The Effects Of FK228 On Activation Of NF-κB Induced By TNFα In Human Hepatoma Cell Line HepG2

Objective: To investigate the effect of FK228 on the TNFα-inducedactivation of NF-κBp65 and transcription of NF-κB-regulatedanti-apoptosis gene Bcl-2, as well as pro-inflammatory cytokines genesuch as IL-6 and IL-8 in human hepatoma cell line HepG2. To explore theanti-neoplastic, potential anti-inflammatory effect of FK228 and possiblemechanisms. Serving the vivo test and clinical application of FK228 withmore and sufficient theoretical evidence.Methods: In vitro cultured HepG2 cells were divided into 3 groups,namely, control group, TNFαstimulation group, and interference group(FK228+TNFα). The expression of NF-κBp65 in the nuclear and IκBαin the cytoplasm were assayed by western blot with special antibody. Cellapoptosis was detected by flow cytomery. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to semi-quantitativeanalyze the changes of IL-6 and IL-8 in mRNA level. Data werepresented as (?)±s and analyzed with ANOVA and LSD by SPSS11.5statistical program. A level of P＜0.05 was considered statisticallysignificant.Results: The increased nuclear translocation of NF-κBp65 inHepG2 was observed 30 min after the stimulation of TNFαat the doseof 20ng/ml,reached the highest level at 1h, then began to descend.NF-κBp65 in nuclear raised with the increased dose of TNFα30min after stimulating. Various concentration of HDAC inhibitor FK228(4ng/ml,8ng/ml,16ng/ml,32ng/ml) inhibited TNFα-induced nucleartranslocation of NF-κBp65. The interference groups (FK228+TNFα) weresignificantly different from TNFαstimulation group (P＜0.05) and FK228decreased the degradation of IκBα, each group differents fromothers(P＜0.01). The flow cytometry profiles revealed that: The apoptosisrate(15.51±0.39%) of interference group (FK228+TNFα) is higher thanTNFαstimulation group with the rate of 4.05±0.30%(P＜0.01). FK228down-rugulate TNFα-induced mRNA level of Bcl-2, IL-6 and IL-8measured by RT-PCR.Conclusions:(1) Transcription of anti-apoptosis protein Bcl-2 and pro-inflammatory cytokines such as IL-6, IL-8 were increased by the TNFαstimuli,which may be regulated by the activation of NF-κB induced byTNFαin HepG2.(2) FK228 can reduce degradation of IκBαand inhibit the activationof NF-κB induced by TNFα.Through the NF-κB inhibitory effect ofFK228, It down-regulates mRNA level of anti-apoptosis protein Bcl-2 andincreases apoptosis ratio of HepG2.In addition, FK228 down-regulatestranscription of pro- inflammatory cytokines such as IL-6,IL-8,imply itsanti-neoplastic and anti- inflammatory effect.