ABSTRACT
Objective To investigate the different regulation of TLR4 between NF-κB and IRF3 signaling pathways which are triggered by LPS. Methods LPS was used to stimulate mouse peritoneal macrophages. Immunofluores-cence was performed to find the translocation of TLR4 and the transcription factors p65 and IRF3. The phosphoryla-tion level of transcription factors was examined by Western blot. Results With stimulation of LPS in 30 minutes, the fluorescence intensity of TLR4 on the cytomembrane was increased (P<0.01),while its fluorescence intensity on the endosome in cytoplasm was increased more robustly(P<0.01),and TLR4 in cytoplasm was found to co-lo-cate with EEA1. When the stimulation reached 90 minutes and 180 minutes,the fluorescence intensity of TLR4 in both cytomembrane and cytoplasm decreased obviously (P<0.01),and the co-location with EEA1 was attenuated present (P<0.01). In the rest state,the fluorescence intensity of the downstream molecule p65 and IRF3 was al-most totally in the cytoplasm. After the stimulation of LPS, fluorescence intensity both in nucleus was increased gradually (P<0.05),but the fluorescence intensity of p65 in nucleus was enhanced earlier and lasted longer than that of IRF3. The phosphorylation of p65 was also earlier and lasted longer than that of IRF3. Conclusions The changed location of TLR4 after being activated can make the transfer time of IRF3 pathway longer and the lasting time shorter than that of NF-κB robustly.
ABSTRACT
Objective To investigate the different regulation of TLR4 between NF-κB and IRF3 signaling pathways which are triggered by LPS. Methods LPS was used to stimulate mouse peritoneal macrophages. Immunofluores-cence was performed to find the translocation of TLR4 and the transcription factors p65 and IRF3. The phosphoryla-tion level of transcription factors was examined by Western blot. Results With stimulation of LPS in 30 minutes, the fluorescence intensity of TLR4 on the cytomembrane was increased (P<0.01),while its fluorescence intensity on the endosome in cytoplasm was increased more robustly(P<0.01),and TLR4 in cytoplasm was found to co-lo-cate with EEA1. When the stimulation reached 90 minutes and 180 minutes,the fluorescence intensity of TLR4 in both cytomembrane and cytoplasm decreased obviously (P<0.01),and the co-location with EEA1 was attenuated present (P<0.01). In the rest state,the fluorescence intensity of the downstream molecule p65 and IRF3 was al-most totally in the cytoplasm. After the stimulation of LPS, fluorescence intensity both in nucleus was increased gradually (P<0.05),but the fluorescence intensity of p65 in nucleus was enhanced earlier and lasted longer than that of IRF3. The phosphorylation of p65 was also earlier and lasted longer than that of IRF3. Conclusions The changed location of TLR4 after being activated can make the transfer time of IRF3 pathway longer and the lasting time shorter than that of NF-κB robustly.