Gingival Bacteria Porphyromonas Restructuring Pilus Protein A And Collagenase Proinflammatory Effects In Vitro And In Vivo Studies Of Lipopolysaccharide On The Synthesis Pathway Of Pge <sub> 2 </ Sub>

Chronic periodontitis(CP)is one of the common oral diseases in human beings which is affected by multiple factors.It has been demonstrated that periodontitis is elicited by suppression of periodontal immune defenses,colonization and overgrowth of periodontal bacterial pathogens which produce an array of virulence factors,release of pro- inflammatory cytokines and chemokines by host cells,initiation of cytotoxic or immunopathological events,and subsequently periodontal tissue breakdown.Being one of the well confirmed putative periodontal pathogens,Porphyromonas gingivalis expresses a number of potential virulent factors that enable it to evade innate immmune defense system and destroy host cells,including lipopolysaccharides (LPS),fimbriae,collagenase,gingipain,hemagglutinin and a superoxide dismutase, etc.Fimbriae are important virulent factors involved in adherence of P.gingivalis to host cells.The inflammatory cytokine profiles induced by P.gingivalis strains with different fimA genotypes are not the same.But the capability of typeâ… FimA protein in inducing inflammation and its role in the pathogenesis of periodontitis remains controversial.Collagenase plays a critical role in periodontal tissue destruction and progression of periodontitis.The collagense produced by P.gingivalis is encoded by prtC gene. But study on its structure and function as an enzyme show the prtC gene product is unique.However,previous studies on the prtC gene product of P.gingivalis mainly concentrate on its collagenolytic activity.Its ability on inducing host cells to produce inflammatory cytokines has yet not been examined.If this protein could have direct effect on inducing inflammation,then it may work in combination with true bacterial or host collagenases and play a role in periodontal tissue destruction.LPS is another important inflammation-causing component of P.gingivalis.LPS could induce host cells to produce prostaglandin E₂(PGE₂).As an important inflammatory mediator,PGE₂ was catalyzed from its progenitor-arachidonic acid by multiple enzymes,including phospholipidase A₂,cyclooxygenase and some terminal prostaglandin E synthase.It has been demonstrated that the structure of LPS of P. gingivalis(Pg-LPS)is particular,and the inflammatory reaction of host cells to Pg-LPS is different from LPS of Escherichia coli(E-LPS).But most researches on LPS induced PGE₂ synthesis used E-LPS.If Pg-LPS plays specific role on PGE₂ bio-synthesis pathway needs to be elucidated.The objective of the present study is:â‘ to understand the inflammation-causing effect of recombinant FimA and PrtC protein in vivo and in vitro;â‘¡to observe the effect of Pg-LPS on release of arachidonci acid and PGE₂ levels and its influence on mRNA and protein expression levels of the key enzymes in PGE₂ bio-synthesis pathway,and to elucidate the associated signaling pathway.The clarification of these aspects would help to further understand the role of Pg in the initiation and progress of periodontitis.The main contents and results are listed below:Partâ… Levels of FimA and PrtC of Porphyromonas gingivalis in subgingival plaque samples from chronic periodontitis patients and their effect on inflammatory cytokine expression in human umbilical vein endothelial cells ECV304 strainIn experiment 1,two prokaryotic expression systems pET-32a-fimA BL21DE3 and pET-32a-prtC BL21DE3 were constructed.Ni-NTA affinity chromatography was used to extract and purify rFimA and rPrtC.Western blot was applied to determine immunoreactivity and antigenicity of rFimA and rPrtC by using rabbit antisera against whole cell of P.gingivalis and the recombinant proteins as the first antibodies, respectively.It was found that the homologies of the sequences of cloned fimA and prtC genes were from 98.5%to 99.7%compared to the corresponding nucleotide and putative amino acid sequences from GeneBank(GenBank:D17795,AB006973). Target fragments from the constructed expression vectors pET-32a-fimA and pET-32a-prtC could be seen to locate at the expected positions after the two vectors had been treated with double restriction endonucleases digestion and agarose eletrophoresis.Correct sequences and directions of the two inserted target genes were confirmed by nucleotide sequence analysis.Purified rFimA or rPrtC protein showed only one band in SDS-PAGE.Western blots showed rFimA and rPrtC both could combine with the rabbit antiserum against whole cell of P.gingivalis and induce rabbits to produce specific antibodies,respectively,which indicated rFimA and rPrtC have good antigenicity and immunoreactivity.In experiment 2,using rabbit antisera against recombinant FimA or PrtC proteins as the first antibodies,ELISAs were established to detect FimA and PrtC levels in 221 subgingival plaque samples from 49 chronic periodontitis patients and 25 individuals with healthy periodontium.The association between FimA and PrtC levels and clinical parameters was also analyzed.It was shown that the FimA and PrtC levels in CP samples were significantly higher than those in healthy samples,and initial periodontal therapy could remarkbly reduced the FimA and PrtC levels(P＜0.05).The FimA level in subgingival samples could be correlated mainly with BOP,while PrtC level was associated with BOP,AL and PD.FimA level in subgingival plaque samples from CP patients was in large part correlated with periodontal tissue inflammation, while PrtC level was associated with periodontal inflammation and tissue destruction.In experiment 3,rFimA and rPrtC were applied to human umbilical vein endothelial cells originated ECV304 strains.The induced secretions of IL-1α,IL-8, TNF-αand ICAM-1 were detected by ELISA kits.The results showed that the IL-1αlevels in supernatants did not increase untill incubation with 1,5 and 10αg P. gingivalis rFimA for 48h(P＜0.05).But the expression of ICAM-1 increased remarkably after 12h stimulation with 1,5 and 10μg rFimA,reached its peak at 24h, and then decreased at 48 h which remained to be higher than the negative control (P＜0.05).No similar results were noted for IL-8 and TNF-αin the study(P＞0.05). After co-incubation with 1μg rPrtC for 24h and with 5 or 10μg rPrtC for 12h,the levels of IL-1α,IL-8 and TNF-αsecreted by ECV304 cells increased significantly (P＜0.05),among which the IL-1αlevel peaked at 24h and the IL-8 and TNF-αlevels increased gradually with time.No effect on the expression of ICAM-1 was observed for rPrtC(P＞0.05).Conclusion:1.The recombinant typeâ… FimA protein showed strong effect on inducing expression of intercellular adhesion molecule,which suggested it might play a role in P. gingivalis -related periodontal inflammation and some systemic diseases.2.The recombinant PrtC protein could efficiently stimulate endothelials to express inflammatoy cytokines,which indicated that rPrtC might exert destructive effect on periodontal tissue by its inflammation-causing effect on host cells.Partâ…¡Effect of lipopolysaccharides of Porphyromonas gingivalis on PGE₂ bio-synthesis pathway in human monocyte cells strain THP-1In experiment 1,Pg-LPS was extracted and purified from P.gingivalis ATCC 33277 strain according to a modified hot-phenol-water method.Using Tachypleus Amebocyte Lysate(TAL)kit to test the bio-activity of purified Pg-LPS.TAL test indicated the extracted Pg-LPS was biologically active(≧15.0ng/ml),and can be used in this study.In experiment 2,Pg-LPS and commercialized lippopolysacchride of Escherichia coli(E-LPS)strain O111:B4 was applied to a human monocyte cells strain THP-1. PGE₂ concentration in supematants was determined by an enzyme immunoassay kit.It was found that level of PGE₂ increased with time when treated with 1μg/ml E-LPS for 1-48h.But for Pg-LPS,elevated expression of PGE₂ could only be observed after incubation for 6h,which peaked at 24h,and declined a little at 48h.The stimulated secretion of PGE₂ were different for E-LPS and Pg-LPS,and higher concentration of PGE₂ was noted for E-LPS(P＜0.05).In experiment 3,the release of tritium labeled arachidonic acid before and after E-LPS and Pg-LPS incubation was detected by a liquid scintillation counter.The results showed that release of[³H]labeled arachidonic acid increased after incubation with E-LPS for 1-6h,with its highest level at 2h.The CPM value of[³H]labeled arachidonic acid increase at 4-6h for Pg-LPS,and the highest value was at 6h.The peak value induced by E-LPS was significantly higher than Pg-LPS(P＜0.05).In experiment 4,reverse transcription polymerase chain reaction(RT-PCR)was used to analyse the mRNA levels of cytosolic phospholipase A₂(cPLA₂)enzyme, cyclooxygenase-2(COX-2),and membrane-bound form of prostaglandin E₂ synthase-1(mPGES-1).It was demonstrated that the mRNA transcription of cPLA₂ began to increase after 1h incubation of E-LPS,and at 2-6h,it was significantly stronger than blank control.But Pg-LPS showed effect on mRNA transcription of cPLA₂ after treatment for 4-6h.Higher transcription level of COX-2 was observed after 1-16h for E-LPS and 6-16h for Pg-LPS.Elevated level of the mRNA of mPGES-1 could be seen at 4-24h induction of E-LPS or at 6-24h of Pg-LPS.In experiment 5,western blot was adopted to analyse the expression of cytosolic phospholipase A₂(cPLA₂)enzyme,cyclooxygenase-2(COX-2),and membrane-bound form of prostaglandin E₂ synthase-1(mPGES-1)proteins.The expression of cPLA₂ protein started to increase at 1h treatment of E-LPS,and at 4h it was obviously stronger than blank control.Pg-LPS could also induce expression of cPLA₂ protein at 4h.It returned to baseline level at 8h for both LPS.Induced expression of COX-2 protein could be observed at 4-16h for E-LPS or at 8-16h for Pg-LPS.The strongest band was seen at 8h for E-LPS or at 16h for Pg-LPS.The specific band for mPGES-1 protein was shown after induction of E-LPS for 4-24h or Pg-LPS for 8-24h.The strongest signals were revealed both at 16h.In experiment 6,specific inhibitors were used before applying Pg-LPS or E-LPS to THP-1 cells,including ERK inhibitor PD98059,and P38 MAPK inhibitor SB203580,JNK inhibitor SP600125,cPLA₂ inhibitor AACOCF3,phospholipase C (PLC)inhibitor U73122,phosphatidylinositol 3-kinase(PI3-K)inhibitor LY294002, COX-2 inhibitor NS-398 and NF-κB inhibitor SN50,and then the variation in level of PGE₂,released arachidonic acid,expression of cPLA₂,COX-2 and mPGES-1 were detected.It was shown that after pretreatment with specific inhibitors,E-LPS and Pg-LPS induced secretion of PGE₂,increased expression of COX-2 and mPGES-1 mRNA and protein could be completely or partially inhibited by PD98059,SB203580, SP600125 and SN50.LY294002 could also exhibit a weak inhibitory effect.But U73122 and AACOCF3 did not block the expression of PGE₂.COX-2 and mPGES-1. E-LPS and Pg-LPS stimulated release of arachidonic acid and enhanced expression of cPLA₂ mRNA and protein was inhibited by PD98059,SB203580,AACOCF3, U73122,LY294002 and SN50,while pretreatment of SP600125 and NS-398 could not affect them all.A strong inhibitory effect of NS-398 was noted for COX-2 transcription and protein expression.Conclusions:1.Compared with E-LPS,Pg-LPS shows weaker and postphoned effect on PGE₂ bio-synthesis pathway.2.The signaling pathway for E-LPS and Pg-LPS induced PGE₂ bio-synthesis is similar,which mainly controlled by the three MAPKs-ERK,P38,JNK,and NF-κB. PI3-K might also be involved.3.E-LPS and Pg-LPS induced PGE₂ bio-synthesis maybe the result of LPS activated multiple signaling pathway,which promoted expression of the two coupled protein, COX-2 and mPGES-1,thus facilitate the production of PGE₂.Among this,the role of cPLA₂ may not be critical.