Studying On The Molecular Mechanism Of7b Suppressing Inflammatory Responses

Amonafide was one of the most active naphthalimide-based topo Ⅱ inhibitors and had been tested in clinical trials. On account of toxicity in solid tumors, the clinical development was regrettably terminated. Cause of its tosicity is mainly due to the amino group at the5-position of benzen ring.7b, as a novel naphthalimide derivative, has showed well anti-tumor activities, which is mediated by ROS-mediated apoptosis and p21-mediated G1cell arrest. Meanwhile,7b also has potential anti-inflammatory activity, but the molecular mechanism is not clear. In the present study, we used mouse macrophage cell lilne RAW264.7, which can be stimulated with LPS to mimic a state of inflammation, to demonstrate the molecular mechanism of inflammatory activity.In the initial experiments, the production of NO and prostaglandin E2(PGE2) was inhibited by7b pretreatment and suggested the possibility of down-regulating their respective genes, iNOS and COX-2. Reverse transcription-polymerase chain reaction and Western blot analysis revealed that7b can inhibit mRNA and protein expression of COX-2and iNOS. In addition,7b reduced the production of pro-inflammatory cytokines, including those of tumor necrosis factor-α (TNF-α), interleukin-1β, and interleukin-6(IL-6), and increased the production of anti-inflammatory cytokines interleukin-10(IL-10). Pretreatment with7b resulted in the reduction of LPS-induced expression of COX-2and iNOS promoter activity without affecting COX-1promoter activity. The nuclear factor-κB (NF-κB) promoter gene transfection experiment supported the idea that inhibition of iNOS and COX-2is caused by the down-regulation of transcriptional activity of the NF-κB. In addition, it was found that pretreatment with7b significantly inhibited the nuclear translocation of the p65subunits of NF-κB, and these inhibitions were found to be associated with decreases in the phosphorylation or degradation of inhibitory kappa B-a (IκBα). Moreover,7b suppressed the phosphorylation of p38MAP kinase and extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase (JNK). Taken together, these results indicated that the anti-inflammatory effects of7b by suppressing of COX-2, iNOS and cytokines by blocking NF-κB and MAPK signalling in LPS-induced RAW264.7cells.