Effect of Porphyromonas gingivalis PrtC on cytokine expression in ECV304 endothelial cells and its level in subgingival plaques from patients with chronic periodontitis
Abstract
Aim: To investigate the effect of the collagenase gene (prtC) product of Porphyromonas gingivalis on inducing host cells to secrete inflammatory cytokines, and to discuss the correlation between the PrtC level in subgingival plaque samples and clinical parameters.
Methods: A prokaryotic expression system pET32a-prtC-Escheria coli BL21DE3 was constructed. Antigenicity and immunoreactivity of the recombinant PrtC protein (rPrtC) was identified by Western blotting. ELISA was applied to detect interleukin (IL)-1α, IL-8, and TNF-α levels in supernatants from rPrtC-induced human umbilical vein endothelial cells (HUVEC) originated ECV304 cells. Clinical parameters recorded at baseline and after treatment included bleeding on probing (BOP), probing depth (PD), and attachment loss (AL). ELISA was established to measure the PrtC level in 196 subgingival plaque samples from 49 patients with chronic periodontitis.
Results: After coincubation with 1 μg/mL rPrtC for 24 h and with 5 or 10 μg/mL rPrtC for 12 h, the levels of IL-1α, IL-8, and TNF-α secreted by the ECV304 cells increased significantly (P<0.05). The PrtC level in the BOP-positive or the ≥5 mm AL or >6 mm PD sites was higher than that in the BOP-negative or the ≤2 mm AL or ≤6 mm PD sites (P<0.05), respectively. Compared with baseline, the PrtC levels in different AL sites or in the ≤6 mm PD pockets decreased remarkably after treatment (P<0.01), but in the BOP-positive or in the >6 mm PD sites, the PrtC levels changed insignificantly (P>0.05).
Conclusion: rPrtC is able to directly induce host cells to synthesize and secrete IL-1α, IL-8, and TNF-α. The PrtC level in subgingival samples is correlated with BOP, AL and PD.
Keywords:
Methods: A prokaryotic expression system pET32a-prtC-Escheria coli BL21DE3 was constructed. Antigenicity and immunoreactivity of the recombinant PrtC protein (rPrtC) was identified by Western blotting. ELISA was applied to detect interleukin (IL)-1α, IL-8, and TNF-α levels in supernatants from rPrtC-induced human umbilical vein endothelial cells (HUVEC) originated ECV304 cells. Clinical parameters recorded at baseline and after treatment included bleeding on probing (BOP), probing depth (PD), and attachment loss (AL). ELISA was established to measure the PrtC level in 196 subgingival plaque samples from 49 patients with chronic periodontitis.
Results: After coincubation with 1 μg/mL rPrtC for 24 h and with 5 or 10 μg/mL rPrtC for 12 h, the levels of IL-1α, IL-8, and TNF-α secreted by the ECV304 cells increased significantly (P<0.05). The PrtC level in the BOP-positive or the ≥5 mm AL or >6 mm PD sites was higher than that in the BOP-negative or the ≤2 mm AL or ≤6 mm PD sites (P<0.05), respectively. Compared with baseline, the PrtC levels in different AL sites or in the ≤6 mm PD pockets decreased remarkably after treatment (P<0.01), but in the BOP-positive or in the >6 mm PD sites, the PrtC levels changed insignificantly (P>0.05).
Conclusion: rPrtC is able to directly induce host cells to synthesize and secrete IL-1α, IL-8, and TNF-α. The PrtC level in subgingival samples is correlated with BOP, AL and PD.