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(0.5μg/m L)刺激组和不同剂量(5、25、125μg/m L)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i NOS m RNA表达,Western blot检测胞浆i NOS蛋白和胞浆与胞核NF-κB/p65蛋白表达,Trans AMTM NF-κB/p65活性检测试剂盒测定NF-κB/p65 DNA结合活性。结果显示,0.5μg/m L LPS以及不同剂量(5、25、125μg/m L)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i NOS m RNA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。
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 μg/mL） or different doses of Sal （5, 25, 125 μg/mL） ＋ LPS （0.5μg/mL）. CCK-8 colorimetric method was used to detect the cell activity. The enzyme-linked immunosor- bent assay （ELISA） was used to detect the contents of TNF-a, MCP-1 and MIP-2 in the supematant, and the content of NO in the supematant 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-kB/p65） protein in both cyto- plasm and nucleus, and DNA binding activity of NF-kB/p65 was detected by using TransAMTM NF-kB/p65 activity assay kit. The results showed that the treatment with 0.5μg/mL LPS and different doses of Sal （5, 25, 125 μg/mL） for 12 h had no effect on cell viability. Compared with LPS stimulation group, pretreatment with Sal significantly reduced the contents of TNF-a, MCP-1, MIP-2 and NO in culture supematant 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-kB/p65 protein in nucleus （P 〈 0.05） while accordingly increased that in cytoplasm （P 〈 0.05）, and decreased DNA binding activity of NF-kB/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-kB signaling pathway, thereby reducing the excessive expression and secretion of inflammatory mediators and cytokines.
作者
黄倩
胡晓兰
HUANG Qian HU Xiao-Lan(Department of Physiology, Zhejiang University School of Medicine, Hangzhou 310058, China)
出处
《生理学报》
CAS
CSCD
北大核心
2017年第1期41-46,共6页
Acta Physiologica Sinica
基金
supported by grants from the National Natural Science Foundation of China(No.81173466 and 81072415)
the Natural Science Foundation of Zhejiang Province
China(No.Y15H180019)
关键词
红景天苷
脂多糖
巨噬细胞
炎症介质
salidroside
lipopolysaccharide
macrophage
inflammatory mediator
