Qing Rui, Huang Zezhi, Tang Yufei, Xiang Qingke, Yang Fan
Division of Pathogenic Biology, Department of Laboratory Medicine, Shaoyang University, Shaoyang, PR China.
Department of Basic Medicine, Xiangnan University, Chenzhou, PR China.
Int Immunopharmacol. 2017 Jun;47:20-27. doi: 10.1016/j.intimp.2017.03.002. Epub 2017 Mar 27.
The present study is to investigate the effect of cordycepin on the expression of heme oxygenase-1 (HO-1) in lipopolysaccharide (LPS)-activated microphages, as well as its mechanism of action.
Mouse RAW264.7 cells were treated with different concentrations of cordycepin for 0-16h. Western blotting was used to determine the expression of HO-1 and the phosphorylation of c-Src and the p47 subunit of NADPH oxidase. Intracellular reactive oxygen species (ROS) level was determined using HDCFDA as fluorescent probe. Laser-scanning confocal microscopy was used to visualize the nuclear translocation of NF-E2-related factor 2 (Nrf2). Enzyme-linked immunosorbent assay was performed to measure the inhibitory effect of cordycepin on LPS-induced secretion of tumor necrosis factor-α and interleukin-6.
Cordycepin induced the phosphorylation of c-Src and p47 subunit of NADPH oxidase in RAW264.7 cells. Cordycepin increased the secretion of ROS by activating NADPH oxidase. In addition, cordycepin enhanced the expression of HO-1 in RAW264.7 cells in both dose- and time-dependent manners. Of note, elevated HO-1 expression induced by cordycepin treatment was regulated by c-Src/NADPH oxidase/ROS pathway. HO-1 expression induced by cordycepin was dependent on the activation of Nrf2, which was regulated by c-Src/NADPH oxidase/ROS. Cordycepin reduced LPS-induced secretion of proinflammatory cytokines through up-regulation of HO-1.
The present study demonstrates that cordycepin induces the expression of HO-1 in RAW264.7 cells via c-Src/NADPH oxidase/ROS/Nrf2 pathway, and plays an anti-inflammatory role by inhibiting the secretion of cytokines from macrophages.
本研究旨在探讨虫草素对脂多糖（LPS）激活的巨噬细胞中血红素加氧酶-1（HO-1）表达的影响及其作用机制。
用不同浓度的虫草素处理小鼠RAW264.7细胞0至16小时。采用蛋白质免疫印迹法测定HO-1的表达以及c-Src和NADPH氧化酶p47亚基的磷酸化水平。以2',7'-二氯二氢荧光素二乙酸酯（HDCFDA）作为荧光探针测定细胞内活性氧（ROS）水平。利用激光扫描共聚焦显微镜观察NF-E2相关因子2（Nrf2）的核转位情况。采用酶联免疫吸附测定法检测虫草素对LPS诱导的肿瘤坏死因子-α和白细胞介素-6分泌的抑制作用。
虫草素可诱导RAW264.7细胞中c-Src和NADPH氧化酶p47亚基的磷酸化。虫草素通过激活NADPH氧化酶增加ROS的分泌。此外，虫草素以剂量和时间依赖性方式增强RAW264.7细胞中HO-1的表达。值得注意的是，虫草素处理诱导的HO-1表达上调受c-Src/NADPH氧化酶/ROS途径调控。虫草素诱导的HO-1表达依赖于Nrf2的激活，而Nrf2受c-Src/NADPH氧化酶/ROS调控。虫草素通过上调HO-1减少LPS诱导的促炎细胞因子分泌。
本研究表明，虫草素通过c-Src/NADPH氧化酶/ROS/Nrf2途径诱导RAW264.7细胞中HO-1的表达，并通过抑制巨噬细胞分泌细胞因子发挥抗炎作用。
