Signal Transduction System For Interleukin-1βmRNA And Interleukin-6 MRNA Synthesis Stimulated By Lipopolysaccharides Extracted From Porphyromonas Endodontalis In Osteoblasts

ObjectiveThe root canals is infected by bacteria in chronic periapical periodontitis,among which Porphyromonas endodontalis is almost only present in infected root canals and is called the special bacterium in endodontic infection.The biological behaviour of osteoblasts is very critical in the rebuilding of bonc tissuc,which is not only possible for forming bone tissue but also producing inflammatory factors to accelerate bone rcsorption of osteoclasts.This study is to quantify the IL-1βmRNA and IL-6 mRNA expression induced by Lipopolysaccharidcs(LPS) extracted from Porphyromonas endodontalis(P.e) in osteoblasts and the cell signal transduction system,and to relate P.e-LPS to the bone resorptivc pathogenesis in the lesions of chronical apical periodontitis.Expectcd to provide theoretical and experimental directions for clinical treatment.Methods1.Cultures of Porphyromonas endodontalisPorphyromonas endodontalis was cultured in solid Brain heart infusion(BHI) medium at 37℃in a anaerobic atmosphere(80%N₂,10%CO₂,10%H₂).The bacteria that were multiplied in fluid BHI medium were collected and then stored at -20℃.2.Extraction of LPSLPS was extracted from Porphyromonas endodontalis by hot-phenol-water method.The tachypleus amebocyte lysate(TAL) was used for qualitative analysis of LPS.3.Cultures of cellsMG63 cells was cultured in RPMI 1640 containing 10%fetal boving serum(FBS), 100u/ml penicillin and 100u/ml streptomycin at 37℃in a humidified atmosphere containing 5%CO₂ in air.Cells of the third or fourth genetation growing well were used for experiments.4.Groups of experimentsThe experiment was divided into three groups.Osteoblasts was treated with P.e-LPS repectively at 0,1,5,10,20,50μg/ml for 6 h in the dose group and at 10μg/ml for 0,1,3,6,12,24h in the time group.For studying the effect of inhibitors of ERK1/2 kinases and p38MAPK,osteoblasts was pretreated with PD98059 or SB203580 for 1h and then added P.e-LPS at 10μg/ml for 6h.For the experiments to examine gene expression,cells were collected when they were grown to confluence for extraction of total RNA and stored at -80℃.5.Detection of IL-1βmRNA and IL-6 mRNATotal RNA was extracted from MG63 cells.cDNA was synthesized using random primers by reverse transcription(RT)reaction,followed by polymerase chain reaction(PCR) amplification using synthetic gene primers specific for human IL-1β, IL-6 andβ-actin.PCR products were electrophoresed on 1.5%agarose gel and detected on a fluoroimage analyzer for the average gray scale value of IL-1β,IL-6 andβ-actin.6.Statistical analysisOne-way analysis of variance(ANOVA) and Dunnett-t test was performed in SPSS11.0 software for statistical analysis.Significance level:P＜0.05 is considered statistically significant,P＜0.01 is considered highly statistically significant.Results1.The production of IL-1βmRNA and IL-6 mRNA increased markedly after treatment with P.e-LPS at 1,5,10,20,50μg/ml compared with 0μg/ml(P＜0.01).2.The production of IL-1βmRNA and IL-6 mRNA increased markedly after treatment with P.e-LPS at 10μg/ml following the raise of hours.3.The production of IL-1βmRNA induced by P.e-LPS decreased in osteoblasts pretreated with PD98059.4.Both of the production of IL-1βmRNA and IL-6 mRNA induced by P.e-LPS decreased in osteoblasts pretreated with SB203580.Conclusions1.P.e-LPS stimulated osteoblasts to express IL-1βmRNA and IL-6 mRNA in a dose dependent manner.2.P.e-LPS stimulated osteoblasts to express IL-1βmRNA and IL-6 mRNA in a time dependent manner.3.The synthesis of IL-1βmRNA stimulated by P.e-LPS in MG63 probably occur via ERK1/2 and p38MAPK signal transduction system.4.The synthesis of IL-6 mRNA stimulated by P.e-LPS in MG63 probably occur via ERK1/2 signal transduction system.