The Experimental Study Of Interleukin-10 And Nitric Oxide Inhibiting Activation Of Nuclear Factor-κB And Production Of Tumor Necrosis Factor In Lipopolysaccharide-induced Pulmonary Alveolar Macrophages

Abstract: acute lung injury (ALI) is a acute progressive ischemic respiratory failure induced by various reasons except cardiogenic factors, and its serious stage is acute respiratory distress syndrome (ARDS). At present, there are still no satisfactory therapeutic methods and its mortality is often highly to 50%-70%. Lipopolysaccharide (LPS), major component of the outer membrane of gram-negative bacteria, has an important role to ALI/ARDS. As confirmed, the pulmonary alveolar macrophage(PAM) is primary inflammatory cell during the earlier period of ARDS, which induced by LPS release generous proinflammatory cytokines, mainly tumor necrosis factor- a (TNF- a), and lead to uncontrolled inflammatory reaction. The nuclear factor- k B (NF- k B) is a sequence-specific DNA-binding protein that initiates transcription from a variety of genes that are all involved in immune response, acute phase, and inflammatory processes, whose relations to ALI have been paid close attention. IL-10 can inhibit the release of multiple inflammatory mediums and reduce the inflammatory reaction, but there is unknown about its mechanism of anti-inflammation about PAM; although NO is known as the newly method to ARDS, there are not many reports about its affections to the inflammation of ALL So we observed activation of NF- K B and the expression of TNF- a in LPS-induced PAM stimulated with IL-10 and NO to investigate the effects of IL-10 and NO on ALI/ARDS.Methods: PAM of rabbits collected by BALT were cultured and divided into four groups: (1) control group; (2) LPS group(induced by LPS 10 u g ?mL-1); (3) IL-10+LPS group(induced by rhIL-10 10 ng ?mL-11 and LPS 10 u g ?mL-1); (4) NO+ LPS group (induced by InM SNAP and LPS 10u g ?mL-1). The NF- k B activity of nuclear protein extract from the PAM and the concentration of TNF- a in the supernatant were measured by electrophoretic mobility shift assay (ESMA)and ELISA after induced in (K 0.5, 1, 2> 4hour.Results: CD The activation of NF- K B and concentration of TNF- a in LPS group during 0.5-4h are obviously increased. Especially at lh,the NF- k B activity is increased from 658.3?7.8 to 4267.9?9.9(P<0.01), concentration of TNF-a from 134.1 ?.8 to 499.9+18.6 pg ?mL-1(P<0.01), and the color of shifted band becomes more deep; (2) Compared with LPS group, NF-k B activity were both significantly lowered in IL-10+LPS group and NO+ LPS group at Ih, IL-10+LPS group is lowered to 2618.4+ 148.8(P<0.01) and NO+LPS group to 3046.2 + 80.3 (P<0.01), the color of shifted bands are weakened; (3)the concentrations of TNF- a have the similar results.Conclusion: Based on these results, we conclude that LPS might active NF-k B in the PAMs and lead to the increasement of transcription and expression of TNF- a gene, which may be the key point of releasing of various inflammatory mediums in ALI; IL-10 and NO could lower the release of TNF- a by inhibiting the activation of NF- K B to improve the LPS-induced ALI.