Mechanisms Of Lanthanum Inhibitting The Production Of Nitric Oxide In Macrophages Of Mice Induced By Lipopolysaccharide

ObjectiveNitric oxide is recognized as a mediator and regulator of inflammatory responses. It possesses cytotoxic properties that are aimed against pathogenic microbes, but it can also have damaging effects on host tissues. NO is synthesized from L-arginine by the nitric oxide synthase (NOS) enzymes. No iNOS (inducible nitric oxide synthase) expression is found in most resting cells. Exposure to microbial products, such as lipopolysaccharide or proinflammatory cytokines, induces the expression of iNOS gene in various inflammatory and tissue cells. High amounts of NO produced by iNOS may result in various pathophysiological effects, such as endotoxin shock and multiple organ failure. Thus, iNOS inhibitors show promising therapeutic potential. Lanthanum, one of the rare earths elements, possesses various biologic effects. Our previous study showed that lanthanum can bind LPS and inhibit the biologic effects of LPS. In the present study, we explored the effects and possible mechanisms of lanthanum chloride on the expression of iNOS in RAW264.7 macrophages with lipopolysaccharide induction.Methods1. The effects of different doses of Lanthanum chloride on proliferation of RAW264.7 macrophages were measured by MTT assay and the optimal dose was chosen.2. The iNOS protein subcellur location and expression were measured by immunofluorescence and western blotting. iNOS gene expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). NO production in culture supernatant was detected by measuring nitrite.3. The effects of Lanthanum chloride on NF-κB activation in RAW264.7 macrophages with Lipopolysaccharide induction were measured by immunofluorescence and western blotting.Results1. 0-40μmol/L of LaCl₃ had no obvious effects on proliferation of RAW264.7 macrophages.2. Immunofluorescence analysis and western blotting detection showed LaCl₃ in low concentration (2.5μmol/L) can remarkably inhibit the expression of iNOS protein in RAW264.7 macrophages induced by LPS.3. The results of RT-PCR showed the expression of iNOS mRNA in RAW264.7 macrophages induced by LPS can be decreased by LaCl₃ significantly.4. NO production assay indicated LaCl₃ can reduce the NO production significantly in RAW264.7 macrophages induced by LPS.5. LaCl₃ inhibited the translocation of NF-κB /p65 from cytoplasm to nuclear in RAW264.7 macrophages induced by LPS.Conclusion1. LaCl₃ downregulates LPS-induced overexpression of iNOS at both gene and protein levels and reduces NO production markedly.2. LaCl₃ inhibits the activation of NF-κB /p65 in RAW264.7 macrophages induced by LPS, which may be the possible mechanism responsible for the inhibition of LaCl₃ on the expression of iNOS and the production of NO.