Porphyromonas gingivalis LPS-stimulated BMSC-derived exosome promotes osteoclastogenesis via miR-151-3p/PAFAH1B1.

OBJECTIVES: Porphyromonas gingivalis-LPS regulated bone metabolism by triggering dysfunction of osteoblasts directly, and affecting activity of osteoclasts through intracellular communication. Exosome, as the mediator of intercellular communication, was important vesicle to regulate osteogenesis and osteoclastogenesis. This research was designed for investigating the mechanism of BMSCs-EXO in modulating osteoclastic activity under the P. gingivalis-LPS. MATERIALS AND METHODS: The cytotoxicity and osteogenic effects of P. gingivalis-LPS on BMSCs was evaluated, and then osteoclastic activity of RAW264.7 co-cultured with exosomes was detected. Besides, Affymetrix miRNA array and luciferase reporter assay were used to identify the target exosomal miRNA signal pathway. RESULTS: BMSCs' osteogenic differentiation and proliferation were decreased under 1 and 10 µg/mL P. gingivalis-LPS. Osteoclastic-related genes and proteins levels were promoted by P. gingivalis-LPS-stimulated BMSCs-EXO. Based on the miRNA microarray analysis, exosomal miR-151-3p was lessened in BMExo-LPS group, which facilitated osteoclastic differentiation through miR-151-3p/PAFAH1B1. CONCLUSIONS: Porphyromonas gingivalis-LPS could regulated bone metabolism by inhibiting proliferation and osteogenesis of BMSCs directly. Also, P. gingivalis-LPS-stimulated BMSCs-EXO promoted osteoclastogenesis via activating miR-151-3p/PAFAH1B1 signal pathway.

Acevaltrate promotes apoptosis and inhibits proliferation by suppressing HIF-1α accumulation in cancer cells.

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.

Periodontitis exacerbates pulmonary hypertension by promoting IFNγ+ T cell infiltration in mice.

Uncovering the risk factors of pulmonary hypertension and its mechanisms is crucial for the prevention and treatment of the disease. In the current study, we showed that experimental periodontitis, which was established by ligation of molars followed by orally smearing subgingival plaques from patients with periodontitis, exacerbated hypoxia-induced pulmonary hypertension in mice. Mechanistically, periodontitis dysregulated the pulmonary microbiota by promoting ectopic colonization and enrichment of oral bacteria in the lungs, contributing to pulmonary infiltration of interferon gamma positive (IFNγ+) T cells and aggravating the progression of pulmonary hypertension. In addition, we identified Prevotella zoogleoformans as the critical periodontitis-associated bacterium driving the exacerbation of pulmonary hypertension by periodontitis, and the exacerbation was potently ameliorated by both cervical lymph node excision and IFNγ neutralizing antibodies. Our study suggests a proof of concept that the combined prevention and treatment of periodontitis and pulmonary hypertension are necessary.

Efficient Nyström-type method for the solution of highly oscillatory Volterra integral equations of the second kind.

Highly oscillatory Volterra integral equations are frequently encountered in engineering applications. The Nyström-type method is an important numerical approach for solving such problems. However, there remains scope to further optimize and accelerate the Nyström method. This paper presents a novel Nyström-type method to efficiently approximate solutions to second-kind Volterra integral equations with highly oscillatory kernels. First, the unknown function is interpolated at Chebyshev points. Then the integral equation is solved using the Nyström-type method, which leads to a problem of solving a system of linear equations. A key contribution is the technique to express the fundamental Lagrange polynomial in matrix form. The elements of the matrix, which involves highly oscillatory integrals, are calculated by using the classical Fejér quadrature formula with a dilation technique. The proposed method is more efficient than the one proposed in the recent literature. Numerical examples verify the efficiency and accuracy of the proposed method.

Modified interproximal tunneling technique with customized sub-epithelial connective tissue graft for gingival papilla reconstruction: report of three cases with a cutback incision on the palatal side.

BACKGROUND: Gingival papilla defects, which cause an unpleasant appearance and involve the upper anterior teeth, may be triggered by several factors. Several noninvasive and invasive techniques have been proposed for gingival papilla reconstruction. The combination of interproximal tunneling and customized connective tissue grafts (CTGs) has shown promise in papilla augmentation. However, due to the narrowness and limited blood supply of the gingival papilla, the long-term outcomes of these techniques remain unpredictable. Therefore, achieving tension-free coronal advancement of the interdental papilla and proper placement of the CTG is crucial for successful long-term outcomes and could provide widely applicable methods for papilla augmentation. CASE REPORT: In this study, we enrolled three patients with gingival papilla defects in the maxillary anterior teeth. For reconstruction, we proposed a modified interproximal tunneling (MIPT) technique combined with a CTG. A crucial modification based on previous studies involved adding a cutback incision to the base of the palatal vertical incision, resulting in tension-free healing. Additionally, the CTG was sutured upright to further enhance the height of the gingiva papilla. To evaluate the efficacy of the MIPT technique, the clinical parameters-including the Jemt papilla index and the distance from the tip of the papilla to the interproximal contact point-were examined using a periodontal probe (UNC15, Hu-friedy) at baseline and 12 months after surgery. All three patients achieved satisfactory papilla reconstruction 12 months after the surgery. These three cases were used to evaluate the efficacy of the MIPT technique combined with the customized CTG. An average increase in the Jemt papilla score from 1.6 to 2.8 and a reduction in the distance from the papilla tip to the contact point of adjacent teeth from 2 mm to 0.08 mm were observed 12 months after surgery. CONCLUSION: The preliminary results confirmed that this technique holds promise for gingival papilla augmentation between tooth/tooth or tooth/implant.

The miR-223-3p Regulates Pyroptosis Through NLRP3-Caspase 1-GSDMD Signal Axis in Periodontitis.

Salivary exosomes contain various components and may play important roles in oral diseases. The purpose of this study was to verify the possible function of miR-223-3p from salivary exosomes in periodontitis. We isolated the salivary exosomes and found that the miR-223-3p content of salivary exosomes from periodontitis was less than the healthy control. Furthermore, we performed dual-luciferase reporter assay and real-time PCR to verify that (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) was the target of miR-223-3p. When we knocked down the miR-223-3p expression in THP-1-derived macrophages, the expression of NLRP3 and the downstream inflammatory mediators interleukin-1ß (IL-1ß) and IL-6 were upregulated. By using integrated bioinformatics analysis, we found that pyroptosis and cytokine secretion participated in inflammatory gingival tissues. In addition, NLRP3, and the pyroptosis executioner, gasdermin D (GSDMD) was highly active in inflammatory gingival tissues compared with healthy controls by western blotting and immunohistochemistry. In summary, we speculated that miR-223-3p in salivary exosomes might regulate GSDMD-mediated pyroptosis by targeting NLRP3 in periodontitis. Detection of miR-223-3p expression in salivary exosomes could be used as an important non-invasive method to diagnose and evaluate the severity of periodontitis.

[Effects of eicosapentaenoic acid on biological activity and inflammatory factor expression of human gingival fibroblasts].

PURPOSE: To explore the effects of eicosapentaenoic acid (EPA) on biological activity and inflammatory factor expression of human gingival fibroblasts (HGFs). METHODS: The effects of EPA on the activity, morphology and cell cycle of HGFs were observed by living and dead cell staining, immunofluorescence staining and flow cytometry, respectively. HGFs were stimulated by lipopolysaccharides (LPS) of Porphyromonas gingivalis (P. gingivalis) or heat inactivated P. gingivalis, after which the effects of EPA on mRNA and protein expression of IL-6, IL-8 and IL-1ß were observed by real-time PCR and ELISA, respectively. The gene and protein expression of heme oxygenase-1(HO-1) was also detected by real-time PCR and Western blotting, respectively. The data were analyzed with SPSS 22.0 software package. RESULTS: 200 µmol/L EPA inhibited cell activity of HGFs; 100 µmol/L EPA did not affect cell activity and morphology of HGFs, and had no significant effect on cell cycle (P>0.05). EPA significantly downregulated gene expression of IL-6 and IL-1ß, and protein expression of IL-6 stimulated by P. gingivalis LPS and heat-killed P.gingivalis(P<0.05), in a dose-dependent manner. EPA increased gene expression of HO-1 in a dose dependent manner(P<0.05), and upregulated HO-1 protein expression. CONCLUSIONS: EPA significantly inhibits the expression of inflammatory factors without affecting the biological activity of HGFs, which may be related to the induction of HO-1, suggesting the potential role of EPA in the prevention and treatment of periodontitis.

LPS stimulates gingival fibroblasts to express PD-L1 via the p38 pathway under periodontal inflammatory conditions.

OBJECTIVE: The overall aim of this research was to investigate the differences in the expression of programmed death ligand 1 (PD-L1) in human gingival fibroblasts (HGFs) between a periodontal healthy group and a periodontal inflammatory group. and explore the possible mechanism involved. METHODS: Differences in PD-L1 mRNA and protein expression in HGFs from a periodontal healthy group and a periodontal inflammatory group were examined by qPCR and western blotting, respectively, and were further tested after lipopolysaccharide (LPS) stimulation in both groups. The effects of a p38 pathway inhibitor on the changes in p38 phosphorylation levels and PD-L1 expression after LPS stimulation were investigated in both groups. RESULTS: PD-L1 mRNA and protein levels in HGFs in the periodontal inflammatory group were significantly higher than those in the periodontal healthy group (p < 0.05). After 10 µg/mL LPS stimulation, PD-L1 mRNA levels in HGFs from both groups increased significantly (p < 0.05), peaking at 4 h, and the peak was significantly higher in the periodontal inflammatory group than in the periodontal healthy group (p < 0.05). However, PD-L1 protein expression was upregulated only in the inflammatory group (p < 0.05). Inhibition of the p38 pathway in HGFs decreased p38 phosphorylation in both groups (p < 0.05) but this treatment reversed the LPS-induced increase in PD-L1 mRNA and protein levels only in the inflammatory group (p < 0.05). CONCLUSION: In the periodontal inflammatory state, the expression of PD-L1 in HGFs is more easily activated, and may be influenced by the p38 pathway.

Nanophase-Separated, Elastic Epoxy Composite Thin Film as an Electrolyte for Stable Lithium Metal Batteries.

The design of solid polymer electrolytes (SPE) with high ionic conductivity and excellent mechanical properties is challenging because these two properties are often conflicting. To achieve both, a reaction-controlled strategy is proposed based on the nanophase separation of an ionic transport pathway and a supporting matrix to balance ionic mobility and mechanical properties. Specifically, an elastic epoxy polymer electrolyte (eEPE), synthesized via two-step polymerization, combines outstanding mechanical strength (toughness of 3.4 MJ m-3) and high ionic conductivity (3.5 × 10-4 S cm-1 at 25 °C). The nanostructured eEPE is both tough and flexible, therefore promotes uniform deposition of Li even under a high current density (2 mA cm-2 and 2 mAh cm-2). Importantly, eEPE composite films greatly improve the safety performance of the LiFePO4/Li pouch cells: safe operations are achieved under several abusive conditions. This work highlights an alternative route for high-safety solid-state lithium metal batteries of the next generation.

Dual-mode visible light-induced aptasensing platforms for bleomycin detection based on CdS-In2S3 heterojunction.

CdS-In2S3 heterojunction with enhanced photoelectrochemical (PEC) performance was synthesized to construct dual-mode visible light-induced biosensors for highly sensitive and selective detection of bleomycin (BLM). Due to improved absorption in the visible region and suppressed recombination of electron-hole pairs in the heterojunction, CdS-In2S3 composite exhibited enhanced photocurrent response under visible light illumination. Using CdS-In2S3 as photoactive materials and BLM-binding aptamer as recognition element, a PEC aptasensor displaying a declined photocurrent response to BLM was facilely constructed, which was linear to BLM concentration in the range of 5.0-250 nM. On the other hand, the CdS-In2S3 photoanode was employed to construct a photofuel cell (PFC). In such a PFC, the oxidation of water on CdS-In2S3 photoanode under visible light illumination and the reduction of oxygen on Pt cathode led to the generation of electricity. When BLM-binding aptamer was immobilized on CdS-In2S3 photoanode, the output power of the PFC was inversely proportional to the logarithm of BLM concentration from 10 to 250 nM, offering a visible light-induced self-powered sensing platform for BLM detection. Both of the proposed sensors showed high selectivity, good reproducibility and high stability. They were successfully applied to the determination of BLM in human serum samples.

One-pot synthesis of a CdS-reduced graphene oxide-carbon nitride composite for self-powered photoelectrochemical aptasensing of PCB72.

Graphitic carbon nitride (C3N4) is a carbon-based metal-free semiconductor, which has been widely explored as a photoactive material. In this work, the CdS, reduced graphene oxide (rGO) and C3N4 (CdS-rGO-C3N4) composite was synthesized by a simple one-pot hydrothermal method and utilized to construct a photoelectrochemical (PEC) sensor. Compared with CdS, C3N4 and CdS-C3N4, the CdS-rGO-C3N4 composite exhibited enhanced photoelectrochemical (PEC) performance, due to the expanded absorption of C3N4 in the visible region by CdS and promoted the charge carrier separation of a photoelectrode by rGO. Based on a glassy carbon electrode (GCE) modified with CdS-rGO-C3N4 and a PCB72-binding aptamer (ap/CdS-rGO-C3N4/GCE), a PEC aptasensor for the detection of 2,3',5,5'-tetrachlorobiphenyl (PCB72) was developed. When H2O2 was added into the electrolyte, the PEC sensor exhibited an amplified response toward PCB72, and could be operated in a self-powered mode at a potential of 0 V. Under optimum conditions, the constructed PEC aptasensor exhibited a wide linear range of 10 to 1000 ng mL-1 for PCB72 detection, with a low detection limit (S/N = 3) of 1.0 ng mL-1. Moreover, this aptasensor exhibited high selectivity, good reproducibility and high stability. The applicability of the developed PEC strategy was demonstrated by determining PCB72 in environmental water.

An RNA-seq screen of P. gingivalis LPS treated human gingival fibroblasts.

BACKGROUND: In gingival tissues, lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) is the most critical stimulator for inducing inflammatory response. Human gingival fibroblasts (HGFs) are the major constituents of gingival connective tissues. The aim of this study was to investigate P. gingivalis LPS induced whole transcriptional profile in HGFs and the potential crosstalk between microRNAs (miRNAs) and inflammatory cytokines. METHODS: RNA-seq was performed on HGFs with and without P. gingivalis LPS treatment. The gene expression of selected inflammatory cytokines and miRNAs induced by LPS at different time points was evaluated by quantitative RT-PCR. The protein expression of chemokines was further confirmed by ELISA. RESULTS: Interestingly, most of the significantly changed genes (198/204) were up-regulated at 4 h after 10 µg/ml LPS stimulation, including inflammatory cytokines and miRNAs. Confirmed by quantitative RT-PCR, the mRNA levels of IL-1ß, IL-6 and IL-8 showed single up-regulation peak (4 h/6 h) after 1 µg/ml and 10 µg/ml LPS treatment. Similarly, 1 µg/ml LPS induced single up-regulation peak (8 h) of miRNA-146a, -146b and -155 expression. However, 10 µg/ml LPS induced the increased expression of miRNA-146a and -155 at both early stage (2 h/4 h) and late stage (24 h). CONCLUSION: Taken together, we investigated P. gingivalis LPS induced whole transcriptional profile, and the different behaviors of miRNA expression induced by different doses of LPS in HGFs.

mTOR Inhibition Rejuvenates the Aging Gingival Fibroblasts through Alleviating Oxidative Stress.

The aging periodontium may be vulnerable to periodontal pathogens and poor response to inflammation and susceptible to tumorigenesis. Human gingival fibroblasts (hGFs) through continuously replicative culture served as an in vitro surrogate for aging. To investigate the effects of the mechanistic target of rapamycin (mTOR) inhibition on the aging gingiva, we stimulated the high-passage hGFs with rapamycin (20 nmol/L) for 3 days and 30 days. The cellular and biological changes were examined by immunofluorescence, real-time PCR, ELISA, Western blotting, and flow cytometry. The data demonstrated that the inhibition of mTOR signaling led to fewer senescence-associated beta-galactosidase- (SA-ß-Gal-) positive cells, delayed the onset of senescence, preserved the capability of proliferation, and lowered the expression levels of relevant senescence-associated markers, such as p16INK4a, p21CIP1a, interleukin-6 (IL-6), and IL-8. In addition, when infected by prominent periodontal pathogens, Porphyromonas gingivalis (ATCC 33277), rapamycin-pretreated groups decreased the expression of inflammatory cytokines (IL-6 and IL-8) compared with the control group. mTOR inhibition upregulated the gene expression of antioxidant components (Cat, Sod2, and Prdx3; P < 0.05) and consequently neutralized the excessive reactive oxygen species (ROS). In conclusion, our results indicated that mTOR inhibition might rejuvenate the aging gingiva to some extent and relieve inflammation through eliminating oxidative stress.

Antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm growing Streptococcus mutans.

The aim of this study was to evaluate the potential antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm modes of Streptococcus mutans (S. mutans). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The effects on planktonic growth and biofilm metabolic activity were evaluated by growth curve determination and MTT assay, respectively. Then, colony forming unit (CFU) counting, scanning electron microscopy (SEM) and real-time PCR were performed to further investigate the actions of DHA and EPA on exponential phase-S. mutans. Confocal laser scanning microscopy (CLSM) was used to detect the influences on mature biofilms. The MICs of DHA and EPA against S. mutans were 100 µM and 50 µM, respectively; the MBC of both compounds was 100 µM. In the presence of 12.5 µM-100 µM DHA or EPA, the planktonic growth and biofilm metabolic activity were reduced in varying degrees. For exponential-phase S. mutans, the viable counts, the bacterial membranes and the biofilm-associated gene expression were damaged by 100 µM DHA or EPA treatment. For 1-day-old biofilms, the thickness was decreased and the proportion of membrane-damaged bacteria was increased in the presence of 100 µM DHA or EPA. These results indicated that, DHA and EPA possessed antibacterial activities against planktonic and biofilm growing S. mutans.

Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 µM and 200 µM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 µM, and a respective minimum bactericidal concentration (MBC) of 12.5 µM and 25 µM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 µM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 µM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 µM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 µM EPA. For exponential-phase bacteria, 100 µM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 µM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of periodontal diseases.

Ultrathin cerium orthovanadate nanobelts for high-performance flexible all-solid-state asymmetric supercapacitors.

Ultrathin CeVO4 nanobelts were successfully synthesized by a hydrothermal method. The thickness of a single nanobelt is about 2.4 nm, which can effectively shorten the ion diffusion and fasten the charge pathway. More importantly, ultrathin CeVO4 nanobelts and graphene are easily assembled as a flexible all-solid-state asymmetric device, which shows a highly flexible property and achieves a maximum energy density of 0.78 mW h cm(-3) and a high life cycle of >6000 cycles.

Endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli: alternations in Toll-like receptor 2 and 4 signaling pathway.

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a hyporesponsive state to subsequent challenge, which is termed endotoxin tolerance. In this experiment, we studied the cytokine production in THP-1 cells upon single or repeated Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or Escherichia coli (E. coli) LPS stimulation by ELISA. In addition, the protein expression profiles of Toll-like receptor 2 (TLR2), TLR4, IL-1 receptor-associated kinase 4 (IRAK4) and IRAK-M and the gene expression changes of Toll-interacting protein (Tollip) and suppressor of cytokine-signaling-1 (SOCS1) were explored to identify possible mechanisms for changes in cytokine secretion. After repeated stimulation with P. gingivalis LPS or E. coli LPS, secretions of TNF-α and IL-1ß were decreased significantly compared with those following single challenge, while the levels of IL-10 were increased (p < 0.05). Only comparable levels of IL-8 were confirmed in P. gingivalis LPS-tolerized cells (p > 0.05). In addition, severe downregulation of TLR2 was detected in THP-1 cells retreated with P. gingivalis LPS, and the reduction of TLR4 expression was observed in cells restimulated with E. coli LPS (p < 0.05). Precondition with P. gingivalis LPS or E. coli LPS also led to an enhancement of IRAK-M and SOCS1, while maintaining the expressions of IRAK4 and Tollip. This pattern of cytokine production indicates the different effects of endotoxin tolerance triggered by P. gingivalis LPS and E. coli LPS, which might contribute to limiting inflammatory damage. Moreover, TLR2, TLR4, IRAK-M, and SOCS1 might play important roles in developing tolerance.

Effects of aging on endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

BACKGROUND: Periodontitis is a bacterially induced chronic inflammatory disease. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, termed endotoxin tolerance. Aging has a profound effect on immune response to bacteria challenge. The aim of this study was to explore the effects of aging on endotoxin tolerance induced by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and Escherichia coli (E. coli) LPS in murine peritoneal macrophages. METHODOLOGY/PRINCIPAL FINDINGS: We studied the cytokine production (TNF-α and IL-10) and Toll-like receptor 2, 4 (TLR2, 4) gene and protein expressions in peritoneal macrophages from young (2-month-old) and middle-aged (12-month-old) ICR mice following single or repeated P. gingivalis LPS or E. coli LPS stimulation. Pretreatment of peritoneal macrophages with P. gingivalis LPS or E. coli LPS resulted in a reduction in TNF-α production and an increase in IL-10 production upon secondary stimulation (p<0.05), and the markedly lower levels of TNF-α and higher levels of IL-10 were observed in macrophages from young mice compared with those from middle-aged mice (p<0.05). In addition, LPS restimulations also led to the significantly lower expression levels of TLR2, 4 mRNA and protein in macrophages from young mice (p<0.05). CONCLUSIONS/SIGNIFICANCE: Repeated LPS stimulations triggered endotoxin tolerance in peritoneal macrophages and the ability to develop tolerance in young mice was more excellent. The impaired ability to develop endotoxin tolerance resulted from aging might be related to TLR2, 4 and might lead to the incontrollable periodontal inflammation in older adults.