P < 0.05). The results suggested that Sal pretreatment can reduce macrophage inflammatory activation induced by LPS, and the mechanism may be through the LPS/TLR4/NF-κB signaling pathway, thereby reducing the excessive expression and secretion of inflammatory mediators and cytokines.

Key words: salidroside; lipopolysaccharide ; Macrophage ; inflammatory mediator

收稿日期：2016-08-27　　录用日期：2016-10-31

通讯作者：胡晓兰　　E-mail: huxiaolan@zju.edu.cn

引用本文：

黄倩, 胡晓兰. 红景天苷对脂多糖诱导小鼠巨噬细胞炎性介质分泌的影响[J]. 生理学报 2017; 69 (1): 41-46.

HUANG Qian, HU Xiao-Lan. Effects of salidroside on the secretion of inflammatory mediators induced by lipopolysaccharide in murine macrophage cell line J774.1.. Acta Physiol Sin 2017; 69 (1): 41-46 (in Chinese with English abstract).