| Periodontitis is a chronic infectious disease which is characterized by destruction of periodontal tissue.Bacterial plaque is considered as the primary inducer in periodontitis.Although dental plaque and dental calculus is common in 90%of the world’s population(Pihlstrom et al.,2005a),most of the patients only stay in gingivitis or light/moderate periodontitis state and without further development.Severe periodontitis just account for 10-15%(Grossi et al.,1994).Among the risk factors of periodontitis,dental plaque accounts for only 20%(KINANE.and BARTOLD.,2007),suggesting that host factors play an important role in the pathogenesis and prognosis of periodontitis.The strength of host immune response is the key factor for the outcome of periodontitis(PRESHAW,2008).However,the current study lack of knowledge about periodontitis host factors,and hardly to fully explain the above phenomena.The self-regulation in the process of host immune inflammatory response results in the difference of host response.Therefore,the knowledge of the self-regulation in the process of host immune inflammatory response is still the important theoretical problem need to be solved.Matrix metalloproteinases(MMPs)is a key point in host response of periodontitis.MMPs is closely related to the tissue reconstruction and degradation,which regulate the degradation of of extracellular matrix(ECM)and affect the extent of periodontal tissue destruction(Page et al.,1997).Extracellular matrix metalloproteinase inducer(EMMPRIN)can induce the synthesis and activation of many MMPs.EMMPRIN-MMPs-Periodontitis pathway is an important mechanism that causes the destruction of periodontal tissue.Studies found that the molecular weights of EMMPRIN in the periodontal tissue were different and periodontal tissue destruction also showed varying degrees(Wang et al.,2014),suggesting that the differences of EMMPRIN molecular weights were closely related to the extent of the subsequent periodontal destruction.EMMPRIN is a highly glycosylated immunoglobulin,and studies in the field of cancer show that the degree of glycosylation affects the biological role of EMMPRIN.Only high-glycosylated membrane-EMMPRIN has the ability to induce MMPs expression and degradation of ECM(Sun and Hemler,2001).The most common glycosylation of EMMPRIN is N-terminal glycosylation,and the changes of sugar chain branches affect the variation of the molecular weight.The key enzyme that causes the change of sugar chain branches is N-acetyl glucosamine transferase(GnT)-V.GnT-V can modulate the conversion of low-glycosylated EMMPRIN(LG-EMMPRIN)to high-glycosylated EMMPRIN(HG-EMMPRIN)through promote the synthesis of β1,6-GlcNAc branch.The N-terminal glycosylation accounts for nearly half of the molecular weight of mature EMMPRIN(Tang et al.,2004a;Yu et al.,2006).Silencing GnT-V or using drugs could specifically suppress the synthesis of β 1,6 N-acetylglucosamine chain(Yu et al.,2016).However,in periodontal tissue,the level of EMMPRIN glycosylation remains unclear,and the role and mechanism of EMMPRIN glycosylation on periodontitis still not clear.To further understand above problems,this study systematically explored the level of EMMPRIN glycosylation,the mechanism of regulation and self-regulatory effects in EMMPRIN-MMPs-Periodontitis pathway,aiming to explain the phenomenon of varying degrees of periodontal destruction caused by EMMPRIN-MMPs-Periodontitis pathway,and then seek the possibility of intervention.The study mainly includes the following three aspects:1)In vivo and vitro study on the expression of EMMPRIN glycosylation;2)Constructing the co-culture model of oral epithelial cells/gingival fibroblasts;Exploring the regulatory mechanism of EMMPRIN glycosylation in EMMPRIN-MMPs-periodontitis pathway by silencing GnT-V and Pg.LPS stimulation to reduce and increase the level of EMMPRIN glycosylation in the co-culture model;3)Exploring the role and mechanism of swainsonine in intervening EMMPRIN glycosylation through the experiments in vivo and vitro.Part Ⅰ The level of EMMPRIN glycosylation and the expression of GnT-V in periodontal tissue.Objective:The purpose of this study is to explore the expression of EMMPRIN glycosylation,GnT-V and MMP-2,9 in periodontal tissue;Investigating the level of EMMPRIN glycosylation in human immortalized oral epithelial cells(HIOEC)and human gingival fibroblasts(HGF).Materials and methods:Collecting the gingival tissues from 10 patients with surgical crown lengthening(healthy periodontal tissue)and gingival tissues from 12 patients with severe periodontitis.The age and plaque index,gingival index,probing depth and clinical attachment loss of these subjects were recorded.Collecting mandibular periodontal tissue from 5 male SD rats and 5 male SD rats with periodontitis(the experimental periodontitis rat model was established by using the silk to ligate the first molar of the mandible),HE staining was used to observe the histology of the above samples.Immunohistochemistry(IHC)was used to detect the expression and location of EMMPRIN and MMP-2,9.Double-labelling immunofluorescence were used to detect the expression of EMMPRIN and GnT-V in the gingival tissue.In vitro,HGFs and HIOEC were cultured,and the expression of EMMPRIN in the primary HGFs and HIOEC was detected by Western Blot.And different concentrations of Pg.LPS were used to stimulate oral epithelial cell line at different times.Western Blot was used to examine the level of EMMPRIN glycosylation.Results:In the gingival tissue from periodontal healthy patients and rats,HE staining showed no obvious abnormalities in the epithelial spikes.IHC can be seen that EMMPRIN is mainly expressed in the cytoplasm of epithelial basal cells.Western Blot showed that the expression of EMMPRIN in HIOEC was significantly higher than that of HGFs,which was more than 20 times(P<0.001).In the gingival tissue of periodontitis patients,HE staining showed that the epithelial spikes were elongated and lots of inflammatory cells were infiltrated.IHC showed that EMMPRIN expression was expressed in the whole layer of gingival epithelium and connective tissue,and EMMPRIN expression was found in cytoplasm and cell membrane,and expression of MMP-2,9 significantly increased in the gingival connective tissue.In the mandibular periodontal tissue of the rats with periodontitis,HE staining showed that the adjacent area between the first molar and the second molar of mandible were obviously damaged,and the inflammatory cells were infiltrated.IHC showed that EMMPRIN is expressed in connective tissue.The results of double-labelling immunofluorescence showed that in gingival tissue of periodontitis patients,the expression of GnT-V and membrane-EMMPRIN were significantly increased,and GnT-V and EMMPRIN were co-located in gingival epithelial basal cells.In vitro,Western Blot results showed that the level of EMMPRIN glycosylation was significantly increased in a concentration dependent manner after the stimulation of Pg.LPS(P<0.01),and the level of EMMPRIN glycosylation was the highest in 12 h with 10 μg/ml Pg.LPS stimulation.Conclusions:In healthy periodontal tissue,EMMPRIN was expressed in the cytoplasm of gingival epithelial basal cells;During periodontitis,EMMPRIN was found in the cell membrane of the whole layer of gingival epithelium and connective tissue,and its expression was stronger in basal cells,and EMMPRIN was co-localized with GnT-V.In vitro,the expression of EMMPRIN was different in different cells,EMMPRIN expression in HIOEC was much higher than that in HGFs,and the level of EMMPRIN glycosylation was increased with Pg.LPS stimulation.Part Ⅱ The mechanism of EMMPRIN glycosylation in regulating EMMPRIN-MMPs-Periodontitis pathway.Experiment Ⅰ Construction of HIOEC/HGFs co-culture model for determination of the interaction between EMMPRIN and MMPs.Objective:This part aims to build a human immortalized oral epithelial cells(HIOEC)and human gingival fibroblasts(HGF)co-culture model which simulate the cellular interactions in the periodontal environment for objectively studying the relationship between EMMPRIN glycosylation and MMPs.Materials and methods:HIOEC and HGF were simultaneously adnministered in the same hole at a ratio of 1:1 and cultured with a-MEM + 10%FBS and serum free cultured KGM medium to making direct contact between the two kinds of cells.The cell adherence time,cell morphology and survival time were observed and pictures were recorded under inverted phase contrast microscope(every 6 hours).Using the Pg.LPS of 10 μg/ml to stimulate HIOEC/HGFs co-culture model,the level of EMMPRIN glycosylation,MMP-2 and MMP-9 in the co-culture model was detected by Western Blot and real-time PCR.In addition to the HIOEC/HGFs co-culture model,we also constructed HIOEC and HGFs individual culture model,and the HIOEC and HGF were respectively cultured in different holes,and the proteins and RNA of the 2 cells were collected and mixed.HIOEC/HGFs co-culture model,and individual culture model were treated with Pg.LPS for 4 hours respectively,using Western Blot and real-time PCR to detect the expression of EMMPRIN glycosylation,MMP-2 and MMP-9 in 2 culture systems.Results:Western Blot results showed that EMMPRIN was mainly expressed in HIOEC,MMP-2,9 were mainly expressed in HGFs(P<0.001).In HIOEC/HGFs co-culture model,HIOEC adherence time was shorter than that of individual incubation time.In the HIOEC/HGFs co-culture model,HIOEC and HGFs cells cultured in the KGM medium without serum showed no obvious epithelial aging.In the HIOEC/HGF co-culture model,the Pg.LPS stimulation of 10 μg/ml can significantly increase the level of EMMPRIN glycosylation as well as the secretion and activity of MMP-2,9(P<0.001).In the co-culture model and individual culture model,with Pg.LPS stimulation,the level of EMMPRIN glycosylation were significantly increased but without significant difference between the two groups.Compared with the individual culture model,the results of Western Blot and real-time PCR showed that Pg.LPS stimulation could lead to a significant increase in the synthesis and expression of MMP-2,9 in the co-culture model(P<0.05).Conclusions:Compared with the individual culture model,the increased expression of MMP-2,9 in the co-culture model suggesting that this result was caused by the interaction between HIOEC and HGFs.The co-culture model simulated the internal environment more scientifically and objectively.HIOEC/HGFs co-culture model was successfully constructed,which laid a foundation for the research about the effect of EMMPRIN glycosylation on MMPs.Experiment Ⅱ GnT-V reduces the activation of MMP-2,9 and degradation of ECM caused by EMMPRIN glycosylationObjective:Extracellular matrix metalloproteinase inducer(EMMPRIN)is a transmembrane glycoprotein and can induce the activation of matrix metalloproteinases(MMPs),leading to the degradation of ECM and destruction of periodohtal tissue eventually.EMMPRIN glycosylation may involve in the above process.The purpose of this study was to explore the role and mechanism of EMMPRIN glycosylation in regulating the expression and activity of MMP-2,9 as well as the ECM degradation.Materials and methods:The human oral epithelial cells(HIOEC)are stably transfected with 4 groups of GnT-V RNAi lentivirus and Negative Control lentivirus(Iv3-negative Control).0.1 μg/ml of puromycin was used to screen HIOECs after the transfection of lentivirus,and the successfully transfected cells were sorted by FCM.The expression of GnT-V gene was detected by Real-time PCR,and the glycosylation level of EMMPRIN was detected by Western Blot.Immunofluorescence(IF)staining was used to observe the location of EMMPRIN in HIOEC with or without the virus transfection.According to the above results,the cell lines with the highest silence efficiency of GnT-V gene were selected.Using 10 μg/ml Pg.LPS to stimulate HIOEC with or without GnT-V silence,and then using flow cytometry to detect the expression of β1,6 N-acetylglucosamine branches.The expression level of EMMPRIN was detected by using Western Blot and Real-time PCR.In order to detect whether the reduction of EMMPRIN glycosylation by GnT-V silence can inhibit the expression of MMPs,we co-cultured the transfected HIOEC with HGFs.The HIOEC/HGF co-culture model was exposed to 10 μg/m,of Pg.LPS for 4 h.Then using Western Blot and IF to detect EMMPRIN glycosylation and the expression of MMP-2,9,using Real-time PCR to detect EMMPRIN and MMP-2,9 mRNA expression and using the gelatin degradation experiment to test the effect of reduction of EMMPRIN glycosylation on the degradation of gelatin.Results:The results of FCM showed that the transfection efficiency of HIOEC was more than 97%after puromycin screening.The results of real-time quantitative PCR showed that the expression level of GnT-V was significantly decreased,accounting for only 1/3 of the control group(P<0.01)in Silence#1(MGAT5-homo-165)group.Western Blot results showed that the glycosylation level of EMMPRIN in the HIOEC group was obviously decreased,accounting for only 1/3 of the control group(P<0.01)in Silence#1 group.Immunofluorescence showed that EMMPRIN expression in epithelial cell membrane significantly decreased in Silence#1 group.Therefore,we chose the cells of Silence#1 as the GnT-V gene silent group for subsequent experiments.FCM analysis showed that after treated with 10 μg/ml Pg.LPS stimulation for 12h,expression of β 1,6 N-acetylglucosamine in HIOEC was significantly lower than the control group(P<0.0001).Real-time PCR also showed that the expression level of EMMPRIN mRNA in silence group was significantly lower than that in the control group(P<0.05).Western Blot showed that GnT-V silence did not significantly reduce the increased expression of high-glycosylated(HG)-EMMPRIN caused by Pg.LPS stimulation(P>0.05),but low-glycosylated(LG)-EMMPRIN expression significantly increased,and there existed a lower molecular weight bands(P<0.05),EMMPRIN glycosylation levels also significantly decreased(P<0.05).In the HIOEC/HGF co-culture model,after treated with 10 μg/ml Pg.LPS stimulation for 4 h,Western Blot showed that the expression of HG-EMMPRIN of silence group,the level of EMMPRIN glycosylation and the secretion and activation of mmp-2 and 9 were significantly lower than that in the control group(P<0.05).The results of Real-time PCR and IF also proved that the expression of EMMPRIN and MMP-2,9 in silence group was not significantly increased with Pg.LPS stimulation(P<0.05).In addition,the gelatinization experiments showed that inhibition of EMMPRIN glycosylation was effective in inhibiting the degradation caused by Pg.LPS(P<0.01).Conclusions:Knockdown of GnT-V can significantly reduce the level of EMMPRIN glycosylation in HIOECs.The glycosylation of EMMPRIN participated in the regulation of MMP-2 and MMP-9 activity through interaction between HIOECs and HGFs.Inhibition of EMMPRIN glycosylation reduced the activation of MMP-2 and MMP-9 and suppressed the degradation of ECM.In this study,the glycosylation of EMMPRIN may affect the host immune inflammatory response of periodontitis by regulating MMPs.Part Ⅲ Study on the role and mechanism of swainsonine in reducing the destruction of periodontal tissueObjective:Swainsonine(SW)is an immunomodulator that inhibits the synthesis of P 1,6 N-acetylglucosamine branches(GlcNAc),thereby reducing the glycosylation of EMMPRIN.The purpose of this part was to explore the role of SW in regulating EMMPRIN glycosylation in HIOEC/HGF co-culture model and in periodontitis rat,and its role in suppressing matrix metalloproteinases(MMPs),anti-inflammatory response and bone protection in the development of periodontitis.Materials and methods:In vitro,after 0.1,0.2,0.3 μg/ml SW treated co-culture cells for 24 h,then we stimulated with 10 μg/ml Pg.LPS for 4 h.FCM analysis was used to assess the influence of SW for p 1,6 GIcNAc in the HIOEC/HGF co-culture model.Western Blot was used to detect expression of EMMPRIN,MMP-2,9,IL-1β and TNF-a.In vivo,30 mg/kg/d and 60 mg/kg/d of SW were injected intraperitoneally into experimental periodontitis SD rats respectively(from the day before the modeling),and the rats were killed at 7 d.Micro-CT was used to observe the alveolar bone destruction of first molar of rat mandible.HE staining was also used to observe the alveolar bone destruction between the first molar and the second molar of rat mandible,inflammatory infiltration and arrangement of collagen fiber.The Phytohemagglutinin-Leucoagglutination(PHA-L)lectin was used to detect the β 1,6 GlcNAc expression in gingival tissue around the first molar of rat mandible.Western Blot was used to detect level of EMMPRIN glycosylation,activity of MMP-2,9,and detect the cytokines IL-1β,TNF-α,and bone protection factors RANKL,OPG in the gingival tissue,to verify the effect of S W on the treatment of experimental rats with periodontitis.Results:In vitro,the results of FCM showed a significantly lower in β 1,6 GlcNAc expression in the co-cultured model which pretreated with SW and then stimulated with Pg.LPS than the control group and the group with only Pg.LPS treatment(P<0.05).The results of Western Blot showed that the level of EMMPRIN glycosylation and activation of MMP-2,9 were significantly reduced by SW treatment,only half of the group with Pg.LPS treatment alone(P<0.05).The expressions of IL-1β and TNF-αwere also significantly decreased(P<0.05).In vivo,the images of micro-CT three-dimensional reconstruction showed that SW could reduce the alveolar resorption(P<0.001)and increase the relative bone volume(bone volume/tissue volume,BV/TV)(P<0.001)in the periodontitis rats.HE staining showed that in the SW treatment group,the inflammatory infiltration in periodontal tissue was decreased,and the arrangement of collagen fibers was more orderly than that of periodontitis group.The PHA-L lectin showed that the expression of β 1,6 GlcNAc was significantly decreased(P<0.01).The results of Western Blot showed the level of EMMPRIN glycosylation and the activation of MMP-2,9 were significantly lower than periodontitis group(P<0.05).Inflammatory cytokines IL-1β and TNF-a were also significantly decreased(P<0.0001).And the RANKL expression is decreased,the OPG expression is increased,RANKL/OPG ratio decreased significantly(P<0.01).Conclusions:SW can reduce the expression of cytokine in the HIOEC/HGF co-culture model and periodontal tissue of periodontitis rats,and also significantly reduce the level of EMMPRIN glycosylation and the expression of MMPs,as well as the alveolar bone resorption in the periodontitis rats. |
