Cardiac Glycosides Inhibit LPS-induced Activation of Pro-inflammatory Cytokines in Whole Blood through an NF-kB-dependent Mechanism

Summary: The process of hemodialysis (HD) produces a pro-inflammatory state that can lead to an increased risk for cardiovascular disease. In part, this is the result of activation of the pro-inflammatory transcription factor NF-kB in response to uremia as well as in response to HD itself, which not only involves exposure of blood leukocytes to abnormal surfaces but also potentially to any bacterial contamination associated with HD. Previously, we used lipopolysaccharide (LPS) to activate isolated peripheral blood mononuclear cells (PBMC), as a model of HD-induced stress, and demonstrated that specific natural products that are known to inhibit the activation of NF-kB exhibited a broad anti-inflammatory activity. These natural products, however, were not effective when whole blood was used. In the present study, a natural product library (TimTec NPL480) was screened, using whole blood, for the abilities of these natural products to protect against LPS-induced expression and secretion of the pro-inflammatory cytokines TNFa, IL-1b and IL-6. We report here that the cardiac glycosides strophanthidin, ouabain, proscillaridin A, digoxin, digitoxin and lanatoside C are effective natural products that limit the development of a pro-inflammatory state by preventing the activation of these pro-inflammatory signals. These active natural products also inhibited the stress-induced activation of NF-kB in a reporter assay, suggesting that inhibition of NF-kB is at least partly the mechanism by which these natural products protect whole blood leukocytes from activation by LPS. Industrial relevance: Media for hemodialysis is used millions of times annually for patients with end stage renal disease, each use representing a potential pro-inflammatory insult. It would be useful to have a drug that could be added to the media which would protect blood leukocytes from any pro-inflammatory activation that may accompany the dialysis procedure. A natural product, if demonstrated to have low toxicity, could be especially attractive as a drug candidate for this application.