The Effects of LPS With or Without Forskolin on NF-KB and TNF-alpha Expression in RT4-D6P2T Cells
Schwann cells can be used as a model to study injury and inflammation in the peripheral nervous system. For this study, two major pathways were explored: the nuclear factor kappa B (NF-KB) and cyclic adenosine monophosphate (cAMP) pathways. Both pathways play a major role in inflammation. The NF-KB pathway is known to be more pro-inflammatory through the production of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In vitro, the NF-KB pathway can be activated by treating Schwann cells with lipopolysaccharide (LPS), a cell wall immunostimulatory component of Gram-negative bacteria. The cAMP pathway is known to be more anti-inflammatory and promotes cell proliferation. In vitro, the cAMP pathway can be activated by treating Schwann cells with forskolin, an artificial root extract from the plant Coleus forskohlii. Although it is well-known that both the NF-KB and cAMP pathways are involved in inflammation, not much is known regarding the effects of cAMP activation on the expression of NF-KB and downstream effector molecules, like TNF-alpha, in Schwann cells during a LPS-stimulated inflammatory environment. It was hypothesized that cells treated with LPS and forskolin would have less NF-KB and TNF-alpha expression than cells treated with LPS only. For this study, RT4-D6P2T cells received one of the following treatments: 0, 0.1, 1, or 10 ug/mL of LPS, with or without 2 uM of forskolin, for 3 hours. Cell lysates were prepared, and SDS-PAGE gel electrophoresis and Western blot were performed. Protein expression was visualized using enhanced chemiluminescence reagent and quantified via densitometry analysis using Bio Rad Image software. As expected, for cells treated without LPS, forskolin appears to downregulate NF-KB and TNF-alpha expression compared to the control. However, for all doses of LPS, forskolin appears to downregulate NF-KB expression while upregulating TNF-alpha expression. Because TNF-alpha is produced downstream of the NF-KB pathway, the fact that TNF-alpha expression is upregulated even when NF-KB expression is downregulated shows that cAMP-activated TNF-alpha expression in LPS-treated Schwann cells may be independent of the NF-KB pathway.