Abstract:

Objective To investigate the mechanism by which lycorine（Lyc） regulates nuclear factor-kappa B（NF-κB） pathway and inhibits tumor necrosis factor-alpha（TNF-α）-induced intestinal epithelial cell damage. Methods Human colorectal adenocarcinoma cell line Caco2 and human normal intestinal epithelial cell line NCM460 were selected. They were classified into control group（no treatment），Lyc group（treated with Lyc only），TNF-α group（treated with TNF-α only） and TNF-α+Lyc group（treated with TNF-α first and then Lyc）. The effects of different concentrations of TNF-α and Lyc on the viability of Caco2 cells and NCM460 cells were evaluated by CCK-8 assay to determine the appropriate experimental concentrations. The expressions of interleukin（IL）-6，inducible nitric oxide synthase（iNOS），cyclooxygenase-2（COX-2）and IL-1β in each group were determined by real-time fluorescence quantitative polymerase chain reaction. The expressions of zonula occludens-1（ZO-1），occludin（OCLN） and claudin-1（CLDN-1） in each group were determined by immunofluorescence and immunoblotting. The apoptosis of cells was determined by Calcein/PI staining. The expressions of Bax protein，Bcl2 protein and inhibitor of kappa B alpha（IκBα），phosphorylated inhibitor of kappa B alpha（p-IκBα），phosphorylated nuclear factor-kappa B p65（p-NF-κB p65）in each group were determined by immunoblotting. Results Compared with control group and Lyc group，the relative expression levels of IL-6 mRNA，iNOS mRNA，COX2 mRNA and IL-1β mRNA in TNF-α group were increased（P<0.05），and the protein expression levels of ZO-1，OCLN and CLDN-1 were decreased（P<0.05）. Compared with TNF-α group，the relative expression levels of IL-6 mRNA，iNOS mRNA，COX2 mRNA and IL-1β mRNA in TNF-α+Lyc group were decreased（P<0.05），and the protein expression levels of ZO-1，OCLN and CLDN-1 were increased（P<0.05）. The number of PI-positive cells in TNF-α group was higher than that in control group（P<0.05），and the number of PI-positive cells in TNF-α+Lyc group was lower than that in TNF-α group（P<0.05），but still higher than that in control group（P<0.05）. The expression of Bax protein in TNF-α group was higher than those in control group and Lyc group（P<0.05），and the expression of Bcl2 protein was lower than those in control and Lyc group（P<0.05）. The expression of Bax protein in TNF-α+Lyc group was lower than that in TNF-α group（P<0.05），and the expression of Bcl2 protein was higher than that in TNF-α group（P<0.05）. The p-IκBα/IκBα ratio and p-NF-κB/NF-κB ratio in TNF-α group were higher than those in control group（P<0.05）. The ratios of p-IκBα/IκBα and p-NF-κB/NF-κB in TNF-α+Lyc group were lower than those in TNF-α group（P<0.05），but still higher than those in control group（P<0.05）. Conclusions Lyc may inhibit TNF-α-induced intestinal epithelial cell damage by regulating NF-κB pathway.

Key words: Lycorine, Tumor necrosis factor-alpha, Caco2 cell, NCM460 cell, Inflammatory bowel disease