Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury.

OBJECTIVES: To investigate the mechanism of circadian clock protein Bmal1 (Bmal1) on renal injury with chronic periodontitis, we established an experimental rat periodontitis model. METHODS: Twelve male Wistar rats were randomly divided into control and periodontitis groups (n=6, each group). The first maxillary molars on both sides of the upper jaw of rats with periodontitis were ligated by using orthodontic ligature wires, whereas the control group received no intervention measures. After 8 weeks, clinical periodontal parameters, including probing depth, bleeding index, and tooth mobility, were evaluated in both groups. Micro-CT scanning and three-dimensional image reconstruction were performed on the maxillary bones of the rats for the assessment of alveolar bone resorption. Histopatholo-gical observations of periodontal and renal tissues were conducted using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Renal function indicators, such as creatinine, albumin, and blood urea nitrogen levels, and oxidative stress markers, including superoxide dismutase, glutathione, and malondialdehyde levels, were measured using biochemical assay kits. MitoSOX red staining was used to detect reactive oxygen species (ROS) content in the kidneys. The gene and protein expression levels of Bmal1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in rat renal tissues were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. RESULTS: Micro-CT and HE staining results showed significant bone resorption and attachment loss in the maxillary first molar region of the periodontitis group. Histological examination through HE and PAS staining revealed substantial histopathological damage to the renal tissues of the rats in the periodontitis group. The findings of the assessment of renal function and oxidative stress markers indicated that the periodontitis group exhibited abnormal levels of oxidative stress, whereas the renal function levels showed abnormalities without statistical significance. MitoSOX Red staining results showed that the content of ROS in the renal tissue of the periodontitis group was significantly higher than that of the control group, and RT-qPCR and immunohistochemistry results showed that the expression levels of Bmal1, Nrf2, and HO-1 in the renal tissues of the rats in the periodontitis group showed a decreasing trend. CONCLUSIONS: Circadian clock protein Bmal1 plays an important role in the oxidative damage process involved in the renal of rats with periodontitis.

Melatonin-Derived Carbon Dots with Free Radical Scavenging Property for Effective Periodontitis Treatment via the Nrf2/HO-1 Pathway.

Periodontitis is a chronic inflammatory disease closely associated with reactive oxygen species (ROS) involvement. Eliminating ROS to control the periodontal microenvironment and alleviate the inflammatory response could potentially serve as an efficacious therapy for periodontitis. Melatonin (MT), renowned for its potent antioxidant and anti-inflammatory characteristics, is frequently employed as an ROS scavenger in inflammatory diseases. However, the therapeutic efficacy of MT remains unsatisfactory due to the low water solubility and poor bioavailability. Carbon dots have emerged as a promising and innovative nanomaterial with facile synthesis, environmental friendliness, and low cost. In this study, melatonin-derived carbon dots (MT-CDs) were successfully synthesized via the hydrothermal method. The MT-CDs have good water solubility and biocompatibility and feature excellent ROS-scavenging capacity without additional modification. The in vitro experiments proved that MT-CDs efficiently regulated intracellular ROS, which maintained mitochondrial homeostasis and suppressed the production of inflammatory mediators. Furthermore, findings from the mouse model of periodontitis indicated that MT-CDs significantly inhibited the deterioration of alveolar bone and reduced osteoclast activation and inflammation, thereby contributing to the regeneration of damaged tissue. In terms of the mechanism, MT-CDs may scavenge ROS, thereby preventing cellular damage and the production of inflammatory factors by regulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. The findings will offer a vital understanding of the advancement of secure and effective ROS-scavenging platforms for more biomedical applications.

Diosgenin alters LPS-induced macrophage polarization by activating PPARγ/NF-κB signaling pathway.

Diosgenin (DG) is a steroidal saponin derived from plants, and it exhibits anti-inflammatory properties. In this study, we employed an in vitro model of P.g.-LPS-stimulated mouse macrophage cell line RAW264.7 to investigate the anti-inflammatory effects and mechanism of DG under the condition of altered polarization of macrophages. The RAW264.7 cells were subjected to pre-treatment with DG with or without P.g.-LPS. In cultured macrophages, DG inhibited P.g.-LPS-induced pro-inflammatory M1 macrophages, and increased anti-inflammatory M2 macrophages. Notably, DG reduced the expression of phosphorylation levels of NF-κB p65 and IκB while increasing the expression of PPARγ. Further studies revealed that PPARγ inhibitor GW9662 or PPARγ siRNA reversed the inhibitory effect of DG on M1 phenotype. Collectively, the anti-inflammatory mechanism of DG is related to altering macrophage polarization by activating PPARγ and inhibiting NF-κB signaling pathways.

18-α-glycyrrhetinic acid alleviates oxidative damage in periodontal tissue by modulating the interaction of Cx43 and JNK/NF-κB pathways.

Objective: Periodontitis is a common chronic inflammatory disease in which oxidative stress is one of the key pathogenic factors. Connexin43 (Cx43) is the most critical and widely distributed connexin isoform. When the organism undergoes a severe and sustained stress response, Cx43-mediated gap junctions (GJs) are believed to underlie the biology of tissue injury exacerbation and amplification. Notably, 18-α-glycyrrhetinic acid (GA) is a classical pharmacological inhibitor of GJs and has antioxidant potential. However, the regulatory role of GA in the redox signaling of periodontal tissues and the potential mechanisms of Cx43 in the pathogenesis of periodontitis remain uncertain. Methods: In this study, we evaluated the effects and mechanisms of GA in alleviating oxidative damage of periodontal tissues and cells by constructing an H2O2-induced oxidative stress model in human periodontal ligament cells (hPDLCs) and a periodontitis model in rats. Results: Cellular experiments showed that GA effectively attenuated H2O2-induced oxidative damage in hPDLCs by inhibiting the expression and function of Cx43. In addition, pretreatment of hPDLCs with either GA or SP600125 (a JNK inhibitor) inhibited the Cx43/JNK/NF-κB pathway, restored cell viability, and reduced apoptosis. Animal experiment results showed that GA intervention reduced alveolar bone resorption and periodontal tissue destruction, inhibited osteoclast differentiation, improved mitochondrial structural abnormalities and dysfunction in periodontal tissue, and decreased oxidative stress levels and apoptosis in rats with periodontitis. Conclusion: Overall, our findings suggest that the Cx43/JNK/NF-κB pathway may play a vital role to promote periodontitis progression, while GA reduces oxidative stress and apoptosis by inhibiting the interaction of Cx43 and JNK/NF-κB pathways, thus alleviating oxidative damage in the periodontal tissues.

N-Acetyl-l-cysteine-Derived Carbonized Polymer Dots with ROS Scavenging via Keap1-Nrf2 Pathway Regulate Alveolar Bone Homeostasis in Periodontitis.

Periodontitis is a type of chronic inflammatory oral disease characterized by the destruction of periodontal connective tissue and progressive alveolar bone resorption. As oxidative stress is the key cause of periodontitis in the early periodontal microenvironment, antioxidative therapy has been considered a viable treatment for periodontitis. However, more stable and effective reactive oxygen species (ROS)-scavenging nanomedicines are still highly needed due to the instability of traditional antioxidants. Herein, a new type of N-acetyl-l-cysteine (NAC)-derived red fluorescent carbonized polymer dots (CPDs) has been synthesized with excellent biocompatibility, which can serve as an extracellular antioxidant to scavenge ROS effectively. Moreover, NAC-CPDs can promote osteogenic differentiation in human periodontal ligament cells (hPDLCs) under H2 O2 stimulation. In addition, NAC-CPDs are capable of targeted accumulation in alveolar bone in vivo, reducing the level of alveolar bone resorption in periodontitis mice, as well as performing fluorescence imaging in vitro and in vivo. In terms of mechanism, NAC-CPDs may regulate redox homeostasis and promote bone formation in the periodontitis microenvironment by modulating the kelch-like ECH-associated protein l (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. This study provides a new strategy for the application of CPDs theranostic nanoplatform for periodontitis.

The role of Sirt3-induced autophagy in renal structural damage caused by periodontitis in rats.

OBJECTIVE: This research aimed to explore the effect of periodontitis on renal tissues injury in rats and the role of Sirtuin3 (Sirt3) and its regulation of autophagy in this progression. MATERIAL AND METHODS: Thirty Wistar rats were assigned into three groups: control, periodontitis (P), and periodontitis with gavage administration of Sirt3 activator resveratrol (P + RSV). To induce periodontitis, the wire ligature was placed around the cervical region of the rat maxillary first molar. After 8 weeks, micro-computed tomography (Micro-CT) and hematoxylin and eosin (HE) were used to evaluate the alveolar bone resorption and periodontal inflammation. Serum and urine biochemical indicators were measured to assess renal function. The pathological changes of the kidney were observed via HE and periodic acid Schiff (PAS) staining. Autophagosome was viewed by transmission electron microscopy (TEM). Real-time PCR and western blot were used to test expressions of Sirt3 and autophagy indicators in renal and periodontal tissues, including mammalian target of rapamycin (mTOR), phosphor-mTOR (p-mTOR), BECN1 (Beclin-1), and microtubule-associated protein 1 light chain 3 (LC3). RESULTS: Alveolar bone destruction, resorption, and periodontal inflammation were observed in the P group (compared with the control group), and the above indexes were significantly improved after RSV intervention; the obvious changes in renal tissue structure in the P group were partially recovered after RSV intervention, while renal functional status was not affected (among the three groups); in addition, the levels of Sirt3 and autophagy in kidney and periodontal tissues of P group were inhibited, manifested as a decrease in the number of autophagosomes (renal tissue) and expressions of autophagy marker Beclin-1 and LC3 conversion rate and an increase in the expression of p-mTOR. After Sirt3 activation (RSV), the above indicators were significantly improved. CONCLUSION: Periodontitis causes renal structural damage in rats, which may be connected to the effect of Sirt3-induced autophagy.

Resveratrol protects renal damages induced by periodontitis via preventing mitochondrial dysfunction in rats.

OBJECTIVES: Periodontitis is closely associated with kidney disease and reactive oxygen species (ROS) involvement. Mitochondria are the primary source of both endogenous ROS and renal energy. We investigated whether resveratrol (RSV) prevents renal injury and mitochondrial dysfunction in periodontitis rats. METHODS: Thirty male Wistar rats were divided into control, experimental periodontitis (Ep) and Ep-RSV groups. To induce periodontitis, a steel ligature was placed on the cervix of the bilateral first maxillary molars. RSV (50 mg/kg/day) to the Ep-RSV group and vehicle to the Ep and control groups were gavaged. After 8 weeks, alveolar bone loss, pocket depth, gingival blood index and tooth mobility were assessed. Oxidative stress parameters, mitochondrial structure, mitochondrial membrane potential (MMP), mitochondrial ROS, adenosine triphosphate (ATP), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) were analysed in renal. Renal function and histology were also evaluated. RESULTS: Compared with the control group, the Ep group showed renal structural destruction, elevated oxidative stress levels, mitochondrial structure destruction, MMP loss, mitochondrial ROS accumulation, ATP reduction, and decreased SIRT1 and PGC-1α levels. RSV prevented these destruction (p < 0.05). However, there was no significant impairment in renal function (p > 0.05). CONCLUSIONS: Periodontitis induces mitochondrial dysfunction in renal tissues. Resveratrol exerts a preventive effect on periodontitis-induced kidney injury by preventing mitochondrial dysfunction.

Application of Bone Marrow-Derived Macrophages Combined with Bone Mesenchymal Stem Cells in Dual-Channel Three-Dimensional Bioprinting Scaffolds for Early Immune Regulation and Osteogenic Induction in Rat Calvarial Defects.

The host immune response to biomaterials is critical for determining scaffold fate and bone regeneration outcomes. Three-dimensional (3D) bioprinted scaffolds encapsulated with living cells can improve the inflammatory microenvironment and further accelerate bone repair. Here, we screened and adopted 8% methacrylamidated gelatin (GelMA)/1% methacrylamidated hyaluronic acid (HAMA) as the encapsulation system for rat bone marrow-derived macrophages (BMMs) and 3% Alginate/0.5 mg/mL graphene oxide (GO) as the encapsulation system for rat bone mesenchymal stem cells (BMSCs), thus forming a dual-channel bioprinting scaffold. The 8% GelMA/1% HAMA/3% Alginate/0.5 mg/mL GO (8/1/3/0.5) group could form a scaffold with a stable structure, good mechanical properties, and satisfied biocompatibility. When exploring the crosstalk between BMMs and BMSCs in vitro, we found that BMSCs could promote the polarization of BMMs to M2 type at the early stage, reduce the pro-inflammatory gene expression, and increase anti-inflammatory gene expression; conversely, BMMs can promote the osteogenic differentiation of BMSCs. In addition, in the model of rat calvarial defects, the dual-channel scaffold encapsulated with BMMs and BMSCs was more effective than the single-cell scaffold and the acellular scaffold. The paracrine of BMMs and BMSCs in the biodegradable dual-channel scaffold effectively promoted the M2-type polarization of macrophages in the microenvironment of early bone defects, avoided excessive inflammatory responses, and further promoted bone repair. In conclusion, our findings suggested that using 3D bioprinting to simultaneously encapsulate two primary cells of BMMs and BMSCs in a dual-channel system may be an effective way to promote bone repair from the perspective of early immune regulation and late induction of osteogenesis.

Global trends in research on oxidative stress associated with periodontitis from 1987 to 2022: A bibliometric analysis.

Background: Oxidative stress has been implicated in many chronic inflammatory diseases, including periodontitis. To date, however, only a few bibliometric analyses have systematically studied this field. This work sought to visualize research hot spots and trends in oxidative stress associated with periodontitis from 1987 to 2022 through bibliometric approaches. Methods: The Web of Science Core Collection was searched to retrieve relevant publications. HistCite, VOSviewer, and CiteSpace were used to perform bibliometric analysis visually in terms of annual output, active countries, prolific institutions, authors, core journals, co-cited references, and co-occurrence of keywords. Results: A total of 1654 documents were selected for analysis. From 1 January 1987 to 11 June 2022, the number of annual publications related to oxidative stress in periodontitis exhibited an upward trend. The most prolific country was China with 322 documents, but the United States had 11334 citations. Okayama University, University of Birmingham, and Sichuan University were the most active and contributive institutions. The Journal of Periodontology ranked first in terms of numbers of publications and citations. Ekuni was the most prolific author, while Chapple ranked first among co-cited authors. The Role of Reactive Oxygen and Antioxidant Species in Periodontal Tissue Destruction published by Chapple was the most frequently co-cited reference. Keywords co-occurrence showed that oxidative stress was closely related to inflammation, antioxidants, and diabetes. Conclusion: Our research found that global publications regarding research on oxidative stress associated with periodontitis increased dramatically and were expected to continue increasing. Inflammation and oxidative stress, and the relationship between periodontitis and systemic diseases, are topics worthy of attention.

Circadian Rhythm Disorders Aggravate Periodontitis by Modulating BMAL1.

Circadian rhythms regulate the body's homeostasis through the temporal control of tissue-specific circadian rhythm control genes. Circadian rhythm disorders (CRD) affect the expression levels of circadian rhythms-associated genes in brain and muscle aryl hydrocarbon receptor nuclear translocator-like-1(BMAL1), which is thought to contribute to metabolic disorders and an altered immune system. However, the relationship between CRD and the development of periodontitis was poorly reported. Therefore, this study aimed to investigate the role played by BMAL1 in periodontitis. We used a modified multi-platform approach (MMPM) to induce circadian rhythm disturbances in rats to investigate the role of BMAL1 in periodontitis. Our results showed significant downregulation of BMAL1 in the CRD with periodontitis group, significant resorption of alveolar bone, increased osteoclast differentiation, and upregulation of the inflammatory signaling molecule NF-κB. In addition, apoptosis and oxidative stress levels were increased in periodontal tissues. Collectively, our study suggests that BMAL1 is a key regulator in periodontitis exacerbated by CRD and that CRD may lead to the downregulation of BMAL1, thereby exacerbating oxidative stress and apoptosis in periodontal tissues. Our study found that BMAL1 may be associated with the progression of periodontitis and provides a new perspective on the treatment of periodontitis.

Reactive oxygen species/c-Jun N-terminal kinase/nuclear factor kappa-B signaling molecules are involved in pe-riodontitis-induced liver injury by regulating apoptosis.

OBJECTIVES: The occurrence and development of periodontitis and nonalcoholic fatty liver disease (NAFLD) are closely related to the accumulation of reactive oxygen species (ROS). ROS are involved in regulating the activation of c-Jun N-terminal kinase (JNK)/nuclear factor kappa-B (NF-κB) signaling molecules. When the signaling molecules are overactivated by ROS, the internal environment of the body can be disturbed. Therefore, this study aimed to explore the mechanism by which ROS/JNK/NF-κB signaling molecules are involved in periodontitis-induced liver injury. METHODS: Twelve SPF male Wistar rats were randomly divided into control and periodontitis groups. The perio-dontitis model of rats was established by wire ligation in the neck of bilateral maxillary first molars. After 8 weeks, the periodontal clinical indexes of the rats were examined, and the rats were sacrificed. Micro-CT reconstruction of a three-dimensional alveolar bone structure and analysis of alveolar bone absorption were conducted. Pathological changes in the periodontal and liver tissues were analyzed by histopathology. MitoSOX red reagent was used to detect the ROS content in liver tissue. Biochemical kits were used to detect liver function and oxidative stress biomarkers. The mRNA expression levels ofinterleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), NF-κB, BCL2-associated X (Bax), and B-cell lymphoma-2 (Bcl-2) in liver tissue were detected through quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of phosphorylated c-Jun N-terminal kinase (P-JNK), JNK, NF-κB, Caspase-3, Bax, and Bcl-2 in liver tissue were detected by Western blot. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. RESULTS: Micro-CT results showed that the mice in the periodontitis group had obvious alveolar bone resorption and significantly greater distance from the cemento-enamel junction to the alveolar bone crest than those in the control group. Histopathological results showed that a large number of inflammatory cells were infiltrated in the periodontal tissue of the periodontitis group. In addition, the resorption of alveolar ridge bone was obvious and liver tissue structure was destroyed, with balloon-like changes and red lipid droplets. MitoSOX red staining results showed that the ROS level was significantly higher in the liver tissue of the periodontitis group than in that of the control group. Biochemical test results showed that the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the serum of the periodontitis group were higher than those in the serum of the control group. The levels of superoxide dismutase (SOD) and glutathione (GSH) in liver tissue decreased, whereas the that of malondialdehyde (MDA) increased. Western blot and qRT-PCR results revealed that the mRNA levels of IL-6, TNF-α, Bax, and NF-κB and the protein levels of P-JNK/JNK, NF-κB, Caspase-3, and Bax were significantly higher in the liver tissue of the perio-dontitis group than in that of the control group. Meanwhile, the mRNA and protein levels of Bcl-2 were lower in the periodontitis group than in the control group. TUNEL staining showed that the number of apoptotic cells was significantly higher in the periodontitis group than in the control group. CONCLUSIONS: ROS/JNK/NF-κB signaling molecules are involved in periodontitis-induced liver injury by regulating apoptosis.

Effect of peroxisome proliferator-activated receptor-γ coactivator-1α on liver injury induced by periodontitis in rats.

OBJECTIVES: To investigate the effect of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) on liver injury induced by periodontitis in rats. METHODS: Twenty-four male Wistar rats were randomly divided into two groups: control group and periodontitis group, twelve per group. In periodontitis group, the periodontitis models were established for the maxillary first molars in rats by means of "wire ligation+vaccinationwith Porphyromonas gingivalis", the control group was inoculated with the equal volume of 2% sodium carboxymethyl cellulose in the same position, for 6 weeks. The probing depth, tooth mobility and sulcus bleeding index were detected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of liver tissues in rats. The quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to detect the gene and protein expression levels of PGC-1α, nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial transcription factor A (TFAM) in liver tissues of rats. RESULTS: The probing depth, tooth mobility and sulcus bleeding index in periodontitis group were significantly higher than that in control group. HE staining showed in periodontitis group, hepatic cords ranged disorderly and there were vacuoles in cells and inflammatory cells infiltrated in liver tissues of rats, and there was no obvious abnormality in control group. The qRT-PCR results showed that the mRNA expression levels of Pgc-1α, Nrf2 and Tfam in liver tissues of rats in periodontitis group were lower obviously than that in control group. IHC results showed that the protein expression level of PGC-1α in liver tissues of rats in periodontitis group was decreased significantly than that in control group. CONCLUSIONS: PGC-1α may be involved in the process of periodontitis-induced liver injury in rats.

The role of TLR4/MyD88/NF-κB pathway in periodontitis-induced liver inflammation of rats.

OBJECTIVES: The aim of this study was to clarify the immune mechanism of hepatic injury induced by periodontitis using a rat model. METHODS: Twenty-four SPF male Wistar rats were randomly divided into two groups: control group (CG) and periodontitis group (PG). In order to induce experimental periodontitis, we tied the wire ligature around bilateral maxillary first molar of rats. After 8 weeks, the following indicators were valued: gingival index, tooth mobility, probing pocket depth; indexes about oxidative stress and circulating biomarkers; bone retraction by micro-CT analysis; Toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88), and nuclear factor kappa B (NF-κB) by qRT-PCR and Western blotting; tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) by qRT-PCR and immunohistochemical staining; inflammation of periodontal and hepatic tissues by histopathological observation. RESULTS: Periodontal indicators and micro-CT results showed the raised levels of inflammatory response and bone retraction in PG compared with CG. The mRNA and protein levels of TLR4, MyD88, NF-κB, TNF-α, and IL-6 have indicated high values in PG versus CG. Histopathological analysis revealed a correlation between periodontitis and hepatic injury. CONCLUSION: TLR4/MyD88/NF-κB pathway may play a role in periodontitis-induced liver inflammation of rats.

Controllable acidophilic dual-emission fluorescent carbonized polymer dots for selective imaging of bacteria.

Fluorescent materials can be powerful contrast agents in photoelectric devices and for bioimaging. As emerging fluorescent materials, carbonized polymer dots (CPDs) with high quantum yields (QYs), long-wavelength emission and multiple functions are highly desired. Despite great progress in the synthetic methods and QYs of CPDs, multiple emission of CPDs is challenging. Therefore, we developed CPDs with dual-emission fluorescence in terms of inherent blue and red emission. In addition, CPDs with sole blue emission (B-CPDs) and red emission (R-CPDs) were synthesized, respectively, by regulating the reaction conditions to control the quantitative structure and emission centers. The absolute QY of R-CPDs in water was 24.33%. These three types of CPDs with dual/sole emission could be used in optoelectronic and bioimaging applications. With different CPDs coated on a commercially available gallium nitride light-emitting diode chip as a color-conversion layer, LEDs with blue, yellow, and red emission were achieved. Benefiting from the different emission intensities and emission peaks of R/B-CPDs in different pH conditions, they were used (without further modification) to distinguish between Porphyromonas gingivalis, Streptococcus mutans, Escherichia coli and Staphylococcus aureus in dental plaque biofilms (the first time this has been demonstrated). These findings could enable a new development direction of CPDs based on the design of multi-emission centers.

Anti-inflammatory Effects of PMX205 in Mouse Macrophage Periodontitis Model.

BACKGROUND: C5a receptor antagonist PMX205 is a synthetic hexapeptide capable of blocking C5a-C5a receptor (C5aR) axis by simulating C5a active C-terminal amino acid residues. This hexapeptide presents good anti-inflammatory effects in a series of inflammation models. The anti-inflammatory effect of PMX205 on periodontitis is yet to be fully fathomed. OBJECTIVE: To examine the anti-inflammatory effects of PMX205 on RAW264.7 mouse macrophages exposed to gingipain extracts and Porphyromonas gingivalis (P. gingivalis). METHODS: MTT assay was carried out so as to specify the cytotoxicity of PMX205. RAW264.7 cells were co-cultured in vitro with gingipain extracts or P. gingivalis to simulate the periodontitis inflammatory milieu. Real-time quantitative PCR, ELISA and Griess assay were performed in order to detect tumor necrosis factor-α (TNF-α), IL-6, IL-23, nitric oxide (NO), IL-10, transforming growth factor-ß1 (TGF-ß1), andarginase-1 (Arg-1). Furthermore, phagocytosis assay was done to evaluate the phagocytic capacity of RAW 264.7 cells. Finally, western blot analysis was conducted to evaluate myeloid differentiation factor 88 (MyD88). RESULTS: PMX205 increased the expression levels of bacteriostatic substances (NO and IL-23) and anti-inflammatory cytokines (TGF-ß1, IL-10 and Arg-1); however, it reduced the expression levels of proinflammatory cytokines TNF-α and IL-6 once RAW 264.7 macrophages were stimulated via gingipain extracts or P. gingivalis. In addition, PMX205 promoted the macrophage phagocytosis and down-regulated protein expression of MyD88. CONCLUSION: PMX205 has recognizable anti-inflammatory effects in RAW 264.7 cell inflammation model, a finding which probably opens doors to future investigations on new targets for the prevention and treatment of chronic periodontitis.

Gingipain of Porphyromonas gingivalis manipulates M1 macrophage polarization through C5a pathway.

Gingipains secreted by Porphyromonas gingivalis (P. gingivalis, Pg) play an important role in maintaining macrophage infiltrating. And, this study is to evaluate effects of gingipain on M1 macrophage polarization after exposure to Porphyromonas gingivalis (P. gingivalis, Pg) and if these effects are through complement component 5a (C5a) pathway. Mouse RAW264.7 macrophages were exposed to gingipain extracts, Escherichia coli lipopolysaccharides (Ec-LPS), Pg-LPS with or without the C5aR antagonist: PMX-53 for 24 h. Then, gene expressions and protein of IL-12, IL-23, iNOS, IL-10, TNF-α, IL-1ß, and IL-6 were determined by qRT-PCR and ELISA assays. Surface markers CD86 for M1 and CD206 for M2 were also evaluated by flow cytometry. The results show that gingipain extracts alone increased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1ß, and IL-6, but not IL-10. Gingipain extracts plus Ec-LPS decreased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1ß, and IL-6 in which Ec-LPS induced increase. For gingipain extracts plus Pg-LPS-treated RAW264.7, macrophages, gingipain extracts enhanced expressions of IL-12 and IL-23 in which Pg-LPS induced increase, but not iNOS and IL-10 while gingipain extracts decreased expressions of TNF-α, IL-1ß, and IL-6 in which Pg-LPS induced increase. Interestingly, PMX-53 increased expressions of IL-12, IL-23, and iNOS when RAW264.7 macrophages were treated with gingipain extracts plus Ec-LPS or Pg-LPS and PMX-53, while PMX-53 decreased expressions of TNF-α, IL-1ß, and IL-6. Changes of CD86-positive macrophages were consistent with cytokine changes. Our data indicate that gingipain is a critical regulator, more like a promoter to manipulate M1 macrophage polarization in order to benefit P. gingivalis infection through the C5a pathway.