Inhibitors of TLR-4, NF-κB, and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells.

The present study was designed to examine and compare the effects of three suppressors on the cytokine response in tandem with examining: the synthesis of inducible forms of heat shock proteins; HSP72 and HSP90α; activities of NF-κB and SAPK/JNK signaling pathways; and TLR4 expression. Pre-treatment with inhibitors offers promise as protective means to lower the activity of these cascades, thereby circumventing the formation of excessive amounts of pro-inflammatory molecules. Three inhibitors of TLR4, SAPK/JNK, and NF-κB signaling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before the Escherichia coli lipopolysaccharide (LPS) application. Treatments of RAW 264.7 cells with each of the inhibitors resulted in a reduced response to LPS as was visualized by a decrease of TNF-α, IL-1, and IFN-γ production. In addition, inhibitors of the NF-κB and SAPK/JNK signaling reduced IL-6 production in LPS-treated cells, whereas the IKK inhibitor XII also decreased IL-10 production. Further, the NO production in LPS-stimulated macrophages was significantly reduced following application of CLI-095 or IKK inhibitor XII. The results also showed that the inhibitors suppressed TLR4 production and decreased phosphorylation of NF-κB and SAPK/JNK proteins, thereby preventing the activation NF-κB and SAPK/JNK signaling pathways in LPS-activated cells. In addition, the production of inducible heat shock proteins, HSP72 and HSP90-α, was reduced in LPS-stimulated RAW 264.7 cells pre-treated with inhibitors. These results suggest that inhibitors CLI-095, SP600125, and IKK inhibitor XII demonstrate potential effectiveness in the reduction of the inflammatory response by mechanisms involving both the cellular defense system and cellular signaling. In conclusion, suppressor of NF-κB cascade, IKK inhibitor XII, seems to be the most effective anti-toxic agent among studied inhibitors.