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

doi:10.3877/cma.j.issn.1674-1366.2018.01.004

Abstract

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 (F_{24 h}= 15.17, F_{48 h}= 76.37, F_{72 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 (F_mRNA= 36.67, F_protein= 41.24, P<0.05) . OPG mRNA and protein were not changed compared with the control (F_mRNA= 0.652, P= 0.593; F_protein= 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

Qianying Chen, Li Gao, Panpan Wang, Chi Zhang, Xiting Li, Chuanjiang Zhao. The effect of interleukin-22 on the expression of RANKL and OPG in human periodontal ligament fibroblasts[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(01): 19-25.

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Figures/Tables 7

References 17

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基因名称	引物序列
β-actin	F:5′-TGGGACGACATGGAGAAA-3′
?	R:5′-GGGGTGTTGAAGGTCTCAAA-3′
RANKL	F:5′-GCCAAGATCTCCAACATGAC-3′
?	R:5′-GTGCTTCCTCCTTTCATCAG-3′
OPG	F:5′-CAACACAGCTCACAAGAACA-3′
?	R:5′-CCCACTTTCTTTCCCGGTAA-3′

基因名称	引物序列
β-actin	F:5′-TGGGACGACATGGAGAAA-3′
?	R:5′-GGGGTGTTGAAGGTCTCAAA-3′
RANKL	F:5′-GCCAAGATCTCCAACATGAC-3′
?	R:5′-GTGCTTCCTCCTTTCATCAG-3′
OPG	F:5′-CAACACAGCTCACAAGAACA-3′
?	R:5′-CCCACTTTCTTTCCCGGTAA-3′

组别	样本量	相对细胞活力
组别	样本量	24 h	48 h	72 h
5 ng/mL IL-22组	4	93.92 ± 2.51	96.45 ± 1.75	91.30 ± 3.68
10 ng/mL IL-22组	4	95.61 ± 3.63	96.47 ± 2.33	89.39 ± 5.73
25 ng/mL IL-22组	4	89.98 ± 2.75	92.38 ± 0.71	89.49 ± 6.78
50 ng/mL IL-22组	4	90.51 ± 3.24	80.51 ± 3.63	73.21 ± 7.05
100 ng/mL IL-22组	4	65.72 ± 4.53	47.25 ± 3.01	28.83 ± 4.82

组别	样本量	相对细胞活力
组别	样本量	24 h	48 h	72 h
5 ng/mL IL-22组	4	93.92 ± 2.51	96.45 ± 1.75	91.30 ± 3.68
10 ng/mL IL-22组	4	95.61 ± 3.63	96.47 ± 2.33	89.39 ± 5.73
25 ng/mL IL-22组	4	89.98 ± 2.75	92.38 ± 0.71	89.49 ± 6.78
50 ng/mL IL-22组	4	90.51 ± 3.24	80.51 ± 3.63	73.21 ± 7.05
100 ng/mL IL-22组	4	65.72 ± 4.53	47.25 ± 3.01	28.83 ± 4.82

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