The effect of interleukin-22 on the expression of RANKL and OPG in human periodontal ligament fibroblasts

Abstract

Objective To investigate the effect of interleukin-22 (IL-22) on the expression of RANKL and OPG in human periodontal ligament fibroblasts (hPDLFs) , and further explore the synergistic effect of IL-22 and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) . Methods Primary hPDLFs were cultured using combined methods of tissue explant and enzymatic digestion, then identified by immunocytochemical staining. Passage third-fifth cells were treated with different concentrations of IL-22 (0~ 100 ng/mL) , and cell counting kit-8 (CCK-8) assay was used to detect the cytotoxicity at 24, 48, 72 h. 5, 10, 25 ng/mL IL-22 were applied to hPDLFs, and the expression of RANKL and OPG mRNA were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) at 72 h. Furthermore, cells were treated with 1 μg/mL Pg-LPS alone, or together with 10 ng/mL IL-22 for 72 h. The expression of RANKL and OPG mRNA and their coding proteins were measured using qRT-PCR and Western blot. Data were evaluated by One-Way ANOVA. Results The 50 ng/mL and 100 ng/mL IL-22 significantly decreased cell viability of hPDLFs, comparing to the control group (F24 h= 15.17, F48 h= 76.37, F72 h= 24.409, P<0.05) . A number of concentrations of IL-22 (5, 10 and 25 ng/mL) up-regulated the mRNA expression of RANKL in hPDLFs (F= 32.88, P<0.05) , with 10, 25 ng/mL more significantly than 5 ng/mL (P<0.05) . However, the mRNA expression of OPG was not affected (F= 0.719, P= 0.555) . Co-stimulation with 10 ng/mL IL-22 and 1 μg/mL Pg-LPS enhanced both RANKL mRNA and protein expression comparing with those of IL-22 or Pg-LPS stimulation alone (FmRNA= 36.67, Fprotein= 41.24, P<0.05) . OPG mRNA and protein were not changed compared with the control (FmRNA= 0.652, P= 0.593; Fprotein= 1.271, P= 0.313) . Conclusions IL-22 up-regulates RANKL expression in hPDLFs, and has synergistic effect with Pg-LPS, suggesting that IL-22 may participate in periodontal bone destruction. Key words: Interleukin-22; Porphyromonas gingivalis; Lipopolysaccharides; Receptor activa-tor of nuclear factor-kappa B; Ligands; Osteoprotegerin