RESUMEN
Porphyromonas gingivalis has been demonstrated to have the strongest association with periodontitis. Within the host, P. gingivalis relies on acquiring iron and heme through the aggregation and lysis of erythrocytes, which are important factors in the growth and virulence of P. gingivalis. Additionally, the excess obtained heme is deposited on the surface of P. gingivalis, protecting the cells from oxidative damage. Based on these biological properties of the interaction between P. gingivalis and erythrocytes, this study developed an erythrocyte membrane nanovesicle loaded with gallium porphyrins to mimic erythrocytes. The nanovesicle can target and adhere with P. gingivalis precisely, being lysed and utilized by P. gingivalis as erythrocytes. Ingested gallium porphyrin replaces iron porphyrin in P. gingivalis, causing intracellular metabolic disruption. Deposited porphyrin generates a large amount of reactive oxygen species (ROS) under blue light, causing oxidative damage, and its lethality is enhanced by bacterial metabolic disruption, synergistically killing P. gingivalis. Our results demonstrate that this strategy can target and inhibit P. gingivalis, reduce its invasion of epithelial cells, and alleviate the progression of periodontitis.
Asunto(s)
Eritrocitos , Periodontitis , Porfirinas , Porphyromonas gingivalis , Porphyromonas gingivalis/efectos de los fármacos , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/química , Periodontitis/microbiología , Periodontitis/tratamiento farmacológico , Periodontitis/patología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Humanos , Porfirinas/química , Porfirinas/farmacología , Animales , Especies Reactivas de Oxígeno/metabolismo , Galio/química , Galio/farmacología , Ratones , Antibacterianos/farmacología , Antibacterianos/química , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacologíaRESUMEN
Objective: To investigate the effects of Porphyromonas gingivalis derived outer membrane vesicles (Pg OMV) on osteoclast differentiation of macrophages and its underlying mechanisms. Methods: The morphology and the size distribution of Pg OMV were analyzed by transmission electron microscopy and nanoparticle tracing analysis, respectively. The osteoclast precursors were treated with 1, 3 and 10 mg/L Pg OMV (1, 3 and 10 mg/L OMV treatment group) or phosphate buffer solution (PBS)(control group). The formation of osteoclasts was analyzed by tartrate-resistant acid phosphase (TRAP) staining and F-actin staining and real-time quantitative PCR (RT-qPCR) were used to detect the expression of Fos and matrix metallopeptidase 9 (MMP9). Polymyxin B (PMB) was used to block lipopolysaccharide (LPS) and then Pg OMV was used to treat osteoclast precursor (PMB-OMV treatment group), and OMV treatment group was used as control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The effect of Pg OMV on the expression of Toll-like receptor (TLR) 2 and TLR4 in preosteoclasts was detected by Western blotting. The osteoclast precursors were pretreated with 10, 50, 100 and 200 µmol/L C29, an inhibitor of TLR2, and then treated with Pg OMV(OMV+10, 50, 100 and 200 µmol/L C29 treatment group) and OMV treatment group without C29 pretreatment was control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The osteoclast precursor cells were treated with OMV (OMV treatment group) and OMV incubated with PMB (PMB-OMV treatment group) and the expression of TLR2 in osteoclast precursor was detected by Western blotting. Results: Pg OMV showed classical vesicular structures, and the average particle size of Pg OMV were 179.2 nm. A large number of actin rings were observed in the 3 and 10 mg/L OMV treatment groups. The percentages of TRAP-positive osteoclast area in 3 mg/L OMV treatment group [(22.6±2.1)%] and 10 mg/L OMV treatment group [(32.0±2.3)%] were significantly increased compared with control group [(4.9±0.5)%] (P<0.001). Compared with the control group (1.000±0.029), the mRNA relative expression of Fos in 3 mg/L OMV treatment group (1.491±0.114) and 10 mg/L OMV treatment group (1.726±0.254) was significantly increased (P=0.013, P=0.001). Compared with the control group (1.007±0.148), the mRNA relative expression of MMP9 in the group of 10 mg/L OMV (2.232±0.097) was significantly increased (P<0.001). Actin ring formation was less in PMB-OMV treatment groups than in OMV treatment groups. The proportion of TRAP-positive osteoclasts area [(14.8±3.8)%] in PMB-OMV treatment group was significantly lower than OMV treatment group [(31.5±6.7) %] (P=0.004). The relative expression of TLR2 in OMV treatment group (1.359±0.134) was significantly higher than that in the control group (1.000±0.000) (t=4.62, P=0.044). Compared with the OMV treatment group [(29.4±1.7)%], 50, 100 and 200 µmol/L C29 significantly decreased the formation of osteoclasts [(24.0±1.7)%, (18.5±2.1)%, (9.1±1.3) %] (P=0.026, P<0.001, P<0.001). TLR2 protein expression in PMB-OMV group (0.780±0.046) was significantly lower than that in OMV group (1.000±0.000)(t=8.32, P=0.001). Conclusions: Pg OMV can promote osteoclast differentiation by carrying LPS, TLR2 plays an important role in Pg OMV mediated osteoclast differentiation.
Asunto(s)
Lipopolisacáridos , Osteoclastos , Lipopolisacáridos/farmacología , Porphyromonas gingivalis/química , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Actinas/metabolismo , Actinas/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Mensajero/metabolismo , Diferenciación CelularRESUMEN
Diabetes and periodontitis are prevalent diseases that considerably impact global economy and diabetes is a major risk factor of periodontitis. Mitochondrial dynamic alterations are involved in many diseases including diabetes and this study aims to evaluate their relevance with diabetes aggravated periodontitis. Sixty mice are randomly divided into 4 groups: control, periodontitis, diabetes and diabetic periodontitis. Periodontitis severity is evaluated by alveolar bone loss, inflammation and oxidative stress status. Mitochondrial structural and functional defects are evaluated by the mitochondrial fission/fusion events, mitochondrial reactive oxygen species (ROS) accumulation, complex activities and adenosine triphosphate (ATP) production. Advanced glycation end product (AGE) and Porphyromonas gingivalis are closely related to periodontitis occurrence and development. Human gingival fibroblast cells (HGF-1) are used to investigate the AGE role and lipopolysaccharide (LPS) from Porphyromonas gingivalis (P-LPS) in aggravating diabetic periodontitis by mitochondrial dynamic and function alterations. In vivo, diabetic mice with periodontitis show severe bone loss, increased inflammation and oxidative stress accumulation. Among mice with periodontitis, diabetic mice show worse mitochondrial dynamic perturbations than lean mice, along with fusion protein levels inducing more mitochondrial fission in gingival tissue. In vitro, AGEs and P-LPS co-treatment causes severe.
Asunto(s)
Diabetes Mellitus Experimental , Periodontitis , Ratones , Humanos , Animales , Dinámicas Mitocondriales , Diabetes Mellitus Experimental/complicaciones , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Periodontitis/etiología , Periodontitis/metabolismo , Inflamación , Porphyromonas gingivalis/química , Porphyromonas gingivalis/metabolismoRESUMEN
The immune response mediated by Th17 cells is essential in the pathogenesis of periodontitis. Emerging evidence has demonstrated that lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) could promote Th17-cell differentiation directly, while the downstream signaling remains elusive. This study was aimed to explore the role of JMJD3 (a JmjC family histone demethylase) and signal transducers and activators of transcription 3 (STAT3) in Th17-cell differentiation triggered by Pg-LPS and clarify the interaction between them. We found that the expression of JMJD3 and STAT3 was significantly increased under Th17-polarizing conditions. Pg-LPS could promote Th17-cell differentiation from CD4+ T cells, with an increased expression of JMJD3 and STAT3 compared to the culture without Pg-LPS. The coimmunoprecipitation results showed that the interactions of JMJD3 and STAT3, STAT3 and retinoid-related orphan nuclear receptor γt (RORγt) were enhanced following Pg-LPS stimulation during Th17-cell differentiation. Further blocking assays were performed and the results showed that inhibition of STAT3 or JMJD3 both suppressed the Th17-cell differentiation, JMJD3 inhibitor could reduce the expression of STAT3 and p-STAT3, while JMJD3 expression was not affected when STAT3 was inhibited. Taken together, this study found that JMJD3 could promote Pg-LPS induced Th17-cell differentiation by modulating the STAT3-RORc signaling pathway.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji , Células Th17 , Diferenciación Celular/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Receptores Nucleares Huérfanos/metabolismo , Porphyromonas gingivalis/química , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
Background: Clinically, the failure of periodontal therapy stems largely from an inability to control the inflammatory response. Resolution of inflammation is an active, energy-requiring repair process, not merely a passive termination of inflammation. AMP-activated protein kinase (AMPK), a key energy sensor, has been shown to negatively regulate inflammatory signaling pathways. Thus, there is a crucial need for new therapeutic strategies to modulate AMPK and to promote enhanced resolution of inflammation. This study is aimed at investigating the anti-inflammatory effects of ETC-1002 through modulating AMPK in periodontitis. Methods: RAW264.7 cells were infected with Pg-LPS in the presence or absence of ETC-1002, following which the expression levels of proinflammatory cytokines and inflammation signaling-related proteins were evaluated by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. ETC-1002 was applied in a murine model of periodontitis to determine its anti-inflammatory effect in vivo. Histological changes were investigated by hematoxylin and eosin (H&E) staining, the levels of proinflammatory cytokines were detected using immunohistochemistry, and alveolar bone height was measured using micro-CT imaging. Results: ETC-1002 inhibited the production of proinflammatory cytokines, promoted AMPK phosphorylation, and decreased IκBα and NF-κB p65 phosphorylation levels in Pg-LPS-treated RAW264.7 macrophages. The inhibitory effects of ETC-1002 on the production of proinflammatory mediators were significantly abrogated by siRNA-mediated silencing of AMPKα in RAW264.7 cells. In vivo, ETC-1002 inhibited inflammatory cell infiltration, the expression of proinflammatory cytokines, and the inflammation-mediated destruction of alveolar bone in mice with experimental periodontitis. The anti-inflammatory effect of ETC-1002 in the periodontium could be reversed by the administration of Compound C, an AMPK inhibitor. Conclusions: ETC-1002 exerts anti-inflammatory effects in Pg-LPS-treated RAW264.7 cells via the AMPK/NF-κB pathway in vitro and inhibits the progress of experimental periodontitis in mice in an AMPK signaling-dependent manner in vivo. These results provide evidence for the beneficial effects of ETC-1002 in the treatment of periodontitis.
Asunto(s)
Lipopolisacáridos , Periodontitis , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Citocinas/metabolismo , Ácidos Dicarboxílicos , Ácidos Grasos , Humanos , Inflamación/metabolismo , Lipopolisacáridos/efectos adversos , Ratones , FN-kappa B/metabolismo , Periodontitis/tratamiento farmacológico , Porphyromonas gingivalis/química , Porphyromonas gingivalis/metabolismoRESUMEN
To acquire heme, Porphyromonas gingivalis uses a hemophore-like protein (HmuY). HmuY sequesters heme from host hemoproteins or heme-binding proteins produced by cohabiting bacteria, and delivers it to the TonB-dependent outer-membrane receptor (HmuR). Although three-dimensional protein structures of members of the novel HmuY family are overall similar, significant differences exist in their heme-binding pockets. Histidines (H134 and H166) coordinating the heme iron in P. gingivalis HmuY are unique and poorly conserved in the majority of its homologs, which utilize methionines. To examine whether changes observed in the evolution of these proteins in the Bacteroidetes phylum might result in improved heme binding ability of HmuY over its homologs, we substituted histidine residues with methionine residues. Compared to the native HmuY, site-directed mutagenesis variants bound Fe(III)heme with lower ability in a similar manner to Bacteroides vulgatus Bvu and Tannerella forsythia Tfo. However, a mixed histidine-methionine couple in the HmuY was sufficient to bind Fe(II)heme, similarly to T. forsythia Tfo, Prevotella intermedia PinO and PinA. Double substitution resulted in abolished heme binding. The structure of HmuY heme-binding pocket may have been subjected to evolution, allowing for P. gingivalis to gain an advantage in heme acquisition regardless of environmental redox conditions.
Asunto(s)
Hemoproteínas , Porphyromonas gingivalis , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Hemo/química , Hemoproteínas/química , Hemoproteínas/genética , Porphyromonas gingivalis/químicaRESUMEN
The type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Secuencia de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/química , Sistemas de Secreción Bacterianos/genética , Porphyromonas gingivalis/química , Porphyromonas gingivalis/genética , Unión Proteica , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac]0-2)-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane ß-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm. IMPORTANCE Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0-2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Porphyromonas gingivalis/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Carbohidratos , Glicoproteínas/genética , Glicosilación , Humanos , Polisacáridos/química , Polisacáridos/metabolismo , Porphyromonas gingivalis/química , Porphyromonas gingivalis/genéticaRESUMEN
Porphyromonas gingivalis (Pg) a major periodontal pathogen involved in periodontal disease development and progression. Moreover, Pg has two fimbriae surface proteins (FimA and Mfa1) that are genetically distinct and make-up the fimbrial shaft which in-turn form crucial attachment to oral bacteria and multiple host cells. However, unlike FimA, Mfa1 attachment to non-periodontal cells has not been fully elucidated. Considering Pg-associated periodontal disease contributes to pulmonary disease development, we investigated whether Mfa1 can functionally interact with human bronchial epithelial cells and, likewise, trigger a functional response. Initially, we simulated molecular docking and performed both luciferase and neutralization assays to confirm Mfa1-related functional interaction. Subsequently, we treated BEAS-2B cells with purified Mfa1 and performed cytokine quantification through real time-PCR and ELISA to establish Mfa1-related functional response. We found that both Mfa1-TLR2 and Mfa1-TLR4 docking is possible, however, only Mfa1-TLR2 showed a functional interaction. Additionally, we observed that both IL-8 and IL-6 gene expression and protein levels were induced confirming Mfa1-related functional response. Taken together, we propose that BEAS-2B human bronchial epithelial cells are able to recognize Pg Mfa1 and induce both IL-8 and IL-6 inflammatory responses.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bronquios/patología , Células Epiteliales/metabolismo , Proteínas Fimbrias/metabolismo , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Porphyromonas gingivalis/fisiología , Receptor Toll-Like 2/metabolismo , Línea Celular , Fimbrias Bacterianas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Porphyromonas gingivalis/química , Unión Proteica , Mapeo de Interacción de Proteínas , Receptor Toll-Like 4/química , Receptor Toll-Like 4/metabolismoRESUMEN
Low electric current can inhibit certain microbial biofilms and enhance the efficacy of antimicrobials against them. This study investigated the electricidal and bioelectric effects of direct current (DC) against Porphyromonas gingivalis biofilms as well as the underlying mechanisms. Here, we firstly showed that DC significantly suppressed biofilm formation of P. gingivalis in time- and intensity-dependent manners, and markedly inhibited preformed P. gingivalis biofilms. Moreover, DC enhanced the killing efficacy of metronidazole (MTZ) and amoxicillin with clavulanate potassium (AMC) against the biofilms. Notably, DC-treated biofilms displayed upregulated intracellular ROS and expression of ROS related genes (sod, feoB, and oxyR) as well as porin gene. Interestingly, DC-induced killing of biofilms was partially reversed by ROS scavenger N-dimethylthiourea (DMTU), and the synergistic effect of DC with MTZ/AMC was weakened by small interfering RNA of porin gene (si-Porin). In conclusion, DC can exert electricidal and bioelectric effects against P. gingivalis biofilms partially via promotion of oxidative stress and antibiotic transport, which offers a promising approach for effective management of periodontitis.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Porphyromonas gingivalis/química , Porphyromonas gingivalis/efectos de los fármacos , Amoxicilina/farmacología , Electricidad , Humanos , Metronidazol/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Periodontitis/microbiología , Porphyromonas gingivalis/crecimiento & desarrollo , Porphyromonas gingivalis/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Periodontitis is defined as a multifactorial polymicrobial infection accompanied by inflammatory reactions. Porphyromonas gingivalis (Pg) is known as a major pathogen in the initiation and progression of periodontitis, and a major virulence factor is Pg lipopolysaccharide (LPS). Molecular hydrogen (H2) has been reported to act as a gaseous antioxidant, which suppresses periodontitis progression by decreasing gingival oxidative stress. However, no human periodontitis model has examined the anti-inflammatory effects of H2. In this study, we examined the effects of H2 on Pg LPS-induced secretion of 8 types of inflammation markers in a human periodontitis model using human gingival cells with enzyme-linked immunosorbent assays. Our results demonstrated that Pg LPS increased interleukin (IL) 1 alpha (IL-1α) and IL-6 secretion, but H2 significantly suppressed the secretion of both cytokines without cytotoxicity. H2 can suppress the production of IL-1α and IL-6, which are identified as cytokines involved in inflammatory reactions in periodontal disease. Thus, H2 may provide therapeutic applications for periodontitis.
Asunto(s)
Células Epiteliales/metabolismo , Encía/metabolismo , Hidrógeno/farmacología , Interleucina-1alfa/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/toxicidad , Porphyromonas gingivalis/química , Humanos , Lipopolisacáridos/químicaRESUMEN
In obese patients, enhanced serum levels of free fatty acids (FFA), such as palmitate (PA) or oleate (OA), are associated with an increase in systemic inflammatory markers. Bacterial infection during periodontal disease also promotes local and systemic low-grade inflammation. How both conditions concomitantly impact tooth movement is largely unknown. Thus, the aim of this study was to address the changes in cytokine expression and the secretion of human periodontal ligament fibroblasts (HPdLF) due to hyperlipidemic conditions, when additionally stressed by bacterial and mechanical stimuli. To investigate the impact of obesity-related hyperlipidemic FFA levels on HPdLF, cells were treated with 200 µM PA or OA prior to the application of 2 g/cm2 compressive force. To further determine the additive impact of bacterial infection, HPdLF were stimulated with lipopolysaccharides (LPS) obtained from Porphyromonas gingivalis. In mechanically compressed HPdLF, PA enhanced COX2 expression and PGE2 secretion. When mechanically stressed HPdLF were additionally stimulated with LPS, the PGE2 and IL6 secretion, as well as monocyte adhesion, were further increased in PA-treated cultures. Our data emphasize that a hyperlipidemic condition enhances the susceptibility of HPdLF to an excessive inflammatory response to compressive forces, when cells are concomitantly exposed to bacterial components.
Asunto(s)
Fibroblastos/inmunología , Hiperlipidemias/fisiopatología , Inflamación/inmunología , Lipopolisacáridos/farmacología , Ligamento Periodontal/inmunología , Porphyromonas gingivalis/química , Estrés Mecánico , Fuerza Compresiva , Citocinas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Inflamación/inducido químicamente , Inflamación/patología , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/patología , PresiónRESUMEN
Curcumin, a yellow polyphenol extracted from the turmeric root is used as a diet supplement. It exhibits anti-inflammatory, antioxidant, and antitumor properties by modulating different intracellular mechanisms. Due to their low solubility in water, the curcumin molecules must be encapsulated into liposomes to improve the bioavailability and biomedical potential. For the periodontal tissue and systemic health, it is essential to regulate the local inflammatory response. In this study, the possible beneficial effect of liposomes loaded with curcumin (CurLIP) in neural crest-derived human periodontal ligament stem cells (hPDLSCs) and in endothelial-differentiated hPDLSCs (e-hPDLSCs) induced with an inflammatory stimulus (lipopolysaccharide obtained from Porphyromonas gingivalis, LPS-G) was evaluated. The CurLIP formulation exhibited a significant anti-inflammatory effect by the downregulation of Toll-like receptor-4 (TLR4)/Myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa light chain enhancer of activated B cells (NFkB)/NLR Family Pyrin Domain Containing 3 (NLRP3)/Caspase-1/Interleukin (IL)-1ß inflammation cascade and reactive oxygen species (ROS) formation. Moreover, the exposure to LPS-G caused significant alterations in the expression of epigenetic modifiers, such as DNA Methyltransferase 1 (DNMT1) and P300, while the CurLIP treatment showed physiological expression. Overall, our in vitro study provides novel mechanistic insights into the intracellular pathway exert by CurLIP in the regulation of inflammation and epigenetic modifications.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Curcumina/farmacología , Endotelio Vascular/efectos de los fármacos , Inflamación/tratamiento farmacológico , Lipopolisacáridos/farmacología , Cresta Neural/efectos de los fármacos , Células Madre/efectos de los fármacos , Diferenciación Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Liposomas/administración & dosificación , Liposomas/química , Cresta Neural/citología , Cresta Neural/metabolismo , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/química , Especies Reactivas de Oxígeno , Células Madre/citología , Células Madre/metabolismoRESUMEN
Periodontitis is a widespread chronic infectious-inflammatory disease associated with multiple systemic diseases. Visfatin is an adipokine-enzyme that can be locally produced by human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). It can upregulate proinflammatory cytokines and matrix metalloproteinases (MMPs) in various types of cells. However, the effects of visfatin on healthy and inflammatory human periodontal cells as well as the underlying molecular mechanisms remain unclear. This study firstly demonstrated visfatin expression was highly elevated in inflamed human gingiva and Pg LPS-treated hPDLCs. Moreover, recombinant visfatin significantly upregulated the expression of proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and prodegradative factors (EMPPRIN, MMP1, MMP3 and MMP13) in hPDLCs. Next, we found the levels of proinflammatory and prodegradative cytokines were significantly increased in visfatin-overexpressing hPDLCs, and decreased in visfatin-silencing inflammatory hGFs (iGFs) when treated with Pg LPS. In the absence of Pg LPS, visfatin silencing failed to affect the expression of these factors in iGFs, and overexpression of visfatin upregulated MMPs but no other factors in hPDLCs. Furthermore, marked NF-κB pathway activation with increased phosphorylation of p65 was observed in visfatin-overexpressing hPDLCs. BAY11-7082, a specific inhibitor of NF-κB, partially reversed the upregulation proinflammatory and prodegradative factors induced by visfatin overexpression. Taken together, this study showed that visfatin critically regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway. The findings suggest that visfatin is closely involved in the development of periodontitis, and may serve as a promising novel biomarker and therapeutic target for periodontitis management.
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Citocinas/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/química , Adulto , Células Cultivadas , Femenino , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos , Masculino , Ligamento Periodontal/efectos de los fármacos , Adulto JovenRESUMEN
Periodontitis is a set of chronic inflammatory diseases caused by the accumulation of Gram-negative bacteria on teeth, resulting in gingivitis, pocket formation, alveolar bone loss, tissue destruction, and tooth loss. In this study, the contents of ginsenosides isolated from Panax ginseng fruit extract were quantitatively analyzed, and the anti-inflammatory effects were evaluated in human periodontal ligament cells. The major ginsenosides, Re, Ra8, and Rf, present in ginseng fruit were simultaneously analyzed by a validated method using high-performance liquid chromatography with a diode-array detector; Re, Ra8, and Rf content per 1 g of P. ginseng fruit extract was 1.01 ± 0.03, 0.33 ± 0.01, and 0.55 ± 0.04 mg, respectively. Ginsenosides-Re, -Ra8, and -Rf inhibited the production of pro-inflammatory factors and the expression of important cytokines in periodontitis by inducing the expression of heme oxygenase 1 (HO-1), promoting osteoblast differentiation of periodontal ligament cells, suppressing alveolar bone loss, and promoting the expression of osteoblast-specific genes, such as alp, opn, and runx2. An inhibitory effect of these ginsenosides on periodontitis and alveolar bone loss was observed via the regulation of HO-1 and subsequent epidermal growth factor receptor (EGFR) signaling. Silencing EGFR with EGFR siRNA confirmed that the effect of ginsenosides on HO-1 is mediated by EGFR. In conclusion, this study evaluated the contents of ginsenosides-Re, -Ra8, and -Rf isolated from P. ginseng fruit extract. Therefore, these results provide important basic data for future P. ginseng fruit component studies and suggest that ginsenosides Re, Ra8, and Rf have potential as future treatment options for periodontitis.
Asunto(s)
Antiinflamatorios/farmacología , Receptores ErbB/metabolismo , Ginsenósidos/aislamiento & purificación , Ginsenósidos/farmacología , Hemo-Oxigenasa 1/metabolismo , Osteogénesis/efectos de los fármacos , Panax/química , Ligamento Periodontal/citología , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Frutas/química , Regulación de la Expresión Génica/efectos de los fármacos , Ginsenósidos/química , Humanos , Mediadores de Inflamación/metabolismo , Límite de Detección , Lipopolisacáridos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Extractos Vegetales/química , Porphyromonas gingivalis/química , Análisis de Regresión , Transducción de Señal/efectos de los fármacosRESUMEN
The enhancer of zeste homolog 2 (EZH2) represents a potential target for periodontitis treatment; however, its role in the development of periodontitis remains unclear. The current study aimed to elucidate the role of EZH2 in osteoclasts (OCs) growth as well as the mechanism underpinning the related process. The potential interaction among EZH2, microRNA-101 (miR-101), and vascular cell adhesion molecule 1 (VCAM-1) was evaluated using chromatin immunoprecipitation and dual-luciferase reporter gene assay. The expressions of EZH2 and miR-101 in OCs were examined by Western blot analysis and reverse transcription squantitative polymerase chain reaction. Loss- and gain-function assays were then performed to determine the role of EZH2/miR-101/VCAM-1 in periodontitis and OCs proliferation, followed by OC growth and proliferation detected using tartrate resistant acid phosphatase (TRAP) and 5-ethynyl-2'-deoxyuridine staining. Enzyme-linked immunoassay was conducted to determine the expression of interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). A periodontitis rat model was established to investigate the effect of EZH2 and VCAM-1 in vivo. EZH2 was overexpressed, while miR-101 was downregulated in the OCs of periodontitis. Silencing of EZH2, VCAM-1 repression, or miR-101 elevation suppressed the growth and proliferation of OC while acting to encumber the release of IL-1ß and TNF-α. EZH2 negatively targeted miR-101, while miR-101 negatively targeted VCAM-1. Moreover, silencing of EZH2 or VCAM-1 was observed to attenuate periodontitis which was evidenced by an increase in BMD, BV/TV, and BS/BV as well as reduction in TRAP and cathepsin K in vivo. Taken together, the key findings of the current study demonstrate that EZH2 knockdown inhibited OC formation by elevating the expression of miR-101 via suppression of VCAM-1, ultimately attenuating periodontitis.
Asunto(s)
Regulación hacia Abajo/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Osteoclastos/metabolismo , Periodontitis/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Secuencia de Bases , Proliferación Celular , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Inflamación/patología , Lipopolisacáridos , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Porphyromonas gingivalis/química , Regiones Promotoras Genéticas/genética , Unión Proteica , Células RAW 264.7 , Ratas Sprague-Dawley , Regulación hacia Arriba/genéticaRESUMEN
Periodontitis is usually sustained from microorganism of oral cavity, like Porphyromonas gingivalis (P. gingivalis). Periodontal disease is an infectious disease that afflicts a large number of people. Researches are investigating on the mesenchymal stem cells (MSCs) response to inflammatory events in combination with antioxidant substances. In particular, ascorbic acid (AA) increased cell proliferation, upregulated the cells pluripotency marker expression, provide a protection from inflammation, and induced the regeneration of periodontal ligament tissue. The purpose of the present research was to investigate the effects of AA in primary culture of human periodontal ligament stem cells (hPDLSCs) exposed to P. gingivalis lipopolysaccharide (LPS-G). The effect of AA on hPDLSCs exposed to LPS-G was determined through the cell proliferation assay. The molecules involved in the inflammatory pathway and epigenetic regulation have been identified using immunofluorescence and Western blot analyses. miR-210 level was quantified by qRT-PCR, and the ROS generation was finally studied. Cells co-treated with LPS-G and AA showed a restoration in terms of cell proliferation. The expression of NFκB, MyD88, and p300 was upregulated in LPS-G exposed cells, while the expression was attenuated in the co-treatment with AA. DNMT1 expression is attenuated in the cells exposed to the inflammatory stimulus. The level of miR-210 was reduced in stimulated cells, while the expression was evident in the hPDLSCs co-treated with LPS-G and AA. In conclusion, the AA could enhance a protective effect in in vitro periodontitis model, downregulating the inflammatory pathway and ROS generation and modulating the miR-210 level.
Asunto(s)
Ácido Ascórbico/farmacología , Epigénesis Genética/efectos de los fármacos , Periodontitis/genética , Células Madre/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Humanos , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/patología , Ligamento Periodontal/fisiología , Periodontitis/inducido químicamente , Periodontitis/microbiología , Periodontitis/patología , Porphyromonas gingivalis/química , Células Madre/patología , Células Madre/fisiologíaRESUMEN
Porphyromonas gingivalis fimbriae play a critical role in colonization. Elucidation of the fimbrial structure in atomic detail is important for understanding the colonization mechanism and to provide means to combat periodontitis. X-ray crystallography is a technique that is used to obtain detailed information of proteins along with bound ligands and ions. Crystallization of the protein of interest is the first step toward structure determination. Unfortunately it is not possible to predict the crystallization condition of a certain protein or even if the protein can be crystallized. Protein crystallization is, on the contrary, a matter of trial and error. However, the best strategy for success is to focus on the protein purification step to obtain a sample that is pure, stable, homogeneous and of high concentration. This chapter addresses general methods for crystallization of fimbrial proteins.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Fimbrias/química , Fimbrias Bacterianas/química , Porphyromonas gingivalis/química , Proteínas Recombinantes/química , Adhesión Bacteriana/fisiología , Cristalización/métodos , Periodontitis/microbiologíaRESUMEN
OmpA-like proteins located in the outer bacterial membrane are potential virulence factors from the major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia. Our previous studies have shown that OmpA-like proteins are glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) and are strongly reactive to wheat germ agglutinin (WGA) lectin, which shows sugar specificity to GlcNAc. Utilizing this property, we have developed a separation method for OmpA-like proteins by affinity chromatography using WGA lectin-agarose. The purity of enriched native OmpA-like proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. More importantly, the purified OmpA-like proteins formed a unique trimeric structure keeping their bioactivity intact. In this chapter, we describe a detailed procedure to separate OmpA-like proteins, which may be used to further progress the biological studies of OmpA-like proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad/métodos , Porphyromonas gingivalis/química , Tannerella forsythia/química , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas Bacterianas/análisis , Infecciones por Bacteroidaceae/microbiología , Electroforesis en Gel de Poliacrilamida/métodos , Glicosilación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Multimerización de Proteína , Aglutininas del Germen de Trigo/químicaRESUMEN
Periodontitis is a multifactorial chronic infectious disease leading to a host immune response involving inflammatory cytokines, especially IL-1ß, which is the main reason for further developing this disease. IL-1 receptor antagonist (IL-1ra) binds IL-1 receptor, inhibiting IL-1ß signaling and reducing the levels of other cytokines closely related to periodontitis, such as IL-6 and TNF-α. Therefore, the use of IL-1ra to inhibit periodontitis development in a system, ensuring its sustained release, might be an effective way to combat this disease. Hence, in this study, a novel IL-1ra-loaded dextran/PLGA microsphere was developed to allow the sustained release of IL-1ra and enhance the anti-inflammatory properties. Therefore, this study's purposes were to develop a novel periodontal treatment for inhibition and treatment of periodontitis and evaluate the sustained-release effect and anti-inflammatory properties of IL-1ra-loaded dextran/PLGA microspheres in vitro by cell experiments and in vivo by animal experiments. The results showed that IL-1ra-loaded dextran/PLGA microspheres were non-toxic both in vitro and in vivo and could be used as a safe and effective treatment. In addition, these microspheres could significantly prolong the half-life of IL-1ra drug, exerting a useful anti-inflammatory effect in macrophages stimulated with P. gingivalis lipopolysaccharide and in rats with periodontitis. In conclusion, IL-1ra-loaded dextran/PLGA microsphere might be a useful tool to combat periodontal disease.