Humic acid (HA), a fluorescent allomelanin, has been implicated as an etiological agent of Blackfoot disease (BFD), a peripheral vascular disease prevailing in the southwest of Taiwan. Clinical and pathological studies reveal that it is similar to atherosclerosis. In this report, the effect of HA on human neutrophils is studied because prolonged and enhanced activation of neutrophils adhered on endothelium may damage the endothelium and initiate the process of thrombosis and vasculitis. Methods: Neutrophils, treated with various concentrations of HA, were added to culture plates, cultured human umbilical vein endothelial cells (HUVECs), or human umbilical vein endothelium tissue culture for 15 or 30 min. The adhesion of neutrophils was measured qualitatively and quantitatively. The mechanism of neutrophil activation was studied with free radical production and various kinase measurements and their activities' assays. Results: HA was shown to enhance, in a dose-dependent manner, the adhesion of neutrophils on the culture plates, cultured human umbilical vein endothelial cells, and human umbilical vein endothelium tissue culture. The adhesion-enhancing ability of HA is elicited through activation of ERK, P38 mitogen-activated kinase (P38MAPK), and phosphoinositide 3 kinase (PI3K) in neutrophils. HA also induces the NF-κB activation in neutrophils. Conclusion: HA treatment markedly enhanced adhesion and superoxide radical production of neutrophils, the characteristics of activated neutrophils; and all these stimulation effects were blocked by several kinase inhibitors, reflecting the involvement of the ERK, P38MAPK, and PI3K on the activation of neutrophils. The induction of NF-κB implied that the consequence of neutrophil activation by HA were similar to other stimulants. The prolonged neutrophil activation will further damage endothelium cell and cause thrombosis, vaculitis, as well as arteriosclerosis. This may partially explain why HA consumption will cause BFD.
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