Aspirin 15cH has Different Effects on Morphology and Function of Lipopolysaccharide- Challenged RAW 264.7 Macrophages In Vitro Compared to a Pharmacological Dose of Aspirin

Introduction Aspirin is one of the most commonly used drugs worldwide. It is known to present antipyretic, anti-inflammatory and anti- thrombotic actions, making it extremely useful in a wide range of clinical contexts. Interestingly, homeopathically prepared Aspirin 15cH has been found to have a pro- thrombotic effect in rats, raising the hypothesis that Aspirin 15cH could also modulate the activity of inflammatory cells in different pathological processes. Objective Our objective was to assess what effect Aspirin 15cH has on RAW 264.7 macrophages in vitro. Methods The effects of Aspirin 15cH on biochemical and morphological activities of lipopolysaccharide (LPS)- stimulated RAW 264.7 macrophages were evaluated. These effects were compared with unchallenged macrophages (negative control), untreated LPS- stimulated macrophages, macrophages treated with succussed water (vehicle control), or aspirin 200 mu g/mL (pharmacological inhibitor of LPS activity). Cell morphology (adhered cell area and cytoskeleton arrangements), cell viability, toll-like receptor-4 (TLR-4) expression, and the production of nitric oxide, cytokines and intracellular reactive oxygen species were assessed. Results Aspirin 15cH reduced the number of cells expressing TLR-4 on the surface (p = 0.03) and induced a "columnar" morphology of macrophage pseudopods, indicating changes in cytoskeleton arrangement. When cells were treated with both Aspirin 15cH and LPS, cell morphology became heterogeneous, suggesting that sub-populations of cells had differing sensitivities to LPS or Aspirin 15cH. Exposure of the cells to LPS alone, succussed water or aspirin 200 mu g/mL produced effects consistent with the literature. Conclusion Aspirin 15cH, aspirin 200 mu g/mL, LPS and succussedwater appear to act as independent stimuli able to induce different patterns of macrophage response. Aspirin 15cH induced changes suggestive of M2 polarization of the macrophages (i.e., toward a wound healing or tissue repair, rather than inflammatory, phenotype). These preliminary findings need to be confirmed in further specific studies.