Table 1.
PCR primers used for the amplification of genes.
Figure 1.
P. gingivalis GroEL induces IL-6 and IL-8 production in PDL cells, potentially via NF-κB activation.
(A) Cellular cytotoxicity of recombinant P. gingivalis GroEL was analyzed by MTT assay. PDL cells were treated with serum-free medium (0 µg/mL; control), 25–100 µg/mL GroEL or 100 µg/mL GST for 24–72 h; an MTT assay was performed, and the absorbance was recorded using a microplate reader. (B and D) PDL cells were treated with serum-free media containing 0–50 µg/mL GroEL or 50 µg/mL GST for 24 h. The levels of IL-6 (B) and IL-8 (D) in the culture media were quantified via ELISA assay, and the absorbance was recorded using a microplate reader. (C and E) PDL cells were treated with serum-free medium (0 µg/mL; control), 50 µg/mL GroEL or 100 µM pyrrolidine dithiocarbamate (PDTC) plus 50 µg/mL GroEL for 12 h. The expression of IL-6 (C) and IL-8 (E) mRNA was analyzed using quantitative real-time PCR. Data are expressed as the mean ± SEM of three independent experiments performed in triplicate. *p<0.05 was considered significant. (F) PDL cells grown on slides were exposed to serum-free medium (0 µg/mL; control), GroEL (25 or 50 µg/mL) or 50 µg/mL GST for 120 min and then imaged on a confocal microscope after immunofluorescence staining against NF-κB p65 was performed. The lower images are merged pictures of the upper images with Hoechst staining for nuclei. White arrows indicate activated NF-κB p65 in the nuclei. The scale bar indicates 10 µm.
Figure 2.
P. gingivalis GroEL increases PDL cell migration, possibly through activation of integrin α1 and α2 expression, as well as cytoskeletal reorganization.
(A) A wound-healing assay was used to evaluate the effect of GroEL on PDL cell migration. PDL cells were cultured with serum free medium (0 µg/mL; control), 10–50 µg/mL GroEL or 50 µg/mL GST for 24 h before wound scraping using a pipette tip. Images were taken 24 h after wound scraping. PDL cells migrating to the denuded area were counted based on the black base line. (B) PDL cells that migrated into the denuded area were quantified, and the magnitude of PDL cell migration was evaluated by counting the migrated cells in six random areas under high-power microscope fields (×100). (C) PDL cells were treated with serum-free medium (0 µg/mL; control), 50 µg/mL GroEL or 50 µg/mL GST for 12 h. The expression of integrin α1, α2, and β1 mRNA was analyzed using quantitative real-time PCR. Data are expressed as the mean ± SEM of three independent experiments and expressed as the percentage of control. *P<0.05 was considered significant. (D) F-actin was stained with rhodamine-phalloidin, and the staining was evaluated using confocal microscopy at 400× magnification. The visible parallel stress fibers are indicated as white arrows. DAPI was used to identify the nuclei of PDL cells.
Figure 3.
P. gingivalis GroEL may be involved in osteoclastogenesis via RANKL activation and ALP inhibition in PDL cells.
(A and C) PDL cells were treated with serum-free media containing 0–50 µg/mL GroEL or 50 µg/mL GST for 12 h. (B and D) PDL cells were treated with 50 µg/mL of GroEL for 0–24 h or 50 µg/mL of GST for 24 h. The levels of ALP and RANKL mRNA were quantified using quantitative real-time PCR. (E) PDL cells were treated with 50 µg/mL GroEL or 100 µM pyrrolidine dithiocarbamate (PDTC) plus 50 µg/mL GroEL for 6 h. The levels of RANKL mRNA were quantified using quantitative real-time PCR. Data are expressed as the mean ± SEM of three experiments performed in triplicate. *p<0.05 compared with control cells (0 µg/mL GroEL).
Figure 4.
P. gingivalis GroEL induces bone loss in rat gingival.
(A) Maxilla sections were stained using H&E. The black arrow indicates the cemento-enamel junction (CEJ) and the triangle arrowhead indicates the alveolar bone. (B) The specimens were scanned by micro-CT at a resolution of 35 µm, and the results were quantified as the ratio of residual bone volume (BV) per total volume (TV). The data represent the mean ± SEM of four animals (n = 4). *p<0.05 indicated a significant difference compared with the PBS injection control.
Figure 5.
GroEL induces cytokines expression and macrophages as well as osteoclasts infiltration in rat gingival.
(A) TRAP histochemistry was used to identify active osteoclasts in maxilla sections. The first molar is labeled by the star symbol, and the black arrows indicate the osteoclasts. (Lower panel) The image highlights a high-power view (200x) of osteoclasts in the connective tissues isolated from the images in the middle panel. The right graphs show the quantitative data of TRAP-positive cells in high-power field (HPF, magnification of 200x). Data are expressed as the mean ± SEM of three slides. *p<0.05 compared with control cells. (B) The expression of IL-6 and IL-8 were stained using specific antibodies and hematoxylin. The periodontal membrane (periodontal ligament) is indicated with the black arrow. (C) Negative control analyses were performed using rabbit pre-immune serum to replace specific antibodies. (D) Representative images (40x) of infiltrated macrophages stained with anti-CD 68 in the gingiva. The black arrowheads indicate the infiltrated macrophages. The right graphs show the quantitative data of CD68-positive cells in HPF (magnification of 200x). Data are expressed as the mean ± SEM of three slides. *p<0.05 compared with control cells.