| Background and ObjectivesPeriodontitis is a chronic-inflammatory disease with a high incidence,which is initiated by microorganisms in the dental biofilm and leads to the destruction of tooth-supporting tissues and eventually tooth loss.Human periodontal ligament fibroblasts(HPDLF),as the most predominant cells of periodontal ligament,play an important role in maintaining the dynamic stability and function of the periodontium.Cultivating HPDLF as a model in vitro is an important mean to study periodontal disease.Periodontal pathogenic bacteria,such as Porphyromanas gingivalis and Actinobacillus actinomycetemcomitans,are likely to serve major roles in the pathogenesis of periodontitis.Lipopolysaccharide(LPS),a cell wall component of gram-negative organisms,is regarded as a potent inducer of the proinflammatory response and initiates numerous host-mediated destructive processes.Previous studies have shown that LPS can get through the broken epithelial and has obvious toxic effects on HPDLF cells.HPDLF cells can synthetise various kinds of cytokines to mediate some cellular behavior such as striking the balance of bone formation by osteoblasts and bone resorption by osteoclast,and contributing to the destruction of periodontal system.Smoking is identified as a risk factor for periodontitis.Smoking can not only accelerate the development of periodontal disease,but also affect the effect and prognosis of periodontal treatment.Nicotine,the main component of tobacco,inhibits the proliferation and adhesion of HPDLF,which reduces the protein synthesis of cells.It also changes the microcirculation of periodontal tissue and aggravates the absorption of alveolar bone.In recent years,the toxicity of nicotine to periodontal support.tissues has been paid more and more attention.In tobacco-smoking patients,various kinds of cytokines are synthetised and mediate the bone resorption,such as cyclooxygenase 2(cox-2),prostaglandin E2(PGE2),nuclear factor activated factor receptor ligands(RANKL).PGE2 expression is associated with bone resorption during the progression of periodontal diseases.COX-2 is the rate-limiting enzyme of prostaglandin E2(PGE2)synthesis.Following the discovery of RANKL and its decoy receptor osteoprotegerin(OPG),it has been hypothesized that the balance of the OPG/RANKL axis is critical in the regulation of osteoclast differentiation and function.During the pathological process of periodontitis,these inflammatory cytokines play an important role in the destruction of the alveolar bone;thus,they are regarded as targets for the suppression of inflammation in tobacco-smoking patients with periodontitis.Heme oxygenase-1(HO-1),the most responsive of the known induced enzymes,which can be induced to high levels within hours by cytokines,hemoglobin,oxygen,hyperoxia,heat shock,endotoxins,hydrogen peroxide,ultraviolet radiation,heavy metal and nitric oxide,has become a focus of medical research.Studies have demonstrated that HO-1 has cell-protective functions.With heme as its substrate,HO-1 metabolizes and produces carbon monoxide(CO),biliverdin and Fe2+CO has been confirmed to serve a notable role in the biological processes of many cells,including the inhibition of cell proliferation and apoptosis,and the suppression of the immune inflammatory response.CO-releasing molecules(CORMs)are a new class of compounds,typically transition metal carbonyl complexes,are capable of liberating CO under the appropriate conditions.Therefore,CORMs may be of therapeutic interest due to their capacity to modulate ongoing inflammatory reactions by delivering CO in a controllable fashion.In addition,CORMs have been widely used to increase understanding of the biological function of CO.In previous research of our research group,we found that CORMs can inhibit inflammatory factors of human gingival fibroblasts,inhibit the expression of adhesion molecules,lower immune-active cells to the periodontal tissue infiltration,thus reduce the host inflammatory pathological reaction.Preliminary results suggested that CORM can provide a potential method for the treatment of periodontal disease.Based on the above research background,in the current study,human PDLCs were used as an in vitro model to investigate the influence of CORM-3 on COX-2,PGE2,OPG and RANKL expression in cells stimulated with LPS and nicotine.The possible mechanism by which CORM-3 exerts this effect was also investigated.Materials and MethodsPart 1 The effects of CORM-3 on the toxicity,proliferation and apoptosis in HPDLFs.HPDLCs were isolated with an explant culture technique from normal periodontal ligament tissues obtained from impacted wisdom teeth being removed or the teeth of patients undergoing orthodontic treatment.Keratin positive and vimentin negative results proved that the cultured cells derived from mesenchymal tissue.The cells were divided into five groups and were treated with CORM-3 at 0,100,200,400 and 800 ?M for 24 H.The toxicities of different concentrations of CORM-3 to the human PDLCs were assessed using a CCK-8(WST-8)assay.Then cells were divided into four groups and were treated with CORM-3 at 0,100,200,400 ?M for 24 H,48H,72H and 96H.The activity of different concentrations of CORM-3 to the HPDLCs was assessed using a CCK-8 assay.To establish the inflammatory model of HPDLF cells stimulated by LPS and nicotine,the apoptosis-inhibited role of CORM-3 to HPDLF cells was detected by application flow cytometry using Annexin V/PI.Part 2 The effects of CORM-3 on the expression of inflammatory and osteoclastogenic cytokines in nicotine and lipopolysaccharide stimulated HPDLF cells and the underlying mechanism.1.The effects of CORM-3 on the expression of inflammatory and osteoclastogenic cytokines in nicotine and lipopolysaccharide stimulated HPDLF cells.HPDLF cells were pretreated with 400?M CORM-3 for 6 hours,then cells were stimulated with 1?g/ml LPS and 5 mM nicotine for 24 hours,realtime PCR and Western blot were performed to confrim the level of COX-2,PGE2,OPG and RANKL.With deactivated CORM-3 pretreated,observed the level of COX-2,PGE2,OPG and RANKL to determine whether the role of CORM-3 is mediated by CO.2.The influence of CORM-3 on the expression of HO-1 in HPDLF cellsHPDLF cells were pretreated with 400?M CORM-3 for 6 hours,realtime PCR and Western blot were performed to confrim the level of HO-1.With deactivated CORM-3 pretreated,observed the level of HO-1 to determine whether the role of CORM-3 is mediated by CO.HPDLF cells were pretreated with 400?M CORM-3 for 6 hours,then stimulated with 1?g/ml LPS and 5 mM nicotine for 24 hours,realtime PCR and Western blot were performed to confrim the level of HO-1.With deactivated CORM-3 pretreated,then stimulated with 1?g/ml LPS and 5 mM nicotine for 24 hours,observed the level of HO-1 to determine whether the role of CORM-3 is mediated by CO.3.The underlying mechanism of CORM-3 on the expression of inflammatory and osteoclastogenic cytokines in the nicotine-and lipopolysaccharide-stimulated HPDLF cells.After HO-1 gene silence using LIP03000,cells were pretreated with 400?M CORM-3 for 6 hours,then were stimulated with 1?g/ml LPS and 5 mM nicotine,the level of HO-1,COX-2,PGE2,OPG and RANKL were detected by real-time quantitative PCR and WB.We determined whether the role of CORM-3 suppressing inflammatory and osteoclastogenic cytokines in nicotine and LPS-stimulated HPDLF cells via the HO-1 pathway.ResultsPart 1Primary HPDLFs were isolated through tissue and cultivated successfully.Keratin positive and vimentin negative results proved that the cultured cells derived from mesenchymal tissue.The results of CCK-8 showed the number of cells and activity in 0,100,200 and 400?M CORM-3 groups had no obvious difference compared with normal control group.But the concentration of 800?M CORM-3 group has obvious toxicity to the HPDLF cells.Cells were cultured with 0,100,200 and proliferation for 0,24,48,72 and 96 hours,then detected with CCK-8 assay kit.The results showed 400?M CORM-3 had proliferation effect to HPDLFs.Flow cytometry using Annexin V/PI showed 400?M CORM-3 inhibited the apoptosis of HPDLF cells stimulated by nicotine and lipopolysaccharide.Part 21.In the group cells stimulated with 1?/ml LPS and 5 mM nicotine,the expression of inflammatory and osteoclastogenic cytokines COX-2,PGE2,and RANKL was significantly higher than those in the control group(P<0.05),and the OPG expression was inhibited.Pretreated with 400?CORM-3 for 6 hours,then stimulated with 1?g/ml LPS and 5 mM nicotine,the expression of COX-2,PGE2,RANKL were significantly lower in inflammatory group(P<0.05),while OPG increased significantly(P<0.05).However,the deactived CORM-3 did not have the role.So we could summarized the anti-inflammatory effect of CORM-3 was mediated by CO released from the CORM-3 solution but not the substrate.2.The mRNA and protein expression of HO-1 were significantly higher in CORM-3 pretreated group than those in the control group(P<0.01).However,the deactived CORM-3 did not have the role.The expression of HO-1 was increased by stimulation of 1?g/ml LPS and 5mM nicotine.Pretreated with 400?M CORM-3 for 6 hours,then stimulated with 1?/ml LPS and 5 mM nicotine,the expression of HO-1 was significantly higher than that in the control group(P<0.01).The deactived CORM-3 did not have the role.3.Using liposome LIPO 3000 mediated small interference RNA into the HPDLF cells for HO-1 gene silence,with or without pretreated with 400?M CORM-3 for 6 hours,the expression of COX-2,PGE2 and RANKL in the cells stimulated with 1 p.g/ml LPS and 5 mM nicotine were significantly higher than those in the control group(P<0.05).The expression of OPG did not change significantly,but the OPG/RANKL ratio significantly decreased(P<0.05).So we summarized that CORM-3 suppresses inflammatory and osteoclastogenic cytokines in nicotine-and lipopolysaccharide stimulated HPDLF cells via the heme oxygenase-1 pathway.Conclusions1.400?M CORM-3 had an important role in proliferation and apoptosis of HPDLFs.2.400?M CORM-3 inhibited the expression of inflammatory cytokines and osteoclasts in the periodontal ligament cells stimulated by lipopolysaccharide and nicotine.3.400?M CORM-3 can significantly enhance the expression of HO-1 in the periodontal ligament cells.4.CORM-3 suppresses inflammatory and osteoclastogenic cytokines in nicotine-and lipopolysaccharide-stimulated HPDLF cells via the heme oxygenase-1 pathway. |
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