Role of cathepsin D induced by Porphyromonas gingivalis lipopolysaccharide in periodontitis

Periodontitis is an inflammatory disease of tooth-supporting tissues caused by oral bacteria. Periodontal ligament loss and alveolar bone destruction occur in progressive periodontitis. Since gingival crevicular fluids (GCF) reflects the inflammatory environment of the periodontal pocket, it is a very important specimen for developing targets for periodontitis diagnosis. An antibody array was performed using GCF collected from healthy participants and patients with periodontitis to identify the proteolytic enzymes involved in periodontitis. Of 21 targets on the antibody array membrane, kallikrein 6 (KLK6), kallikrein 10 (KLK10), cathepsin A (CathA), and cathepsin D (CathD) showed higher levels in periodontitis GCF than in GCF from healthy participants. Lipopolysaccharide stimulation of Porphyromonas gingivalis (PG-LPS) in immortalized gingival fibroblasts only increased CathD protein levels among the four targets. The substrate cleavage activity of CathD was increased in PG-LPS-treated immortalized gingival fibroblast extract. The PG-LPS-induced substrate cleavage effect was abolished by the CathD inhibitor pepstatin A. Osteoclast formation was promoted by treatment with conditioned media from PG-LPS- treated immortalized gingival fibroblasts but inhibited by the CathD inhibitor pepstatin A. These results suggest that PG-LPS affected the osteoclast formation process by increasing CathD expression in cells around the alveolar bone, thereby participating in periodontitis progression.     

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis

Odontology - Gut dysbiosis induces ‘leaky gut,’ a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium...     

五倍子水提取物对Pg LPS诱导单核细胞分泌IL-6的抑制作用

目的：研究百倍子水提取物对牙龈卟啉菌(Pg)内毒素(LPS)介导的人单核细胞分泌IL-6水平的影响。方法：采用健康人外周血分离培养的单核细胞以25μg/ml牙龈卟啉菌内毒素作为刺激因子，用放射免疫分析法(RIA)测定细胞培养上清中IL-6的水平，观察5种浓度瓦倍子水提取物对单核细胞分泌炎性细胞因子的影响，结果：五倍子水提取物可显著抑制牙龈卟啉菌内毒素诱导人单核细胞分泌IL-6的水平，其作用在一定范围内呈浓度依赖性。结论：五倍子水提取物能够显著抑制牙龈卟啉菌内毒素诱导人单核细胞分泌IL-6的水平，提示五倍子具有一定的抗炎作用，有助于对牙周病的防治。     

Mechanical stress enhances production of cytokines in human periodontal ligament cells induced by Porphyromonas gingivalis

Objective

We have previously reported that human periodontal ligament (hPDL) cells produced many kinds of cytokines as a result of bacterial stimulation, including stimulation with Porphyromonas gingivalis (P. gingivalis). However, the effects of mechanical stress on cytokine production in hPDL cells stimulated by periodontopathogenic bacteria are not clearly understood. In this study, we investigated the effects of mechanical stress on the production of inflammatory cytokines in hPDL cells induced by stimulation with P. gingivalis.

Methods

The hPDL cells were exposed to various levels of mechanical stress (1, 6, 10 and 50 MPa) and costimulated with mechanical stress and P. gingivalis for 24 h. Cytokine mRNA expressions were determined by RT-PCR. Cytokines in the culture supernatant were assessed by ELISA, and morphologic changes in hPDL cells were observed.

Results

The expressions of interleukin (IL)-6, IL-8 and tumor necrosis factor-α mRNA were observed in hPDL cells after exposure to mechanical stress. Moreover, the production of IL-6 and IL-8 increased significantly after exposure to mechanical stress ranging from 1 to 10 MPa. The amount of IL-8 in the culture supernatants of hPDL cells costimulated with P. gingivalis and mechanical stress was significantly higher than the expected additive amount. The morphology of hPDL cells did not change after exposure to 6 MPa, but these cells were partly detached from the Petri dish after exposure to 50 MPa.

Conclusions

These results suggest that local inflammation of the periodontal ligament may be induced mainly by periodontal bacteria, and mechanical stress may promote local inflammation.     

Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14-Toll-like receptor-nuclear factor kappaB pathway

OBJECTIVES: Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor kappaB (NF-kappaB). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14-TLR-NF-kappaB pathway and the cellular mechanism of calprotectin release in human neutrophils. MATERIAL AND METHODS: Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-kappaB, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-kappaB binding activity using electrophoretic mobility shift assays. RESULTS: P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-kappaB inhibitors suppressed P-LPS-induced NF-kappaB binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. CONCLUSION: These results suggest that calprotectin release is induced by P-LPS via the CD14-TLR2-NF-kappaB signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems.     

Induction of calprotectin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils

Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils, monocytes/macrophages, and epithelial cells. This protein is known to be a marker for several inflammatory diseases and is detected in inflammatory gingival tissue with periodontal disease. Recently, we found that the calprotectin level in gingival crevicular fluid from periodontitis patients was significantly higher than that of healthy subjects. However, the regulation of calprotectin in periodontal disease is unclear. In the present study, we investigated the effect of lipopolysaccharides of periodontopathic bacteria on calprotectin release from human neutrophils. Neutrophils from healthy donors were treated with lipopolysaccharides from Porphyromonas gingivalis (P-LPS), Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, and Escherichia coli. Calprotectin of neutrophil was identified by immunoblotting and calprotectin amount was determined by ELISA. Two subunits (10 and 14 kDa) of calprotectin were observed in the cell and medium fractions from neutrophils. P-LPS increased calprotectin release from seven to 16 times the control level after 30 min and its effect appeared in a dose-dependent manner (10-1000 ng/ml). Lipopolysaccharides from A. actinomycetemcomitans, P. intermedia, F. nucleatum, and E. coli also induced calprotectin release from neutrophils. These results suggest that lipopolysaccharides from periodontopathic bacteria induce calprotectin release from human neutrophils.     

葡萄籽原花青素对牙龈卟啉单胞菌内毒素的影响

目的探讨葡萄籽原花青素对牙龈卟啉单胞菌内毒素的影响。方法采用二倍稀释法测定葡萄籽原花青素对牙龈卟啉单胞菌的最小抑菌浓度(MIC);采用鲎试验测定低于MIC 6个浓度的葡萄籽原花青素对牙龈卟啉单胞菌内毒素含量的影响。采用SPSS17.0软件包进行组间比较。结果葡萄籽原花青素对牙龈卟啉单胞菌的MIC为0.8 mg/mL;在0.05~0.4 mg/mL范围内,随着葡萄籽原花青素浓度的升高,对释放内毒素的抑制作用逐渐增强（P<0.05、0.01）。结论葡萄籽原花青素对牙龈卟啉单胞菌内毒素的释放具有抑制作用。     

Cytokine production induced by a 67-kDa fimbrial protein from Porphyromonas gingivalis

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. P. gingivalis ATCC 33277 has two distinctly different fimbriae expressed on the cell surface. The 67-kDa fimbriae differ in size and antigenicity from the earlier reported FimA, a major 41-kDa fimbrial component of P. gingivalis. Expression of the 67-kDa fimbriae on the cell surface of a fimA mutant was investigated by electron microscopy. The 67-kDa fimbrial protein was purified from the fimA mutant by sonication, precipitation, and chromatography on a DEAE Sepharose CL-6B column. The N-terminal amino acid sequence of the 67-kDa fimbrillin was distinct from that of the 41-kDa fimbrillin. Moreover, we have found that the 67-kDa fimbrial protein from P. gingivalis ATCC 33277 induced IL-1alpha, IL-beta, IL-6 and TNFalpha cytokine expression in mouse peritoneal macrophages. These results suggest that P. gingivalis 67-kDa fimbriae may play a part in the inflammatory response during the development of periodontal diseases.     

Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells

ObjectivePorphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) induces pro-inflammatory cytokines, such as interleukin-1 β (IL-1β), IL-6, and IL-8, which induce periodontal tissue destruction. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration and are expected to have future applications in cellular therapies for periodontitis. However, no studies have examined the effects of P. gingivalis LPS on PDLSCs. The aim of this study was to investigate how P. gingivalis LPS affects the osteoblastic differentiation and pro-inflammatory cytokine production of PDLSCs.DesignPDLSCs were obtained from healthy adult human mandibular third molars. The identification of PDLSCs was confirmed by immunohistochemical evaluations of the mesenchymal stem cell markers STRO-1 and SSEA-4. Cell proliferation and osteoblastic differentiation were investigated by culturing the PDLSCs in a normal or osteogenic medium with P. gingivalis LPS (0, 1, or 10 μg/mL) and then measuring the alkaline phosphatase (ALP) activity and the production of collagen type 1 Alpha 1 (COL1A1), osteocalcin production, and mineralisation. Additionally, we examined the production of IL-1β, IL-6, and IL-8 in the PDLSCs.ResultsP. gingivalis LPS inhibited the ALP activity, COL1A1 and osteocalcin production, and mineralisation in the PDLSCs, which are positive for STRO-1 and SSEA-4. P. gingivalis LPS also promoted cell proliferation and produced IL-1β, IL-6, and IL-8.ConclusionsThis study provides the first findings that P. gingivalis LPS inhibits osteoblastic differentiation and induces pro-inflammatory cytokines in PDLSCs. These findings will help clarify the relationship between periodontitis and periodontal tissue regeneration.     

Stimulation of growth of Porphyromonas gingivalis by cell extracts from Tannerella forsythia

OBJECTIVE: In order to examine if Tannerella forsythia stimulates the growth of Porphyromonas gingivalis, an in vitro study was performed. Background: P. gingivalis and T. forsythia are often isolated simultaneously from active periodontitis sites, indicating that these bacteria somewhat interact in the periodontal environment. We reported previously that mixed infection of P. gingivalis and T. forsythia synergistically induced lesion formation in a murine abscess model, and gingipains of P. gingivalis played an important role in this synergism. One of the possible mechanisms of this synergism is growth promotion by coinfection of the two bacteria. METHODS: Cell extracts of T. forsythia were added to the nutrition-decreased medium and the promotion of growth of P. gingivalis was examined. RESULTS: Sonicated extract of T. forsythia stimulated growth of P. gingivalis in nutrition-decreased medium in a dose-dependent manner. Proteins appeared to be the nature of growth-promoting factor, and the cell extract of T. forsythia had no stimulating effect on the growth of P. gingivalis strain devoid of gingipain activities. CONCLUSION: A product or a component of T. forsythia seemed to stimulate growth of P. gingivalis under nutrition-limited conditions. Gingipains are considered to play an important role in digestion or uptake of this growth-promoting factor. The interaction between T. forsythia and P. gingivalis in growth may be in part related with the synergistic virulence in a murine model.     

牙龈卟啉单胞菌脂多糖对人牙周膜成纤维细胞胶原吞噬作用的影响

目的研究牙周病致病菌牙龈卟啉单胞菌脂多糖（LPS）对体外培养的人牙周膜成纤维细胞（hPDLF）胶原吞噬作用的影响。方法将不同质量浓度的LPS加入体外培养的hPDLF 48 h后,采用荧光定位术和流式细胞技术检测hPDLF胶原吞噬率的变化。结果LPS导致hPDLF胶原吞噬率显著增加（P<0.05）。结论牙龈卟啉单胞菌脂多糖具有促进hPDLF吞噬胶原的作用,可能是牙周组织破坏机制之一。     

Immunomodulation of dendritic cells differentiated in the presence of nicotine with lipopolysaccharide from Porphyromonas gingivalis

Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T‐cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte‐derived DCs to elucidate the mechanism of tissue destruction of smoking‐associated periodontal diseases. P. gingivalis LPS‐stimulated DCs differentiated with nicotine (NiDCs) induced lower T‐cell proliferation and human leukocyte antigen (HLA)‐DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon‐γ production but maintained interleukin (IL)‐5 and IL‐10 production in co‐cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)‐type chemokines macrophage chemotactic protein‐1 and macrophage‐derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS‐stimulated DCs.     

五倍子提取物对牙龈卟啉菌内毒素介导白介素-1β活性的影响

目的：研究五倍子提取物对牙龈卟啉菌内毒素介导白介素—lβ活性的影响。方法：采用人外周血分离培养单核细胞，25μg／mL牙龈卟啉菌内毒素作为刺激因子，观察6．25、12．5、25、50、100μg/mL五种质量浓度的五倍子提取物对单核细胞分泌细胞因子白介素—lβ活性的影响，以放射免疫分析法(RIA)测定白介素—lβ的水平。结果：五倍子提取物可显著抑制牙龈卟啉菌内毒素介导单核细胞分泌白介素—lβ的活性，其作用在一定范围内呈浓度依赖性。结论：五倍子提取物抑制牙龈卟啉菌内毒素介导的白介素—lβ的活性，揭示五倍子提取物抗炎作用的机制。     

����߲���������յ���ϸ��������ʵ���о�

目的    探讨牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）对骨细胞凋亡的诱导作用,及其对骨质破坏的影响。方法    2009年8—12月于昆明医学院口腔实验室,选取昆明小鼠60只,随机分为实验组和对照组,每组30只。于实验组小鼠头骨中线两耳之间的部位注射一定浓度的Pg细菌悬液;对照组动物在同样条件下注射等量的0.01 mol／L PBS液。分别于注射后当即及第1、3、5、8、12 天处死,保留整个头颅骨进行固定、脱钙和包埋,原位末端标记（TUNEL）法观察骨细胞的凋亡状况,并计算细胞凋亡率。结果    实验组在注射后第1天,细胞凋亡率较注射前显著增加,第5天达峰值［（35.64% ± 3.69）%］,第8、12 天的细胞凋亡率有所下降;与对照组相比,差异均有统计学意义（P < 0.05）。结论    Pg具有诱导骨细胞凋亡的作用,提示大量骨细胞凋亡可能是牙周炎骨组织破坏产生而重建愈合相对不足的原因之一。     

牙龈卟啉单胞菌脂多糖对泡沫细胞凋亡基因的影响

目的 了解牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)脂多糖(lipopolysaccharide,LPS)在巨噬细胞吞噬氧化低密度脂蛋白(oxidized low density lipopmtein,oxLDL)形成泡沫细胞过程中和形成后对凋亡基因的影响,以期了解Pg影响动脉粥样硬化的可能机制.方法 以oxLDL、oxLDL+Pg-LPS刺激人急性单核细胞白血病单核细胞株THP-1源性巨噬细胞,以及oxLDL诱导巨噬细胞形成的泡沫细胞.采用吖啶橙-溴化乙锭双染色观察细胞凋亡,聚合酶链反应(PCR)芯片检测11种动脉粥样硬化相关凋亡基因的变化,实时PCR检测p53、c-Myc和半胱氨酸天冬氨酸蛋白酶(caspase)-3基因的变化.结果 Pg-LPS提高了巨噬细胞吞噬oxLDL形成泡沫细胞过程中和形成后的细胞凋亡率,分别为(5.47±0.93)%、(7.50 4-0.54)%;PCR芯片检测显示泡沫细胞形成过程中Pg-LPS上调了B细胞淋巴瘤-白血病-2相关蛋白Al的转录(>2倍),泡沫细胞形成后上调了B细胞淋巴瘤-白血病-2和B细胞淋巴瘤-白血病-2相关蛋白A1的转录(>2倍);在泡沫细胞形成过程中提高了p53和caspase-3的转录水平[分别为(4.50×10-3±4.02×10-4)和(5.30×10-2±4.58×10-3)],抑制了c-Myc的转录水平(1.53×10-2±5.77×10-4);在泡沫细胞形成后降低了p53转录水平(4.23×10-3±5.85×10-4),促进了caspase-3的转录水平(6.00×10-2±6.08×10-3),P<0.05.结论 Pg-LPS影响了泡沫细胞形成过程中和形成后细胞中多种凋亡基因的转录,促进了凋亡的发生.     

microRNA-146a对牙龈卟啉单胞菌脂多糖刺激下淋巴细胞分泌细胞因子的调节作用

目的探讨microRNA-146a (miR-146a)对牙龈卟啉单胞菌(P. gingivalis)脂多糖(LPS)刺激下淋巴细胞分泌细胞因子的作用。方法从小鼠脾脏收集淋巴细胞。实时定量聚合酶链反应和酶联免疫反应用于检测淋巴细胞在P. gingivalis LPS、miR-146a模拟物或抑制剂处理后细胞因子的表达。结果与P. gingivalis LPS未刺激组相比,P. gingivalis LPS能促进白细胞介素(interleukin,IL)-1β、IL-6、细胞核因子κB受体活化因子配体(RANKL)和IL-10表达(P<0.05),虽抑制骨保护素(OPG) mRNA水平(P<0.05),但分泌水平差异无统计学意义(P>0.05)。与阴性对照组相比,P. gingivalis LPS处理的淋巴细胞中miR-146a模拟物抑制IL-1β、IL-6和RANKL表达(P<0.05),促进IL-10和OPG表达(P<0.05),miR-146a抑制剂对这些细胞因子产生相反的效果(P<0.05)。结论 miR-146a可通过抑制淋巴细胞IL-1β、...     

低浓度牙龈卟啉单胞菌脂多糖刺激骨唾液酸蛋白的基因表达和转录

目的研究低浓度牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g）脂多糖（Lipopolysaccharide,LPS）对成骨细胞系（ROS17/2.8）中骨唾液酸蛋白（Bonesialoprotein,BSP）基因表达和转录的调节作用,以及细胞外信号调节蛋白激酶（ERK1/2）转导途径阻断剂（U0126）对此调节作用的影响。方法0.01mg/LP.g·LPS作用ROS17/2.8细胞0h、3h、6h和12h后,用Northern杂交观察BSP和骨桥蛋白（Osteopontin,OPN）mRNA的表达;再将ROS17/2.8细胞随机分为四组：空白对照组、LPS（0.01mg/LP.g·LPS）组、U0126（5μmol/L）组、U0126＋LPS组（U0126预刺激细胞30min后,U0126与P.g·LPS共同作用）,各组持续作用12h后,用瞬时转染法分析BSP基因启动子的转录活性。结果0.01mg/LP.g·LPS作用ROS17/2.8细胞12小时后BSP和OPN的mRNA杂交条带增强;0.01mg/LP.g·LPS使BSP基因启动子（pLUC3）转录活性值与空白载体活性值的比值升高1.582（F=5.734,P〈0.05）,U0126使其降低2.693（F=11.500,P〈0.01）,U0126使LPS对比值的升高变化降低2.242（F=6.204,P〈0.05）。结论低浓度（0.01mg/L）P.g·LPS增强ROS17/2.8细胞BSP基因表达和转录,而且其对BSP基因转录活性的上调作用是经由ERK1/2信号转导路径介导的。