Fig. 2. The effect of silibinin on Helicobacter pylori-induced expression of COX-2 and inducible nitric oxide synthase (iNOS) and NF-κB activation. (A) Silibinin (100 µM) was treated for 1 hour to MKN-1 cells prior to infection with H. pylori (500 MOI) for 7 hours. Whole cell lysates were immunoblotted with antibodies against COX-2 and iNOS. (B) Under the same conditions, total RNA was isolated and then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA expression of COX-2 and iNOS. Actin and Glyceraldehyde-3-phosphate dehydrogenase levels were used as loading controls for immunoblot and RT-PCR analyses, respectively. (C) Cells were treated with silibinin for 1 hour prior to H. pylori (500 MOI) infection for 5 hours. The nuclear extracts were subjected to Western blot analysis to examine nuclear translocation. (D) The nuclear extracts from the infected cells were subjected to electrophoresis mobility shift assay to assess DNA binding of NF-κB as described in Materials and Methods. (E) Cells were transfected with a luciferase reporter plasmid construct harboring the NF-κB binging site or β-galactosidase control vector for 48 hours by using lipofectamin 2000 reagent according to the manufacturer’s instructions. Transfected cells were pretreated with silibinin for 1 hour followed by H. pylori (500 MOI) infection for 5 hours and subsequently treated with reporter lysis buffer for measurement of luciferase activity. The luciferase activity was normalized with β-galactosidase activity. Columns, means (n = 3); bars, standard deviation; *significantly different between the numbers compared (P < 0.05).

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