Expression And Effection Of TOLL Like Receptors7and9in Lipopolysaccharide-inuced Inflammatory Effect Of AR42J Cells

Objective To observe the expression changes of Toll Like Receptor7(TLR7), Toll Like Receptor9(TLR9), Nuclear Factor-κB P65(NF-κB P65),Tumor Necrosis Factor-α (TNF-α) in lipopolysaccharide-induced inflammatoryeffect of AR42J cells which is an acute pancreatitis (AP) model in vitro, at thesame time to investigate the roles and the possible mechanism of TLR7, TLR9in the pathogenesis of acute pancreatitis.Methods The logarithmic growth phase AR42J cells were subcultured withthe six-well cell culture plate for24hours. Then the AR42J cells werestimulated with lippolysaccharide at different dosages(0mg/L,1mg/L,10mg/L,100mg/L) respectively. In this way, the acute pancreatitis in vitro modelwas established successfully. After24hours, the cell culture supernatant wascollected, and centrifuged to remove impurities. The AR42J cells were collectedat the same time. Total RNA and total protein of AR42J cells were extractedseparately. Expression of TLR7, TLR9, NF-κB P65mRNAs were determined byReverse Transcription-polymerase Chain Reaction (RT-PCR).Expression of TLR7, TLR9, NF-κB P65proteins were determined by Western Bolt. ELISAwas performed to detect the concentrations of TNF-α in cell culture supernatant.Results Compared with the0mg/L group, AR42J cells stimulated bydifferent dosages lipopolysaccharide expressed TLR7, TLR9, NF-κB P65at thelevel of mRNA and protein in a dose-dependent manner, and reached themaximum value at100mg/L. Difference among the groups were statisticallysignificant (all P＜0.05). Correlation Analysis indicated that there were positivecorrelation between the expression of TLR7and NF-κB P65, TLR9and NF-κBP65at mRNA and protein level (mRNAs: r=0.786,0.762；proteins: r=0.801,0.720；all P＜0.05). The concentration of TNF-α in cell culture supernatant alsoincreased in a dose-dependent manner, difference among the four groups werestatistically significant (P＜0.05).Conclusions TLR7and TLR9are obviously up-regulated in the vitro acutepancreatitis model induced by lipopolysaccharide. It suggests that TLR7andTLR9may play a role in the development of acute pancreatitis. TLR7and TLR9may work by activating NF-κB, then increasing the secretion ofproinflammatory cytokines.