Diversity Of Porphyromonas Gingivalis Lipopolysaccharide On Inducing Different Cells To Secrete Inflammatory Cytokines

Chronic periodontitis(CP)is one of the common oral infectious diseases in human beings which is affected by multiple factors.Recently researches have 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 inflammatory cytokines and chemokines by host cells,initiation of cytotoxic or immunopathological change,and subsequently periodontal tissue breakdown,at last odontoptosis happen.It is a major cause for teeth loss in the adult population.Besides affecting the function of digestive system owing to loss of teeth,periodontitis itself also has been associated with general systemic diseases such as cardiovascular disease,pre-term delivery of low birth weight infants,diabetes,etc.It affected human quality of life.Porphyromonas gingivalis(Pg),a gram-negative,oral anaerobic black-pigmented bacterial species,is one of the received suspected periodontopathic bacteria.It expresses a number of potential virulence factors that enable it to evade host innate immmune defense system and destroy host cells,including lipopolysacharides(LPS),fimbria,gingipains.LPS is considered to be the most important virulence factor in numerous pathogenic materials of LPS.LPS is a major component of the outer membrane of gram-negative bacteria and a key inflammatory mediator.As a potential cell activator,it can activate many kinds of target cells in periodontal tissues,such as immunologic reaction cells(macrophage and lymphocyte),neutrophils which having defensive function,major cells of periodontal tissues(fibroblast),osteoblasts and osteoclast,etc.LPS can also make these cells synthesize and secret various kinds of cytokines and inflammatory mediators for the damage of periodontal tissues,and TNF-a,IL-1β,IL-6和IL-8 are the representative inflammatory cytokines.Pg-LPS is similar to a classical bacterial endotoxin Escherichia coli LPS(E.coli-LPS,E-LPS)in its chemical structure and biological properities,only the variations in phosphyorylation and acylation patterns in biologically active component, lipid A,have some differences.However,although highly conserved,LPS contains important structural differences among different bacterial species that can significantly alter host responses.Some reseachs report that the activity of inflammatory reaction induced by Pg-LPS is weaker than E-LPS, there are great differences of pro-inflammation effects in different target cells.It is well know that epithelial cells and gingival fibroblasts are the main cells of periodontal tissue.As the major constituent of gingival connective tissue,gingival fibroblasts which play an important role in the periodontal disease may directly interact with bacteria and bacterial products including LPS in periodontitis lesion.And accumulating evidence indicates that epithelia are not merely a kind of mechanical barriers but also a kind of the innate immune system.The present study was performed to examine corresponding cytokine responses of oral epithelial cells after infection with Pg.The mononuclear macrophagocytes plays an important role in regulating local inflammatory response to bacterial insult by production of cytokines.According to the result of previous study,we found that inducing cells of periodontal tissues,immunologic reaction cells by Pg-LPS they can secrete inflammatory cytokines,but the studies about the changed process of inflammatory cytokines secreted by those cells we know little.The pathological changes of CP has rest stage and active stage,it is sure that its acute episode is directly correlated to its chronic progress.It is necessary to make further discuss to this study.We also hope to perform some preparative works for thoroughly studying of the generation and development of periodontitis and improving the levels of periodontitis prevention and treatment.ObjectivePg-LPS was extracted and purified from P.gingivalis ATCC 33277 strain according to a modified hot-phenol-water method.Using limulus test and infrared spectrum to test the bio-activity and chemical elementary structure of purified Pg-LPS.To determine the capability and diversity of Porphyromonas gingivalis lipopolysaccharide inducing human oral epithelial cell(KB cells),human gingival fibroblasts(HGF-1 cells)and human mononuclear macrophagocytes cell(THP-1 cells)to secrete inflammatory cytokines TNF-a,IL-1β,IL-6和IL-8.Methods1.Pg-LPS was extracted and purified from P.gingivalis ATCC 33277 strain according to a modified hot-phenol-water method.Limulus test and infrared spectrum analysis were applied to identify the extracted Pg-LPS.2.By using different dosages Pg-LPSs stimulate KB,HGF-1,THP-1 for different duration time,we extracted their supernatants and then used quantitative ELISA kits to test the changes of TNF-a,IL-1β,IL-6 and IL-8 levels.A commercial E.coli strain O111:B4 LPS was used as a control in this study.Results1.The output of Pg-LPS:The output of purified Pg-LPS was 1.3mg/g wet bacterium.2.The minimal dosages of both Pg-LPS and E-LPS to solidify limulus agents were 15 ng/ml and their infrared spectrums were much similar.3.Pg-LPS or E-LPS could induce HGF-1 cells to secrete TNF-a and IL-1βlevels which were remarkably increased with a perk model(P＜0.01), while a continuous enhancement model of the two cytokines for THP-1 cells was presented(P＜0.01).4.Either Pg-LPS or E-LPS had effects to promote HGF-1 cells or THP-1 cells to continuously heighten the secretion of IL-6(P＜0.01).5.Both Pg-LPS and E-LPS were able to induce increase of IL-8 secretion in THP-1 ceils(P＜0.01),but only Pg-LPS showed the similar effect in HGF-1 cells(P＜0.01).6.Neither Pg-LPS nor E-LPS could induce KB cells to secrete the cytokines mentioned above.Conclusions1.The output of Pg-LPS extracted by a modified hot-phenol-water method was higher.2.Pg-LPS has the fundamental characteristic of the endotoxin of the bacteria.The extracted Pg-LPS had higher biologically active(≧15.0ng/ml), and could be used in this study.Infrared spectrums indicate that they are homologous structure.3.Pg-LPS has powerful activity to promote its target cells to increase their secretion of several inflammatory cytokines,which causing initial local inflammation.4.The inflammation-causing effect of Pg-LPS is similar to that of E-LPS but there are much different models to inducing inflammatory cytokines.5.KB cell can not used as the target cells to determine inflammation causing effect of LPS.