摘要
Nuclear factor-κB(NF-κB), a transcription factor, which plays a pivotal role in the expression of a wide variety of genes, involves in systemic inflammatory response syndrome and is the new target for therapy. To explore the effects and mechanism of lanthanum on nuclear translocation of NF-κB in macrophage of mice(Mφ), fluorescent antibody technique, western blotting and ELISA(enzyme-linked immunosorbent assay) were employed. Mφ were divided into five groups at random: one was the control group, in which the cells were cultured in medium without any stimulation, one was LPS (lipopolysaccharide) group, in which the cells were incubated with 1 μg·ml -1 LPS for 30 min and one was LaCl_3 group, in which cells were incubated with 2.5 μm of lanthanum chloride for 30 min. The forth group was named as LaCl_3+LPS group, in which cells were incubated with 2.5 μm of lanthanum chloride for 30 min before stimulated with LPS(1 μg·ml -1, 30 min), and the fifth group was known as LaCl_3/LPS group, in which the cells were cultured in medium containing 2.5 μm of lanthanum chloride at first, then the cells were incubated with LPS(1 μg·ml -1, 30 min) after lanthanum chloride was removed from the media. The location of NF-κB p65 subunit (NF-κB/p65) in Mφ was detected by immunofluorescence and fluorescence microscope. The activation of NF-κB/P65 in nuclei was detected by TransAM TM NF-κB/P65 Transcription Factor assay kit. Meanwhile, the expression of NF-κB/p65 in nuclei and that of IκBα in cytoplasm were measured by Western blotting analysis. The majority of FITC-labeled NF-κB/p65 was located in the nuclei stimulated with LPS. However, there was less fluorescence seen in the nuclei of control group, LaCl_3 group, LaCl_3 + LPS group and LaCl_3/LPS group. Compared with LPS group, the IκBα protein level in cytoplasm increase in LaCl_3 group, LaCl_3+LPS group and LaCl_3/LPS group. The expression and activation of nuclei p65 protein decrease significantly in LaCl_3 group, LaCl_3+LPS group and LaCl_3/LPS group, compare with LPS group. All the results indicate that lanthanum inhibits the nuclear translocation of NF-κB induce by LPS, and the effects are partly due to the upregulation of IκB.
Nuclear factor-κB(NF-κB), a transcription factor, which plays a pivotal role in the expression of a wide variety of genes, involves in systemic inflammatory response syndrome and is the new target for therapy. To explore the effects and mechanism of lanthanum on nuclear translocation of NF-κB in macrophage of mice(Mφ), fluorescent antibody technique, western blotting and ELISA(enzyme-linked immunosorbent assay) were employed. Mφ were divided into five groups at random: one was the control group, in which the cells were cultured in medium without any stimulation, one was LPS (lipopolysaccharide) group, in which the cells were incubated with 1 μg·ml -1 LPS for 30 min and one was LaCl_3 group, in which cells were incubated with 2.5 μm of lanthanum chloride for 30 min. The forth group was named as LaCl_3+LPS group, in which cells were incubated with 2.5 μm of lanthanum chloride for 30 min before stimulated with LPS(1 μg·ml -1, 30 min), and the fifth group was known as LaCl_3/LPS group, in which the cells were cultured in medium containing 2.5 μm of lanthanum chloride at first, then the cells were incubated with LPS(1 μg·ml -1, 30 min) after lanthanum chloride was removed from the media. The location of NF-κB p65 subunit (NF-κB/p65) in Mφ was detected by immunofluorescence and fluorescence microscope. The activation of NF-κB/P65 in nuclei was detected by TransAM TM NF-κB/P65 Transcription Factor assay kit. Meanwhile, the expression of NF-κB/p65 in nuclei and that of IκBα in cytoplasm were measured by Western blotting analysis. The majority of FITC-labeled NF-κB/p65 was located in the nuclei stimulated with LPS. However, there was less fluorescence seen in the nuclei of control group, LaCl_3 group, LaCl_3 + LPS group and LaCl_3/LPS group. Compared with LPS group, the IκBα protein level in cytoplasm increase in LaCl_3 group, LaCl_3+LPS group and LaCl_3/LPS group. The expression and activation of nuclei p65 protein decrease significantly in LaCl_3 group, LaCl_3+LPS group and LaCl_3/LPS group, compare with LPS group. All the results indicate that lanthanum inhibits the nuclear translocation of NF-κB induce by LPS, and the effects are partly due to the upregulation of IκB.
基金
ProjectsupportedbytheNationalNaturalScienceFoundationofChina(30260107)andNaturalScienceFoundationofJiangxProvince(024008)
