Objective: To investigate the influence of lanthanum on lipopolysaccharide (LPS) induced NF-KB activation in murine peritoneal macrophage.
Methods: Peritoneal macrophages were isolated and cultured by routine method, and randomly divided into 5 groups: i. e, control group, LPS group (with LPS stimulation for 30 min), La3+ group (with 2.5 micromol/L La3+ group for 30 min) , La3+ + LPS group( with 1 microg/ml LPS stimulation for 30 min after 30 min incubation with DMEM-F12 containing 2.5 microM of lanthanum.) ; La3+/LPS group (with 2.5 microM of lanthanum stimulation for 30 min, and then with 1 microg/ml of LPS for another 30 min after lanthanum was removed. The location of NF-kappaB p65 subunit (NF-kappaB/p65) in Mphi was detected by immunofluorescence and fluorescence microscope. The binding activity of NF-kappaB/p65 with DNA in nuclei was detected by TransAMTM NF-kappaB/p65 Transcription Factor assay kit. Meanwhile, the expression of NF-kappaB/p65 in nuclei, as well as IkappaBalpha in cytoplasm was measured by Western blotting. TNF-alpha content in culture supernatant were detected by ELISA.
Results: (1) The green fluorescence in control, La3+, La3+ LPS and La+/LPS groups was mainly located in cytoplasm, while that in LPS group was located in nuclei. The fluorescent intensity in LPS group was (116 +/- 14), which was obviously higher than that in other 4 groups (42 +/-7,73 +/-30,48 +/- 11 and 67 +/- 19, respectively, P <0.01). (2) The IkappaBalpha protein level in cytoplasm in control (0.048 +/- 0.027), La3+ group (0.062 +/- 0.049), La3+ + LPS group (0.066 +/-0.031) and La3+/LPS group (0.108 +/- 0.017) was significantly lower than that in LPS group (0.435 +/-0.066, P <0.01). (3) The expression and activation of nucleus p65 protein in Mphi in LPS group was obviously higher than the other 4 groups, but changes in the IkappaBalpha expression between LPS group and other 4 groups was of controversy. (4) TNFalpha level in the culture supernatant in La3+ group was lower than that in control group ( P < 0.05) and below the detection limit (25 pg/ml). Moreover, it in La3+ + LPS group and La3*/LPS group was lower than that in LPS group (P <0.01), but higher than that in control group.
Conclusion: LPS can activate the nucleus translocation of NF-kappaB/p65 in Mphi of mice, increase NF-KB/p65 expression and activity, but reduce IkappaBalpha protein expression, which lead to increase of TNFalpha secretion. Lanthanum can inhibit lipopolysaccharide induced NF-kappaB activation.