Neutrophil extracellular traps induced by Porphyromonas gingivalis lipopolysaccharide modulate inflammatory responses via a Ca2+-dependent pathway.

OBJECTIVE: The aim of this study was to explore the effects and underlying mechanisms of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on the formation of neutrophil extracellular traps (NETs). DESIGN: NETs induced by 1 µg/ml P. gingivalis LPS were observed by a fluorescence microscope and quantified by a microplate reader. Quantities of extra- and intracellular P. gingivalis in neutrophils were determined to assay the bactericidal efficiency of NETs. Intracellular Ca2+ levels in neutrophils were explored by flow cytometry. Expressions of phospho-tumor progression locus 2 (p-TPL2), phospho-mitogen-activated protein kinase kinase1/2 (p-MEK1/2), phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), ORAI1, ORAI2 and peptidylarginine deiminase 4 (PAD4) were detected by Western blot. In addition, neutrophil elastase activities in NETs incubated with macrophages were assayed to evaluate their clearance. RESULTS: P. gingivalis LPS contributed to the formation of NETs and the increased levels of extracellular DNA (p < 0.05), which enhanced bactericidal activity of neutrophils (p < 0.05). Levels of intracellular Ca2+, p-TPL2, p-MEK1/2, p-ERK1/2, ORAI1, ORAI2 and PAD4 were increased in P. gingivalis LPS-treated neutrophils compared with control group (p < 0.05). In addition, inhibition of intracellular Ca2+ by two Ca2+ chelators, and PAD4 knockdown resulted in decreased levels of extracellular DNA (p < 0.05). After co-culture of NETs with macrophages, neutrophil elastase activities were decreased (p < 0.05). CONCLUSION: P. gingivalis LPS induced the formation of NETs via a Ca2+-TPL2-MEK-ERK-PAD4 signaling pathway, which contribute to the elimination of P. gingivalis.

Enhanced Responsive Formation of Extracellular Traps in Macrophages Previously Exposed to Porphyromonas gingivalis.

Tolerance is defined to be a hyporesponsive state following repeated stimulations with bacteria or their virulence factors and has potential impacts on the development of periodontitis. Recently, macrophages have been reported to release chromatin and antimicrobial peptides to form extracellular traps upon bacterial or chemical stimulations. Thus, we explored the roles and mechanisms of tolerance induced by Porphyromonas gingivalis (P. gingivalis) in macrophage extracellular traps (METs). Tolerance in peritoneal macrophages from mice was triggered by repeated P. gingivalis stimulation. METs were observed using fluorescence microscopy, and the levels of extracellular DNA were determined by microplate reader assays. The expression of p-RAF, p-MEK, and p-ERK was examined by Western blot, and reactive oxygen species (ROS) production was explored using flow cytometry. Moreover, the levels of intracellular Ca2+ were also determined by confocal microscopy to identify the possible mechanisms related to the changes in METs in P. gingivalis-pretreated macrophages. Repeated P. gingivalis stimulation contributed to the formation of METs and increased levels of extracellular DNA (p < 0.05). ROS generation and RAF/MEK/ERK phosphorylation were decreased in P. gingivalis-pretreated macrophages compared with non-pretreated cells (p < 0.05), which was inconsistent with the changes in METs. However, in P. gingivalis-pretreated macrophages, the levels of intracellular Ca2+ were significantly increased compared with the single stimulation group. Additionally, inhibition of intracellular Ca2+ resulted in a decrease in the levels of extracellular DNA in P. gingivalis-pretreated cells (p < 0.05). Taken together, P. gingivalis-pretreated macrophages released more METs, possibly related to the increased levels of intracellular Ca2+.

[Effect of curcumin on TGF-ß1 /Smad3 pathway in rat gingival fibroblast treated with cyclosporine A].

PURPOSE: The aim of the present study was to investigate the effect of curcumin (Cur) on TGF-ß1/Smad3 pathway of rat gingival fibroblast treated with cyclosporine A (CsA) in vitro, and to provide theoretical basis for the mechanism of curcumin inhibiting drug-induced gingival hyperplasia induced by CsA. METHODS: Healthy Sprague-Dawley rat gingival fibroblasts were cultured with different concentrations of Cur (0, 5, 10, 20, 30 µmol/L) and Cur (20 µmol/L)+CsA(200 ng/mL), cell proliferation was assessed with CCK-8 assay. The mRNA levels of TGF-ß1, Smad3, α-SMA and collagen type Ⅰ in gingival fibroblasts were detected by real-time PCR under Cur(20 µmol/L)+CsA(200 ng/mL); the protein level of TGF-ß1, Smad3, p-Smad3, α-SMA and collagen type Ⅰ were determined through Western blot. The effect of Cur(20 µmol/L)+CsA(200 ng/mL) on migration ability of gingival fibroblasts was observed through Scratch wound-healing assay. The data were analyzed with SPSS 23.0 software package. RESULTS: Cell proliferation and migration ability of rats gingival fibroblasts were significantly reduced under Cur(20 µmol/L)+CsA(200 ng/mL). 20 µmol/L Cur significantly decreased mRNA expression of TGF-ß1, α-SMA and collagen type Ⅰ in gingival fibroblasts, and Western blot suggested significantly down-regulated expression of TGF-ß1, p-Smad3, α-SMA, and collagen typeⅠ. CONCLUSIONS: Cur may inhibit TGF-ß1/Smad3 signaling pathway of gingival fibroblasts activated by CsA, thereby weakening proliferation and migration, reducing secretion of smooth muscle actin and collagen of gingival fibroblasts, and ameliorating gingival hyperplasia.

[Effects of Compound Aconitum Polysaccharide on Glycosylation of Tumor Cells].

Objective To observe inducing or inhibiting effects of Chinese medicine (CM) poly- saccharides on glycoprotein chain synthetized different glycosyltransferases, thus disclosing targets of CM polysaccharides and its mechanisms. Methods In vivo anti-tumor effects of CM polysaccharides were observed using the inhibiting rate of tumor growth by dividing different Aconitum containing groups. Effects of CM polysaccharides on liver cancer cell SK-HEP-1 glycosyltransferase and tumor related gene expressions were observed. Meanwhile, changes of polylactosamine expression were detected using flow cytometry (FCM) with polylactosamine specific biotin labeling lectin. Results Compared with the model group, the average tumor weight was significantly lower in each medication group (P <0. 01). Compared with the adriamycin group, no significant difference in average tumor weight of the three compound groups (P>0. 05). The expression level of polylactosamine was reduced after adding Aconitum polysac- charide; and CM compound polysaccharides respectively. Conclusions Polysaccharide compound showed similar anti-tumor effect as that of adriamycin. Besides, polylactosamine expression level was reduced in the three compound groups along with increased prepared Aconitum polysaccharide, with more obvious anti-tumor effects shown.

[Effect of cyclosporine A on TGF-ß/Smad3 signaling in rat gingival fibroblasts].

PURPOSE: The aim of the study was to investigate the effect of cyclosporine A (CsA) on TGF-ß/Smad3 signaling in rat gingival fibroblasts and to explore the mechanism of CsA induced gingival overgrowth. METHODS: Healthy Sprague-Dawley rat gingival fibroblasts were cultured with different concentrations of CsA and the cell proliferations were assessed with CCK-8 assay. The mRNA levels of TGF-ß1, Smad3 and collagen I were measured by real-time PCR. The protein level of TGF-ß1, Smad3, p-Smad3 and collagen I were determined using western blot and immumofluorescence. Cell migration ability was detected by cell wound scratch assay. The data was analyzed with SPSS 20.0 software package. RESULTS: The use of 200 ng/mL CsA stimulated proliferation and migration of gingival fibroblasts. The mRNA levels of TGF-ß1 and collagen I were significantly promoted after CsA exposure. The protein syntheses of TGF-ß1, p-Smad3 and Collagen I were also increased by CsA stimulation. CONCLUSIONS: CsA may activate TGF-ß/Smad3 signaling pathway, thus promoting the proliferation and migration of rat gingival fibroblasts as well as collagen accumulation, which eventually lead to gingival overgrowth.

Mitotic arrest defective protein 2 expression abnormality and its clinicopathologic significance in human osteosarcoma.

Osteosarcoma is the most common primary malignancy of bone. Overexpression of mitotic arrest defective protein 2 (MAD2) is found in many human neoplasms, but its role in the oncogenesis of osteosarcoma is an untouched topic. The objective of this research was to observe the expression of MAD2 in human osteosarcoma and explore its clinicopathologic significance. MAD2 expression was analyzed in 48 primary osteosarcoma cases (19 osteoblastic osteosarcomas, 17 chondroblastic osteosarcomas and 12 fibroblastic osteosarcomas) using immunohistochemistry. A total of 20 normal bone specimens formed a control group. MAD2 was commonly overexpressed in human osteosarcoma. Immunopositivity was higher in tumors with lower differentiation and higher clinical stage. Increased expression of MAD2 was associated with earlier metastasis and poorer survival. Our findings provide evidence that MAD2 contributes to the pathogenesis and development of human osteosarcoma, Testing may have a clinical role in predicting prognosis, selecting appropriate chemotherapeutic strategies and providing novel strategies for osteosarcoma therapy.