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 ex...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.展开更多
Nitric oxide（NO）and its reaction products were key players in the pathophysiology of sepsis and shock.The present study was designed to explore the effects of lanthanum chloride（LaCl3）on inducible nitric oxide syn...Nitric oxide（NO）and its reaction products were key players in the pathophysiology of sepsis and shock.The present study was designed to explore the effects of lanthanum chloride（LaCl3）on inducible nitric oxide synthase（iNOS）expression,at both gene and protein levels,in RAW264.7 macrophages induced by Lipopolysaccharide（LPS）.Reverse transcription polymerase chain reaction（RT-PCR）,immunofluorescence,and western blot were employed to measure iNOS gene expression,localization,and protein expression respectively.NO production in culture supernatants was detected by the nitrate reductase method.The results showed that LaCl3 significantly attenuated the iNOS gene and protein expression,as well as NO production in RAW264.7cells induced by LPS.展开更多
文摘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Project supported by the National Natural Science Foundation of China(30660182)the Program for Innovative ResearchTeam of Nanchang University
文摘Nitric oxide（NO）and its reaction products were key players in the pathophysiology of sepsis and shock.The present study was designed to explore the effects of lanthanum chloride（LaCl3）on inducible nitric oxide synthase（iNOS）expression,at both gene and protein levels,in RAW264.7 macrophages induced by Lipopolysaccharide（LPS）.Reverse transcription polymerase chain reaction（RT-PCR）,immunofluorescence,and western blot were employed to measure iNOS gene expression,localization,and protein expression respectively.NO production in culture supernatants was detected by the nitrate reductase method.The results showed that LaCl3 significantly attenuated the iNOS gene and protein expression,as well as NO production in RAW264.7cells induced by LPS.
