| Background and ObjectivePeriodontitis is an inflammatory disease characterized by the presence of pathogenic bacteria in subgingival plaques,resulting in soft tissue destruction,alveolar bone resorption and eventual tooth loss.The most prominent feature of periodontitis is the accumulation of immunoactive cells,such as monocytes/macrophages and lymphocytes,and their cytokines in extravascular periodontal connective tissues.In particular,it has been demonstrated that the pro inflammatory cytokines tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)play an important role in periodontal tissue destruction.It has also been reported that TNF-? and IL-1? enhance leukocyte adhesion by upregulating chemokines,increasing barrier permeability and downregulating tight and adherens junction molecule expression.Human gingival fibroblasts(HGFs)are one of the most abundant cell types in periodontal connective tissue.HGFs function as support cells for periodontal tissues and produce inflammatory mediators in response to proinflammatory stimuli and pathogens.The important role of periodontal connective tissue in maintaining periodontal tissue integrity has been well studied,as well as its role in regulating the local inflammatory response.Within the cell junctional complex,tight junctions are largely responsible for controlling paracellular transport,whereas adherens junctions are primarily responsible for cell-cell adhesion.As the rate-limiting enzyme in heme degradation,heme oxygenase-1(HO-1)induction represents an essential event in cellular responses to proinflammation to maintain cellular homeostasis.HO-1,one of the most common represents of the known induced enzymes,has been proven to act as a cellular biosensor.High levels of HO-1 can be induced within a few hours by many stimulants,such as hemoglobin,cytokines,and endotoxins.The pharmacological or genetic modulation of HO-1 induces nuclear localization and inhibits cell migration,proliferation and invasion.HO-1 metabolizes and produces biliverdin,Fe2+and carbon monoxide(CO).CO has been shown to play important roles in multicellular events;for example,CO can inhibit cell apoptosis,suppress inflammation,and protect organs against ischemia/reperfusion injury.The effect of CO is mediated by HO-1 induction,guanylate cyclase activation,and p38 MAPK signaling pathway regulation.Extensive studies have shown that carbon monoxide releasing molecules(CORMs),which can release CO in a controllable manner under physiological conditions,can increase heme oxygenase-1(HO-1)expression in various animal models and cell types.CORMs belong to two major classes:metal carbonyl complexes containing ruthenium,manganese,or molybdenum,which carry CO bound to the transition metal.Among CORMs,CORM-1 and CORM-2 are lipophilic,they have to be dissolved in organic solvents such as dimethyl sulfoxide(DMSO).CORM-3 is fully water-soluble,can rapidly liberate CO when dissolved in physiological solutions,which shows more promising potential in clinical treatment in the future.By delivering and carrying CO in a controllable way,CORM-3 may exert important pharmacological activities.A previous study by our research group showed that CORM-3 inhibits the expression of adhesion molecules in HGFs stimulated with TNF-? and IL-1?.Thus,the objective of our present study was to investigate the effects of CORM-3 on HGFs barrier function following exposure to the inflammatory cytokines TNF-? and IL-1?,and try to elucidate the mechanism underlying this effect of CORM-3.Materials and Methods1.Isolation,culture and identification of HGFsThe HGFs were isolated from the explants of normal gingival tissues with an explant culture technique.Cells were cultured with 100 U/ml penicillin G,100 mg/ml streptomycin and 20%heat-inactivated FBS in humidified air with 5%CO2 at 37?.HGFs from passages 3?5 were used in the subsequent experiments.2.Effects of CORM-3 on the viability of HGFsCell Counting Kit-8(CCK-8)assays were used to assess the effect of CORM-3 at different concentration on the proliferation of HGFs.HGFs were firstly stimulated with 0,100 ?M,200 ?M,400 ?M and 800 ?M CORM-3 for 6 h and then incubated by TNF-?(10 ng/ml)and IL-1?(2 ng/ml)for 24 h.Then,cell viability was assessed by CCK-8 assays.3.The effects of CORM-3 on the expression of junctional molecules on human gingival fibroblasts incubated by TNF-? and IL-1?To check the expression of junctional molecules on HGFs,cells were divided into five groups.HGFs were firstly stimulated with 0,100 ?M,200 ?M and 400 ?M CORM-3 for 6 h and then incubated by TNF-?(10 ng/ml)and IL-1?(2 ng/ml)for 24 h.Cells stimulated with TNF-? and IL-1? were used as a control.Immunofluorescence staining was used to observe the changes in permeability across HGFs.Cells were observed with confocal laser scanning microscopy.The mRNA expressions of ZO-1,VE-cadherin and ?-catenin were detected by RT-qPCR,and the data were calculated by using the 2-??Ct method.At the same time,the protein expression of ZO-1,VE-cadherin and ?-catenin were detected by Western blot,and the gray value was analyzed by Image J software.Cells stimulated with TNF-? and IL-1? were used as a control.4.The effects of CORM-3 on the permeability of HGFs monolayers stimulated by TNF-? and 1L-1?To determine the changes in permeability across HGFs monolayers,HGFs were seeded at a density of 1.0×105 cells/well on top of 24-well transwell chambers with 0.4 ?m pore size diameter polyester membrane inserts and grown to confluence.HGFs were firstly incubated with increasing concentrations of CORM-3 for 6 h and then incubated with TNF-?(10 ng/ml)and IL-1?(2 ng/ml)for 24 h.Cells stimulated with TNF-? and IL-1? were used as a control.Fluorescein isothiocyanate(FITC)-BSA flux across HGF monolayers were used to measure the changes in permeability across HGF monolayers.Fluorescence intensity was quantified at 494 nm excitation and 520 nm emission.5.The effects of CORM-3 on the expression of HO-1 on human gingival fibroblasts incubated by TNF-? and IL-1?HGFs were firstly stimulated with 200 ?M CORM-3 for 6 h and then incubated by TNF-?(10 ng/ml)and IL-1?(2 ng/ml)for 24 h.Cells stimulated with TNF-? and IL-1? were used as a control.RT-qPCR and Western blot were performed to confirm the level of HO-1.With 200?M inactivated CORM-3 treated,the level of HO-1 was observed to determine whether the role of CORM-3 is mediated by CO.6.The role of HO-1 on the effects of CORM-3 on the expression of junctional molecules on human gingival fibroblasts incubated by TNF-? and IL-1?With HO-1 gene silence using LIP02000,HGFs were firstly stimulated with 200?M CORM-3 for 6 h and then incubated by TNF-?(10 ng/ml)and IL-1?(2 ng/ml)for 24 h.Cells stimulated with TNF-? and IL-1? were used as a control.The mRNA expression of HO-1,ZO-1,VE-cadherin and ?-catenin were detected by RT-qPCR,and the data were calculated by using the 2-??Ct method.At the same time,the protein expression of HO-1,ZO-1,VE-cadherin and ?-catenin were detected by Western blot,and the gray value was analyzed by Image J software.Based on the results,we determined whether the role of CORM-3 suppressing high permeability of HGFs stimulated by TNF-?-and IL-1? via HO-1 pathway.7.The effects of CORM-3 on the expression of junctional molecules on gingival fibroblasts in rats with periodontitis.(1)Establishment of experimental periodontitis in rats32 male wistar rats(200±20 g)were divided into four groups randomly:control group(8 rats),CP group(8 rats),CORM group(8 rats)and ZPP-? group(8 rats).The rats in control group did not receive any procedure.Rats in the other three groups were induced experimental periodontitis on the first molar of maxillary.Rats in CORM group were treated with CORM-3(10 mg/kg every other day).Rats in ZPP-IX group were treated with CORM-3(10 mg/kg every other day)and ZPP-?(zinc protoporphyrin-9,10 ?mol/kg every other day).Rats in CP group were treated with saline solution(NaCl 0.9%every other day).(2)The effects of CORM-3 on the expression of junctional molecules on gingival fibroblasts in rats with periodontitis.All the rats were sacrificed on the 10th day after modeling.The first molars of both maxillary regions were resected from each rat and observed by IHC(immunohistochemistry).Part of the gingiva around the periodontitis model teeth was harvested quickly in liquid nitrogen and kept at-80? to detect the expression of HO-1,ZO-1,VE-cadherin and ?-catenin by RT-qPCR and Western blot.8.Statistical analysis.The values were obtained from at least three independent experiments performed in triplicate.The Turkey's multiple comparisons test was used for difference analysis between two groups.Values of P less than 0.05 were taken as statistically significant.Results1.Isolation and culture of HGFsHGFs were isolated from explants of normal gingival tissues obtained during routine clinical procedures.Primary HGFs were isolated through gingival tissues and cultivated successfully.2.The effects of CORM-3 on the viabilty of HGFsThe results of CCK-8 showed the proliferation of HGFs in 0,100 ?M,200 ?M and 400?M CORM-3 groups had no obvious difference compared with normal control group.But the concentration of 800 ?M CORM-3 group had obvious toxicity to the HGFs and inhibited the proliferation of HGFs stimulated by TNF-? and IL-1?(P<0.001).Therefore,CORM-3 was used at 100 ?M,200 ?M and 400 ?M for the subsequent assays.3.The effects of CORM-3 on the expression of junctional molecules on human gingival fibroblasts incubated by TNF-? and IL-1?Immunostaining for VE-cadherin,ZO-1,and ?-catenin showed a linear staining pattern at the cell borders under control conditions in confluent HGFs cell monolayers.Incubation with TNF-? and IL-1? for 24 h led to intercellular gap formation and pronounced loss of VE-cadherin,ZO-1,and ?-catenin staining at the cell borders.Incubation with increasing CORM-3 concentrations prevented the loss of VE-cadherin,ZO-1,and ?-catenin to different degrees.The inhibitory effects of CORM-3 on VE-cadherin,ZO-1,and ?-catenin loss also showed dose dependency.The most obvious protective effects were observed at the concentration of 200 ?M for every junction molecule.Incubation with TNF-? and IL-1? induced an increase in cell permeability.After TNF-? and IL-1? were added to HGFs for 24 h,VE-cadherin,ZO-1,and p-catenin were significantly decreased at the gene and protein levels compared with those in normal and noninduced cells.With CORM-3 added,the VE-cadherin,ZO-1,and?-catenin levels were reversed significantly at 200 ?M.4.The effects of CORM-3 on the the permeability of HGFs monolayers stimulated with TNF-? and IL-1?Changes in the permeability of HGFs monolayers to CORM-3 were assessed by measuring FITC-BSA flux following 24 h of incubation with or without TNF-?,IL-1?and CORM-3.The result showed that the inflammatory mediators TNF-? and IL-1?significantly increased the permeability of the HGFs monolayer(P<0.001),and CORM-3 was able to reduce this effect by 82.8±6.8%(P<0.001)at 200 ?M,by 25.4±5.7%(P<0.01)at 100 ?M,and by 22.3±7%(P<0.05)at 400 ?M.The greatest inhibitory effect of CORM-3 on permeability was observed at 200 ?M(P<0.001).5.The effects of CORM-3 on the expression of HO-1 on human gingival fibroblasts incubated by TNF-? and IL-1 ?HGFs were incubated with CORM-3(200 ?M)with or without TNF-?(10 ng/ml)and IL-1?(2 ng/ml)for 24 h.Then,HO-1 mRNA and protein expression levels were examined by RT-qPCR and Western blot.The mRNA and protein expression levels of HO-1 induced by CORM-3 were significantly increased compared with those in the TNF-? and IL-1? group.Deactivated CORM-3 did not induce HO-1 expression,which indicated that the effect of CORM-3 mainly resulted in CO.The results showed that TNF-a-and IL-1?-induced HO-1 expression levels were comparable to those in the TNF-? and IL-1? group.Compared with the TNF-? and IL-1? group,CORM-3 significantly increased the mRNA and protein expressions of HO-1 in HGFs.On the other hand,deactivated CORM-3 did not have an inductive effect on HO-1.CORM-3 could promote HO-1 expression by releasing CO.6.The role of HO-1 on the effects of CORM-3 on the expression of junctional molecules on human gingival fibroblasts incubated by tumor necrosis factor-a and interleukin-1?To examine whether the regulatory effect of CORM-3 on HGFs was mediated by HO-1,HO-1 siRNA was transfected into HGFs.HO-1 siRNA transfection decreased the mRNA expression of HO-1 significantly,while scrambled siRNA transfection had no effect on HO-1 expression.In other experiments,HGFs were transfected with HO-1 siRNA for 6 h and then incubated with TNF-? and IL-1? and with or without CORM-3 for 24 h.The mRNA and protein expression level changes in HO-1 were significantly abolished by HO-1 siRNA transfection.HO-1 siRNA or scrambled siRNA transfected cells were used to clarify the influence of HO-1 or scrambled siRNA on the expression of VE-cadherin,ZO-1,and ?-catenin.The results showed that HO-1 siRNA transfection suppressed the anti-inflammatory effects of CORM-3,and VE-cadherin,ZO-1,and ?-catenin expression levels were not increased by CORM-3.The results were shown by the mRNA and protein levels.The expression levels of VE-cadherin,ZO-1,and ?-catenin were lower in the HO-1 siRNA+CORM-3+TNF-?+IL-1? group than in the TNF-?-IL-1? group(P<0.05).The results that CORM-3 was no longer effective in inducing the anti-inflammatory effects of VE-cadherin,ZO-1,and ?-catenin gene and protein expression after HO-1 siRNA transfection suggested that the regulatory effect of CORM-3 was mediated mainly by HO-1 pathway.7.The effects of CORM-3 on the expression of junctional molecules on gingival tissues in rat with periodontitis.(1)Establishment of experimental periodontitis in ratsExperimental periodontitis was induced with orthodontic ligature around the cervix of the first molars of maxillary.The gingiva was injected with the concentration of 1mg/ml in the absence(NL group)or presence(the other three groups)of LPS(Lipopolysaccharide).After ten days,experimental periodontitis in rats were accomplished.(2)The effects of CORM-3 on the expression of junctional molecules on gingival tissues in rat with periodontitis.The gingiva collected from experimental periodontitis teeth was analyzed by RT-qPCR and Western blot.In CP group,VE-cadherin,ZO-1 and ?-catenin were significantly lower than those in NL group at mRNA and protein level(P<0.001).In CORM group,the level of VE-cadherin,ZO-1 and ?-catenin were increased significantly compared with those in NL group in mRNA and protein level(P<0.01).Junctional molecules complex in ZPP-? group decreased compared with those in NL group(P<0.01).The results suggested that the regulatory effect of CORM-3 was mediated mainly by HO-1 pathway.The IHC results showed that compared with the NL group,the expression of VE-cadherin,ZO-1 and ?-catenin in the maxillary regions of the CP group were significantly decreased.The expression of VE-cadherin,ZO-1 and ?-catenin were increased in the CORM group than the NL group.The expression of junction molecules in the ZPP-IX group was significantly decreased due to the inhibition of HO-1.8.Statistical analysis.The values were obtained from at least three independent experiments performed in triplicate.The Turkey's multiple comparisons test was used for difference analysis between two groups.Values of P less than 0.05 were taken as statistically significant.Conclusion1.CORM-3 increased TNF-?-and IL-1?-induced expression of the junctional molecules,and the most significantly effect was at 200 ?M.2.CORM-3 at 200 ?M promoted the expression of HO-1 by releasing CO.3.The effect of CORM-3 on the expression of junctional complex molecule VE-cadherin,ZO-1,and ?-catenin on HGFs was abrogated after HO-1 siRNA transfected into the cells,suggesting that the regulatory effect of CORM-3 was mediated by the HO-1 pathway.4.CORM-3 increased gene and protein expression of VE-cadherin,ZO-1 and(3-catenin in gingival tissues in rats with experimental periodontitis,and this effect was mediated by the HO-1 pathway.Innovation and SignificanceZO-1,VE-cadherin and ?-catenin are the main junctional molecules in maintaining periodontal tissue integrity,and they are also important reference indexes in evaluating cell permeability.Change of HGFs permeability has great effects in the development of periodontitis.On the basis of studies in vivo and in vitro,we investigated the effect of CORM-3 on junctional molecules of HGFs stimulated with inflammatory cytokines and gingival tissues in rats with experimental periodontitis,and explored the importance of HO-1 pathway in this process,which provided a new selection for the treatment of periodontitis.It is the first time to report the effect of CORM-3 on the permeability of HGFs in periodontitis and the underlying mechanism. |
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