Anti-inflammatory Activities Of Compounds Of N-2 And BDM And Their Underlying Mechanisms Of Action

Objective: In this paper,the two compounds of N-2(9-(4-Fluoro-phenyl)-5,8,9,10-tetrahydro-4H-oxa-2,3,9,11b-tetraaza-cyclopenta [a] anthracene)and BDM(9-buty-9,10-dihydrochromeno[8,7-e][1,3]oxazin-2[8H]-one),which were found in the previous experiment,were used to explore the anti-inflammatory activities and their mechanisms.Methods: 1.In this part,we built LPS-stimulated murine RAW264.7 macrophages model in vitro,ELISA was used to detect the expression of TNF-α and IL-6,while western blotting was used to measure the protein of ERK1/2,JNK,p38 MAPK,NF-κB p65,IκBαand its phosphorylation.2.We used a LPS-induced ALI mouse model to investigate the effect of N-2 and BDM in vivo.The production of TNF-α and IL-6 was measured by ELISA.The lung MPO activity and wet-to-dry weight ratios were measured in LPS-induced mice,while the lung histopathologic changes observed via paraffin section were assessed.To further study the mechanism of N-2 effects on ALI,the protein of ERK1/2,JNK,p38 MAPK,NF-κB p65,IκBα and its phosphorylation was detected by western blot.Results: 1.RAW264.7 macrophages treated with LPS alone for 24 h produced significant amounts of TNF-α and IL-6 compared to the control group in vitro.However,the production of TNF-α and IL-6 were significantly inhibited by treating with N-2 and BDM.The results of western blot indicated that the different concentrations of N-2 inhibited the phosphorylation of ERK1/2,p38 MAPK,NF-κB p65 and IκBα degradation,and the expression of non-phosphorylated ERK1/2,p38 MAPK and NF-κB p65 was no changes among groups.Furthermore,treatment of cells with the BDM before LPS stimulated inhibited the proteins of MAPKs,but had no effect on NF-κB pathway.2.The levels of TNF-α and IL-6 in BALF were increased dramatically compared with control group.However,pretreatment with N-2 significantly down-regulated the levels of TNF-α and IL-6 compared with LPS group.Moreover,treatment with N-2 attenuated LPS-induced pulmonary inflammation by suppressing the MPO activity in the lung tissue,reducing the lung wet/dry weight rations,as well as ameliorating the pulmonary histopathological changes by LPS challenge.Furthermore,the results of western blot indicated that the phosphorylation of ERK1/2,p38 MAPK and IκBα degradation was inhibited afterpretreatment with N-2.But,the role of BDM in vivo did not continue to study due to its toxicity in mice.Conclusions: Our study demonstrated that compound of N-2 had better anti-inflammatory effects compared to compound of BDM.At the same time,N-2 exerted anti-inflammatory role in vitro and in vivo and might be provided a theoretical basis and experimental basis for further study.