Free radicals enzymatically triggered by Clonorchis sinensis excretory-secretory products cause NF-κB-mediated inflammation in human cholangiocarcinoma cells

Abstract

Chronic clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis worms and their excretory-secretory products, is associated with hepatobiliary damage, inflammation, periductal fibrosis and even development of cholangiocarcinoma. Our previous report revealed that intracellular reactive oxygen species were generated in C. sinensis excretory-secretory product-treated human cholangiocarcinoma cells; however, their endogenous sources and pathophysiological roles in host cells were not determined. In the present study, we found that treatment of human cholangiocarcinoma cells with excretory-secretory products triggered increases in free radicals via a time-dependent activation of NADPH oxidase, xanthine oxidase and inducible nitric oxide synthase. This increase in free radicals substantially promoted the degradation of cytosolic IκB-α, nuclear translocation of nuclear factor-κB subunits (RelA and p50), and increased κB consensus DNA-binding activity. Excretory-secretory product-induced nuclear factor-κB activation was markedly attenuated by preincubation with specific inhibitors of each free radical-producing enzyme or the antioxidant, N-acetylcysteine. Moreover, excretory-secretory products induced an increase in the mRNA and protein expression of the proinflammatory cytokines, IL-1β and IL-6, in an nuclear factor-κB-dependent manner, indicating that enzymatic production of free radicals in ESP-treated cells participates in nuclear factor-κB-mediated inflammation. These findings provide new insights into the pathophysiological role of C. sinensis excretory-secretory products in host chronic inflammatory processes, which are initial events in hepatobiliary diseases.