Regulatory effects of lanthanum chloride on the activation of nuclear factor kappa B inhibitor kinase beta induced by tumor necrosis factor alpha / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).</p><p><b>METHODS</b>(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-κB/p65 in nuclei, and the protein levels of IκBα, phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.</p><p><b>RESULTS</b>(1) High expression of NF-κB/p65 was observed in cytoplasm of control group. High expression of NF-κB/p65 was observed in nuclei of TNF-α group. The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group. The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-κB/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).</p><p><b>CONCLUSIONS</b>LaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.</p>

Inhibitory effect of lanthanum chloride on the expression of inducible nitric oxide synthase in RAW264.7 macrophages induced by lipopolysaccharide / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the influence of lanthanum chloride (LaCl3) on inducible nitric oxide synthase (iNOS) expression in RAW264.7 macrophages with lipopolysaccharide (LPS) induction, and to investigate its possible mechanisms.</p><p><b>METHODS</b>The RAW264.7 macrophages were randomly divided into four groups: i. e, control group (without treatment), LaCl3 group (with treatment of 2.5 micromol/L of LaCl3 for 24 hrs), LaCl3 + LPS group (with treatment of 2.5 micromol/L LaCl3 for 24h), and LPS group (with treatment of 1 mg/L LPS for 24 hrs). The iNOS protein expression was measured by immunofluorescence and Western blot. iNOS gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). NO production in culture supernatant was assayed by nitrate reductase method.</p><p><b>RESULTS</b>Immunofluorescence analysis showed that iNOS was located mainly in the cytoplasm. RAW264.7 cells with overexpression of iNOS accounted for 44.4%, which was obviously higher than that in LaCl3 + LPS group (11.8%, P < 0.05). There was a faint signal of FITC-labeled green tint in control group or LaCl3 group. The iNOS mRNA and protein expression, and the NO content in LPS group were significantly higher than those in control, LaCl3, and LaCl3 + LPS groups (P < 0.05).</p><p><b>CONCLUSION</b>LaCl3 can suppress LPS-induced iNOS overexpression at mRNA and protein level and reduce NO production, indicating that LaCl3 can antagonize the excessive activation of iNOS induced by LPS.</p>

Effects of lanthanum on inhibition of lipopolysaccharide induced NF-kappaB activation / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of lanthanum on lipopolysaccharide (LPS) induced NF-KB activation in murine peritoneal macrophage.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>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.</p>