Porphyromonas Gingivalis Lipopolysaccharide Promotes The Process Of Chronic Periodontitis Through Thrombospondin-1

Periodontitis,is one of chronic bacterial infectious diseases,and affects a large proportion of population,consituting of the main reasons for teeth loss in adults.Oral infectious agents such as Porphyromonas gingivalis is an initial factor,followed by host-pathogen interaction,will further induce severe inflammation of periodontium tissues.LPS,a major constituent of outer membrane of P.gingivalis,is a powerful immune-activating stimulus,which can modulate host's comprehensive immune responses and activate related signaling pathway.Once activated by microbial products,monocytes/ macrophages may engulf pathogens and simultaneously secrete pro-inflammatory mediators including TNF-? and ILs,which result in persistent inflammation and sustaining injury of local tissues.As a signaling activator molecule,LPS induces macrophages to injure other types of cells,and modulates cell growth,differentiation and expression of cytokines.Human periodontal ligament fibroblasts(h PDLFs),constitute most of the total periodontal ligament cellularity,is the most important population,which responsible for the homeostasis and functions of the periodontium,not only sever as a mechanical barrier,but also a immune regulator.According to different microenvironment,h PDLFs can synthesize collagen fibers and secret proteoglycan which form extracellular matrix,as well as express gelatinase and collagenases which induce degradation of collagen fibers.Engaging in osteogenesis potential,h PDLFs also can regulate the balance between RANKL and OPG,are the basis for periodontal tissues repair and regeneration.Thrombospondin-1(TSP-1)is the best studied member of the thrombospondin(TSP)family.TSP-1 is a multifunction matricellular protein,first discovered by Baenziger in activated platelets as a major component of platelet alpha granules in 1971.However,TSP-1 was not unique to platelets,it was involved in many organs and secreted by various types of cells,capable of binding to a wide range of macromolecules and cell surface receptors that might play important functions.TSP-1 is generally regarded as the first natural angiogenesis inhibitors and a classical activator of transforming growth factor(TGF-?1).Recent studies have identified,TSP-1 might play a significant role in microbial-infection diseases and inflammatoryimmune diseases.TSP-1 might represent a general and important strategy of Gram-positive bacteria during development of invasive diseases through direct interaction to bacterial or as a key co-factor of platelet-bacteria-association.TSP-1 enhanced the pathogenesis by promoting expression of inflammatory cytokines and meanwhile reducing phagocytic capacity of macrophages,resulting in increased pathogenic burden and inflammation persistence.2014,Gokyu demonstrated that TSP-1 m RNA expression level was significantly upregulated in inflamed periodontitis gingival tissues but the specific role of TSP-1 in periodontal diseases is unknown.Objective: As previous studies indicated the effect of TSP-1 related with inflammation,this study focuses on the role of TSP-1 in chronic periodontitis,especially the role of macrophage and h PDLFs growth and cytokines expression,to clarify the related molecular mechanism of periodontitis,and to provid a new target for preventing and treating periodontal diseases.Meterials: In the present study,we first collect human gingiva tissue in periodontitis and normal individuals.HE staining,immunohistochemistry and immunofluorescence staining were used to determine the expression and the main cell sources of TSP-1.Here we employed the THP-1 macrophages and primary cultured h PDLFs to investigate the mechanism how TSP-1 function.We transfected TSP-1 si RNAs or p TT3-TSP-1 plasmids into THP-1 cells by LipofectamineTM 2000,followed by using P.gingivalis LPS to activate cells,and then to verify the affect of TSP-1 on the expression of the pro-inflammatory cytokines(IL-6,IL-1? and TNF-?)by ELISA and q RT-PCR,to investigate the role of TSP-1 in regulating cell cycle and apoptosis by flow cytometric analysis,to determine whether TSP-1 regulates nuclear factor-?B(NF-?B)p65 and I?B by Western Blot in THP-1 cells.On the basis,we transfected p TT3-TSP-1 plasmid and simultaneously used PDTC(a specific inhibitor of NF-?B),to analyze the protein levels of IL-6,IL-1?,TNF-? by ELISA.Secondly,THP-1 cells which transfected p TT3-TSP-1 plasmids and activated by P.gingivalis LPS was subjected to h PDLFs for transwell cocluture to simulate the in vitro effect of the activating macrophages on cell cycle and apoptosis of fibroblast by FAM and production of MMP-2,MMP-9 by Western Blot.At last,we transfected TSP-1 si RNAs or p TT3-TSP-1 plasmid into h PDLFs followed by P.gingivalis LPS to active cells,to verify the affection of TSP-1 on the expression of the cytokines(MMP-2,MMP-9,TIMP-1,RANKL,OPG)by Western Blot and q RT-PCR,to investigate the role of TSP-1 in regulating cell viability by MTT,cell cycle and apoptosis by FAM,to determine whether TSP-1 regulates Mitogen-activated protein kinase(MAPK)related p38,ERK1/2,JNK by Western Blot.Results:(1)TSP-1 expressions was markedly increased in periodontitis group,primarily co-localized with CD68 and vimentin,indicating that CD68+ positive macrophages and vimentin+ positive h PDLFs were the important cell sources of TSP-1 production in human gingiva tissues.(2)With tissue culture method we can obtain human periodontal ligament fibroblasts which vimentin-positive and keratin-negative by cell immunohistochemistry.(3)The Western Blot and q RT-PCR analyses revealed a prominent upregulation of TSP-1 at both protein and m RNA levels after being induced by P.gingivalis LPS treatment in THP-1 cells.We also found that m RNA and protein levels of IL-6,IL-1?,and TNF-? were synchronously elevated in dose-and time-dependent manner used q RT-PCR and ELISA analyses.We knocked down endogenous TSP-1 followed by P.gingivalis LPS treatment,the q RT-PCR analysis demonstrated that depression of TSP-1 strongly inhibited m RNA expression of IL-6,IL-1?,and TNF-? in THP-1 cells following TSP-1-si RNAs interfering.These results were confirmed by the ELISA examination,revealing less protein expression of IL-6,IL-1?,and TNF-? in THP-1 cells.We found that TSP-1 si RNAs transfection gave rise to a significant increase of apoptosis and led to a significant inhibition of G2/M phase of cell cycle in P.gingivalis LPS-treated THP-1 cells.Western Blot demonstrated that after TSP-1 si RNAs treatment,p-p65 and p-I?B? expression was significantly down regulated in THP-1 cells induced by P.gingivalis LPS.And on the contrary,we transfected THP-1 cells with plasmids p TT3-TSP-1 followed by treatment with P.gingivalis LPS.q RT-PCR showed that TSP-1 upregulation potently facilitated IL-6,IL-1?,and TNF-? production induced by P.gingivalis LPS.ELISA assays simultaneously demonstrated the promotive effect.Accordingly,p TT3-TSP-1 transfection markedly inhibited apoptosis and induced G2 /M phase of THP-1 cell treated with P.gingivalis LPS,and resulted in a remarkable increase of p-p65 and p-I?B? expressions.Western Blot analysis showed that THP-1 cells treated with PDTC significantly inhibited P.gingivalis LPS-induced expressions of p-p65 and p-I?B?,while p-p65 and p-I?B? expressions remained unchanged when the cells were treated with p TT3-TSP-1 and PDTC synchronously.Corresponding examinations showed no notable alterations of IL-6,IL-1?,and TNF-? production in THP-1 cells with p TT3-TSP-1 and PDTC treatments.(4)THP-1 cells which transfected p TT3-TSP-1 plasmid into and followed by P.gingivalis LPS treatment,markedly increased cell apoptosis and inhibited(S+G2)/M phase of h PDLFs,and promoted the expression of MMP-2,MMP-9 when used transwell systems in vitro.(5)The Western Blot analyses revealed a prominent upregulation of TSP-1,MMP-2,MMP-9 in dose-and time-dependent manner after being induced by P.gingivalis LPS treatment in h PDLFs.We knocked down endogenous TSP-1 followed by P.gingivalis LPS treatment,the Western Blot analysis demonstrated that depression of TSP-1 strongly inhibited protein expression of MMP-2,MMP-9,RANKL as well as promoted protein expression of TIMP-1 and OPG in h PDLFs following TSP-1-si RNAs interfering.These results were confirmed by the q RT-PCR examination.We found that TSP-1 si RNAs transfection gave rise to a significant inhibition of apoptosis,led to a significant increase of cell viability and(S+G2)/M phase of cell cycle,accompanied by increased protein levels of PCNA and declining protein levels of Caspase-1(p20)in P.gingivalis LPS-treated h PDLFs cells.Western Blot demonstrated that after TSP-1 si RNAs treatment,p-p38,p-ERK1/2,and p-JNK expression were significantly down regulated in h PDLFs cells induced by P.gingivalis LPS.And on the contrary,we transfected THP-1 cells with plasmids p TT3-TSP-1 followed by treatment with P.gingivalis LPS.Western Blot showed that TSP-1 upregulation potently facilitated MMP-2,MMP-9 and RANKL while were adverse to the production of TIMP-1,OPG in h PDLFs induced by P.gingivalis LPS.q RT-PCR assays simultaneously demonstrated the promotive effect.Accordingly,p TT3-TSP-1 transfection markedly induced apoptosis and inhibited cell vitality and cell(S+G2)/M phase of h PDLFs cell treated with P.gingivalis LPS,and resulted in a remarkable increase of Caspase-1(p20)and less PCNA production.Western Blot demonstrated that after p TT3-TSP-1 treatment,p-p38,p-ERK1/2,and p-JNK expression were significantly up regulated in h PDLFs induced by P.gingivalis LPS.Conclusions: In summary,here we presented an evidence that TSP-1 which is high expression in periodontitis,acts as a modulator,is responsible for facilitating pro-inflammatory cytokine profile,affecting cell growth and apoptosis,which is involved in pathogenesis of periodontitis.The present investigation might help for a better understanding of the mechanism in periodontitis,and provide some clinical significance in development of the novel therapeutic strategy.