| Periodontal disease is the most common oral disease,and is the main cause of teeth loss.Periodontal disease is triggered by host inflammatory response against periodontal bacteria and their virulence factors.Lipopolysaccharide(LPS),a major component of the outer membrane of gram-negative bacteria,plays a crucial role in the development of periodontal disease.It stimulates host cells and initiates pro-inflammatory signaling pathways such as the nuclear factor ?B(NF-?B)and mitogen-activated protein kinase(MAPK)pathways,resulting in excessive expression and secretion of a large number of pro-inflammatory cytokines.Ubiquitin proteasome system(UPS)-mediated protein degradation is an important mechanism of regulating protein,which involves in signal transduction,apoptosis,cell cycle and antigen presentation,NF-?B and MAPK signalingpathways are also regulated by UPS.The proteasome inhibitors get widely attention,and have been studied on tumor.Bortezomib(BTZ)is the first proteasome inhibitor that was used for the treatment of multiple myeloma in the clinic.In recent years,BTZ has been used for the study in the field of other diseases.Our previous study showed that BTZ blocked the differentiation of osteoclastsby inhibiting the degradation of cylindromatosis(CYLD)mediated by ?-transducin repeat-containing protein,a ubiquitin-protein ligase.In addition,BTZ was reported to induce cytodifferentiation and mineralization of mouse periodontal ligament cells.These discoveries suggested the potential application of proteasome inhibitors in the treatment of periodontal disease.As a proteasome inhibitor,the divergent effects of BTZ largely depend on its dose and cells type it is administered to.Opposing responses were observed in the same cell line treated with different concentrations of BTZ,and an identical dose of BTZ had opposite effects on different types of cells.Periodontal ligament cells(PDLCs)play an important role in the formation and health of periodontal tissue.The safty,tissue specificity and anti-inflammatory mechanism of proteasome inhibitors must be payed more attention.Therefore,in this study,we investigated the anti-inflammatory activity of BTZ in LPS-stimulated h PDLCs and in a LPS and ligature-induced periodontitis rat model in order to assess the potential clinical application of BTZ in periodontal disease treatment.This study were divided into six parts:Part ? The primary culture and biological characteristics of h PDLCsObjective: To culture primary human periodontal ligament cells(h PDLCs)and provide the cells for experiments in vitro.Methods: The h PDLCs were cultured by explant combined with trypsin-digested technique.The biological characteristics of the h PDLCs were identified by immunocytochemistry staining,immunofluorescence staining,cloning experiments and cell proliferation assay.Results: The h PDLCs were cultured successfully,which showed characteristics of fibroblasts,positive expression for vimentin and negative expression for keratin.The cell clones were observed after 14 days culture.The cell growth curve was S-shaped for nine days.Conclusion: The h PDLCs were cultured successfully by explant combined with trypsin-digested technique.Cells were derived from the embryonic mesoderm,showed extensive self-renewing capacity and proliferation capacity.Part ? The biological safety of bortezomibObjective: To determine the safety of BTZ on h PDLCs and to identify the appropriate concentrations of BTZ for subsequent experiments.Methods: We evaluated the effects of BTZ(0–10 n M)on the proliferation activity,cell cycle and cell apoptosis of h PDLCs by cell proliferation assay and flow cytometry.Following,we chose a relatively safe concentration range of BTZ(0.25 n M,0.5 n M and 1 n M)and determined its effects on cell proliferation for 7 days,cyclin D1 and caspase3 expression in LPS-stimulated h PDLCs using western blot,real-time polymerase chain reaction(PCR)and cell counting kit-8(CCK8).Results: When only BTZ treated h PDLCs,BTZ could not decreased cell viability dose-dependently at concentrations less than 4 n M,but could make the h PDLCs stay in G1 phase.BTZ had little effect on apoptosis of h PDLCs at concentrations less than 10 n M.When LPS stimulated h PDLCs,BTZ had little effects on proliferation of h PDLCs,could not induce the over-expression of cyclin D1 and caspase3,even showed the inhibiting effect on apoptosis.Conclusion: BTZ showed the biosecurity for h PDLCs at low concentration.And we chose relatively safe concentration range of BTZ(0.25 n M,0.5 n M and 1 n M)to carry out the experiments in vitro.Part ? Inhibition effects of bortezomib on inflammatory response in LPS-stimulated h PDLCsObjective: To study the effects of BTZ on m RNA and protein expression of pro-inflammatory cytokines in LPS-stimulated h PDLCs,and to know the anti-inflammatory effects of BTZ.Methods: The h PDLCs were pre-treated with BTZ(0.25 n M,0.5 n M,1 n M)for 1 h prior to co-treatment with LPS(1 ?g/m L)for 3 h,6 h,12 h and 24 h.Relative normalized m RNA expression of tumor necrosis factor-?(TNF-?),interleukin-1 beta(IL-1?),IL-6,and IL-8 was determined using real-time PCR,and protein expression was determined using enzyme-linked immuno sorbent assay(ELISA).Results: m RNA expression of these pro-inflammatory cytokines increased after 3 h to 24 h of LPS treatment,especially when treated with LPS for 6 h.And the protein expression of these pro-inflammatory cytokines increased after 6 h of LPS treatment,but the m RNA and protein expression was both reduced by BTZ pre-treatment.Conclusion: BTZ of 0.25 n M to 1n M could safely down-regulate the production of pro-inflammatory cytokines in LPS-stimulated h PDLCs.These data suggested that the further research value of BTZ in treatment of periodontal disease.Part ? Bortezomib inhibited the inflammatory response in LPS-stimulated h PDLCs via NF-?B signaling pathwayObjective: To study the effects of BTZ on protein and phosphorylation levels of I?Ba,which is an important inhibitor of NF-?B,and effect on nucleus localization of NF-?B/p65 complex in LPS-stimulated h PDLCs,and to elucidate the anti-inflammatory mechanism of BTZ in LPS-stimulated h PDLCs.Methods: The h PDLCs were pre-treated with BTZ(0.25 n M,0.5 n M,and 1 n M)for 6 h or 1 h prior to co-treatment with LPS(1 ?g/m L)for 0.5 h or 6 h.Protein expression and phosphorylation level of I?B? was determined using western blot.And Localization of NF-?B/p65 complex was visualized using immunofluorescence microscopy.Results: Protein concentrations of I?B? and p-I?B? slightly decreased after treatment with LPS for 0.5 h,but there were no statistic difference compared with control group.In contrast,an accumulation of I?B? and p-I?B? was observed after pre-treatment with BTZ for 6 h,the expression of I?B? and p-I?Ba was the highest in the 1 n M BTZ group.When the h PDLCs were pre-treated with BTZ for 1 h prior to co-treatment with LPS for 6 h,LPS significantly induced phosphorylation of I?B?,and slightly increased the total protein concentration of I?B?.Additionally,an increase of BTZ concentration led to an increased expression of I?B? and p-I?B?.Furthermore,we observed that the NF-?B/p65 complex in h PDLCs was localized in the cytoplasm,but was translocated into the cell nucleus upon LPS treatment for 0.5 h and 6 h;its translocation was partly blocked by BTZ.Conclusion: These data indicated that I?B? was regulated by multiple factors besides LPS and that BTZ may suppress NF-?B activation by inhibiting I?B? degradation and preventing the nuclear translocation of NF-?B/p65 complex,and inhibit the transcription of down-stream pro-inflammatory cytokines.Part ? Bortezomib inhibited the inflammatory response in LPS-stimulated h PDLCs via MAPK/AP-1 signaling pathwayObjective: To determine the effects of BTZ on MAPK signaling pathways,mitogen-activated protein kinase phosphatase-1(MKP1)and activator protein-1(AP-1)in LPS-stimulated h PDLCs,and to furtherly elucidate the anti-inflammatory mechanism of BTZ in LPS-stimulated h PDLCs.Methods:(1)The h PDLCs were pre-treated with BTZ(0.25 n M,0.5 n M,and 1 n M)for 6 h prior to co-treatment with LPS(1 ?g/m L)for 0.5 h or 1 h.Phosphorylation and expression of MAPK signaling pathway proteins(p38,Jun N-terminal kinase(JNK),extracellular-signal-regulated kinase 1/2(ERK1/2),and ERK5)were analyzed using western blot.(2)The h PDLCs were pre-treated with BTZ(0.25,0.5 and 1 n M)for 6 h prior to co-treatment with LPS(1 ?g/m L)for 0.5 h,protein expression of MKP1 was analyzed using western blot,while its m RNA level was determined using real-time PCR.(3)Phosphorylation and total protein expression of c-Jun and c-fos were analyzed using western blot.(4)Expression and localization of pc-Jun and pc-fos in the cell nucleus were observed by immunofluorescence staining.Results:(1)The phosphorylation levels of p38,ERK1/2,JNK,and ERK5 increased significantly under 1?g/ml LPS stimulation,and that BTZ selectively inhibited the phosphorylation of p38 and ERK5,but not that of p-JNK and p-ERK1/2.(2)The protein and m RNA levels of MKP1 increased slightly under 1?g/ml LPS stimulation,but there was no statistical difference campared with control group.An increase in BTZ concentration led to an increased expression of protein and m RNA of MKP1.The protein level in 1 n M BTZ group was the highest,the m RNA levels in 0.5 n M and 1 n M BTZ were higher than control group.(3)In LPS-stimuated h PDLCs,the phosphorylation level of c-Jun increased significantly,and the phosphorylation level of c-fos increased slight.But the phosphorylation levels of c-Jun and c-fos were inhibited by BTZ administration,especially for c-Jun.(4)The results of immunofluorescence staining were consistent with that of western blot,1?g/ml LPS induced the expression of pc-Jun and pc-fos in nucleus,but these cellular responses were significantly inhibited by BTZ administration.Conclusion: BTZ inhibited the MAPK pathway not only by inducing the expression of MKP1,but also by blocking its degradation,and then blocked the AP-1 activity.p38,ERK5 and c-Jun are the targets of BTZ for suppressing periodontal inflammation.Part ? Animal experiment: bortezomib ameliorates experimental periodontitis in rats induced by LPS and ligatureObjective: To evaluate anti-inflammatory effect of bortezomib on LPS and ligature-induced periodontitis rat model upon local delivery considering the characteristic of periodontitis.Methods:(1)Periodontitis of the bilateral maxillary second molars of rats was induced by ligature and local LPS injection.Meanwhile,the periodontal clearance of periodontitis models were injected with BTZ(0.01,0.1,and 1 mg/m L,respectively)for 1 h before local injection of 1mg/m L LPS,Three injections were administered per week.After four weeks,all rats were euthanized by cervical dislocation.(2)The linear and volumetric micro-CT data were analyzed after micro-computed tomography(CT)scan.(3)Sections of maxilla samples were stained with HE,the expression of RANKL,OPG,TNF-?,IL-1?,IL-6 and IL-8 in periodontal tissue was detected by immunohistochemical staining.Results:(1)The experimental periodontitis was successfully induced by ligature and topical LPS injection for 4 w.(2)The obvious alveolar bone absorption was observed in experimental periodontitis group.The distance from CEJ to ABC in the experimental periodontitis group was significantly greater than that in control group,in addition,the mean bone volume fraction and bone mineral density were less than those in control group.Topical administration of BTZ partly prevented alveolar bone loss,decrease of the distance from CEJ to ABC and increase of BV/TV values and bone density were followed with increase in BTZ concentration.(3)The infiltration of inflammatory cells in periodontal tissue induced by LPS and ligature was partly suppressed by topical administration of bortezomib using HE staining.(4)BTZ reduced the expression of RANKL,while promoting the expression of OPG in periodontal tissue induced by LPS and ligature.(5)the expression of TNF-?,IL-1?,IL-6,and IL-8 in the periodontal tissue increased in LPS and ligature group,but was partly suppressed by BTZ,which showed that BTZ could inhibit the expression of pro-inflammatory cytokines.Conclusion: Based on the LPS and ligature-induced rat periodontitis model,topical administration of bortezomib reduced the expression of TNF-?,IL-1?,IL-6,and IL-8 in periodontal tissue and the RANKL/OPG ratio,and then inhibited the alveolar resorption.BTZ was shown to be a potent therapy for experimental periodontitis in rats.However,further studies are needed to determine the optimal dosage form and dose for its application. |
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