Plumbagin suppresses chronic periodontitis in rats via down-regulation of TNF-α, IL-1β and IL-6 expression

Abstract

Chronic periodontitis (CP) is one of the most common oral diseases, which causes alveolar bone absorption and tooth loss in adults. In this study we aimed to investigate the potential of plumbagin (PL), a widely-investigated active compound extracted from the traditional Chinese herb Plumbago zeylanica L in treating CP. Human periodontal ligament stem cells (PDLSCs) were used for in vitro studies, whereas an animal model of CP was established in SD rats by ligation+Porphyromonas gingivalis (Pg) stimulation. The rats were injected with PL (2, 4, and 6 mg·kg^-1·d^-1, ip) for 4 weeks. Treatment of PDLSCs with TNF-α (10 ng/mL) markedly stimulated the expression of the proinflammatory cytokines TNF-α, IL-1β and IL-6, as well as the chemokines CCL-2 and CCL-5, which were dose-dependently suppressed by co-treatment with PL (1.25-5 μmol/L). Furthermore, PL (3.75 μmol/L) markedly suppressed TNF-α-induced activation of the MAPK, NF-κB and JAK/STAT signaling pathways in PDLSCs. In consistence with the in vitro studies, PL administration significantly decreased the expression of TNF-α, IL-1β and IL-6 in gingiva of the rat with CP, with the dosage 4 mg·kg^-1·d^-1 showing the best anti-inflammatory effect. Moreover, PL administration decelerated bone destruction in the rat with CP, evidenced by the aveolar bone loss (ABL) and H&E staining results. In conclusion, PL suppresses CP progression in rats by downregulating the expressions of TNF-α, IL-1β and IL-6 and inhibiting the MAPK, NF-κB and JAK/STAT signaling pathways.

Figure 1

Cell viability of PDLSCs after individual treatment with PL (A), OZ (B) and TNF-α (C) for 24 h and 48 h. (D) Chemical structural formula of PL, OZ and TNF-α. The data are presented as the mean±SD. All experiments were repeated at least three times. ^**P<0.01.

Figure 2

The effects of PL and OZ on the TNF-α-induced PDLSCs. (A) TNF-α, IL-1β and IL-6 mRNAs were subjected to real-time PCR analysis after 24 h. The expression levels were normalized to that of β-actin. (B) The levels of TNF-α, IL-1β and IL-6 in the medium were measured using an ELISA kit. (C) The expressions of CCL-5 and CCL-2 mRNA were measured by real-time RT-PCR. The data are presented as the mean±SD. ^*P<0.05, ^**P<0.01 vs the (TNF-α”+”, PL”0”, OZ”0”) group. All data were obtained from at least three independent experiments.

Figure 3

A schematic diagram of the proposed mechanisms on suppression effect of PL on chronic periodontitis via the inhibition of MAPK, NF-κB and JAK/STAT signaling pathways.

Figure 4

The effects of different duration of PL treatment on MAPK, NF-κB and JAK/STAT phosphorylation in TNF-α-induced PDLSCs. PL at a concentration of 3.75 μmol/L stimulated by 10 ng/mL TNF-α was cultured for 0, 1, 2, 4, 6 and 24 h. (A) The levels of p38, ERK, JNK, p65 and STAT3 and phosphorylated p38, ERK, JNK, p65 and STAT3 were examined in whole-cell lysates via Western blotting. (B) The relative expression levels of p-p38/p38, p-ERK/ERK, p-JNK/JNK, p-p65/p65 and p-STAT3/STAT3 were calculated based on the analysis of the gray band intensities. The data are presented as the mean±SD. ^*P<0.05, ^**P<0.01. All data were obtained from at least three independent experiments.

Figure 5

The effects of PL and OZ on MAPK, NF-κB and JAK/STAT phosphorylation in TNF-α-induced PDLSCs. The cells were treated with 10 ng/mL of TNF-α and different concentration of PL (0, 2.5, 3.75 and 5 μmol/L) and OZ (10 μg/mL) for 24 h. (A) The levels of P38, ERK, JNK, p65 and STAT3 and phosphorylated p38, ERK, JNK, p65 and STAT3 were examined in whole-cell lysates via Western blotting. (B) The relative expression levels of p-p38/p38, p-ERK/ERK, p-JNK/JNK, p-p65/p65 and p-STAT3/STAT3 were calculated based on the analysis of the gray band intensities. The data are presented as the mean±SD. ^*P<0.05, ^**P<0.01 vs the (TNF-α”+”, PL”0”, OZ”0”) group. All data were obtained from at least three independent experiments.

Figure 6

(A) Effect of PL on maxilla of cemento-enamel junction (CEJ) to alveolar bone crest (ABC). Buccal and palatal sides of the maxilla, where alveolar bone loss (ABL) was measured from the CEJ to the ABC at four points: mesiolingual (ML), mesiobuccal (MB), distolingual (DL), and distobuccal (DB) regions for first maxillary molar (M1) and second maxillary molar (M2). The overall result was also calculated. (B) Analysis of micro-CT volumetric parameters: bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). (C) Descriptive analysis of micro-CT and H&E staining. The H&E staining images show sagittal sections of each group at 40× magnification. Values are expressed as the mean±SD. ^*P<0.05, ^**P<0.01 vs the (Ligation+Pg”+”, PL”0”) group.

Figure 7

TNF-α, IL-1β and IL-6 expression levels in SD rat model. (A) Real-time PCR analysis was done on SD rat gingiva. The expression levels were normalized to that of β-actin. The data are presented as the mean±SD. ^*P<0.05, ^**P<0.01. (B) Immunohistochemical staining images on periodontal ligament sites at 400× magnification. (C) Immunohistochemical staining images on alveolar bone sites at 400× magnification. Arrows represent the immunoreactive cells.