Cucurbitacin B supplementation reduces inflammatory responses and alveolar bone loss via regulating MPO, COX-2 and RANK/RANKL/OPG signals in a rodent model of ligature-induced periodontitis

2.1 Animals

The Southern Medical University animal care and the ethical committee (Ethical approval number: TAMY05471) approved this research study, and all experimental and surgical protocols were conducted in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals [30]. Male Sprague-Dawley rats (n = 60; weighing 180–220 g and 7–8 weeks of age) from Guangdong Medical Laboratory Animal Co., China, were used in the study. The rats were held in sterile cages and were sustained on 12 h light/dark cycle with a relative humidity of 50–55% and room temperature at 22° ± 1 °C. Study animals were provided with ad libitum access to standard pellet diet and water. The animals were left to be tolerable and to adapt to the lab conditions for five days before initiation of the study.

2.2 Study design

The animals were divided into five experimental groups, with 12 animals per group. Using ligature method periodontitis was induced. The rats were anesthetized under ketamine (70 mg/kg), and xylazine (10 mg/kg) intramuscularly and a 4/0 silk suture (Teleflex Medical OEM, USA) was placed subgingivally positioned around the right mandibular first molars. The ligatures were observed daily to make sure of the subgingival position. The treatment group animals were administered Cucurbitacin B (12.5 mg/kg, 25 mg/kg, and 50 mg/kg) via oral gavage for 12 days starting 2 days prior ligature. The doses were chosen based on the previous results in our laboratory conducted with varying doses of cucurbitacin B (10 mg/ Kg b.wt to 2 g/Kg b.wt). The effects of 10–100 mg/Kg of cucurbitacin B were assessed for anti-inflammatory studies in our laboratory in other experimental models (data not included). The 3 doses of 12.5 mg/kg, 25 mg/kg, 50 mg/kg were found to exhibit anti-inflammatory effects and thus were chosen for the present study against periodontitis.

The control group rats were not induced nor received cucurbitacin B. Separate group of rats that were induced experimental periodontitis and not treated with cucurbitacin B served as an experimental control. The rats were sacrificed following 11 days after ligature under anesthesia and the mandibles were excised and split into two equal pieces in at the incisor teeth by dissection. The right piece of the mandible was removed and fixed in formalin for 24 h and used for analysis, and the alveolar bone loss in the first molars were determined histometrically. Periodontal tissues were excised and examined for histological changes, and the expressions of COX-2, RANKL/RANK, and OPG were determined. MPO activity in the tissues was assessed. The inflammatory mediators (1L-10, 1L-β, and TNF-α) levels were measured.

2.3 Histometrical analysis

The right mandible samples were fixed for 24–72 h in 10% formalin and were decalcified and then integrated in paraffin. Mesio-distal serial sections of 5 μm thickness were made and subjected to hematoxylin and eosin (HE) staining. First and last sections were excluded for observation, and three equally distant sections of each molar were taken. The distance between the alveolar bone crest and the cemento-enamel junction was measured too. Evaluation was done using an image analysis system connected to a light microscope (Olympus BX51, Tokyo, Japan) at 40x magnification. Six linear measurements were noted on the mesial and distal sides (three on each side), and the mean was calculated.

2.4 Histopathological analysis

The excised maxillae along with ligature were formalin (10%) fixed, demineralized in 10% formic acid, dehydrated and were paraffin embedded. The tissues were sectioned (4 μm) in a mesiodistal plane and were stained with HE and were observed and analyzed under light microscopy. The sections were taken from the area where the ligature was made between the first and second molars. Parameters as inflammatory cell influx, alveolar bone, and cementum integrity were determined. The level of inflammation was scored from 0 to 3, as previously standardized (Lima et al., 2000). Score 0 – absence of or inflammatory cell infiltration restricted to the marginal gingiva and preserved alveolar process and cementum; score 1- moderate cellular infiltration observed all over the insert gingiva and minor alveolar process resorption with intact cementum; score 2 – severe infiltration of inflammatory cells into the gingiva and periodontal ligament, degradation of the alveolar process, and partial destruction of cementum; score 3 indicates severe cellular infiltration with complete resorption of the alveolar process and intense destruction of cementum.

2.5 Immunohistochemical analysis

Immunohistochemical analysis was performed to evaluate the expressions of COX-2, RANKL, RANK, and OPG. 4 µm sections of the periodontal tissue were made and were transferred to slides that were coated with gelatin. The tissue sections were then deparaffinized, rehydrated, and washed with Triton X-100 (0.3%) in phosphate buffer. The sections were then incubated with hydrogen peroxide (3%) to reduce endogenous peroxidase reactions and then incubated overnight with specific primary antibodies at 4 °C for COX-2, RANK-L, RANK, and OPG (1:1000 dilution; Santa Cruz Biotechnology, CA, USA). The slides were washed with phosphate buffer and further incubated with secondary antibodies (streptavidin-HRP-conjugated; Biocare Medical, CA, USA) for 40 min at room temperature. Immunoreactivity was measured using TrekAvidin- HRP Label + Kit (Biocare Medical).

2.6 Determination of TNF-α, 1L-10, and IL-1β

The gingival tissues that were excised and stored at −70 °C were used for the assay. The tissues sections were processed as previously described (Safieh-Garabedian et al., 1995). The levels of TNF-α, IL-1β, and 1L-10 - the inflammatory mediators were assayed using commercially available ELISA kits from R&D Systems in accordance with the instructions provided.

2.7 Myeloperoxidase assay

The level of concentration of neutrophils into the tissues was determined by measuring myeloperoxidase (MPO) activity. The gingival samples (n = 6) from all the experimental groups were removed and stored at −70 °C. The tissues were homogenized and centrifuged (2000xg for 20 min). The supernatant was collected and assayed for MPO by the colorimetric method as described by Souza et al. (2003). The absorbance was measured at 450 nm using a Beckmann DU-65 spectrophotometer, and the results were presented as units of MPO/mg of tissue.

2.8 Statistical analysis

Statistical analysis of the obtained data was done using SPSS (Statistical Package for the Social Sciences) version 22.0 (SPSS, IBM, IL, USA). One-way analysis of variance was performed, followed by Duncan’s Multiple Range Test (DMRT) post hoc analysis for multiple group comparisons. Values at p < 0.05 were regarded statistically important.

3.1 Cucurbitacin B reduces alveolar bone loss

The histometrical analysis revealed alveolar bone loss in rats presenting periodontal disease (Fig. 1) while no bone loss was seen in control animals. The observation indicated that administration of cucurbitacin B at all the three tested doses (12.5 mg/kg, 25 mg/kg, and 50 mg/kg) effectively prevented alveolar bone loss. The highest tested dose of 50 mg was said to be the most efficient dose in reducing a bone loss compared to lower doses. The results indicate the efficacy of cucurbitacin B supplementation in preventing alveolar bone loss.

Close

Fig. 1

Cucurbitacin B reduced alveolar bone loss. Values are represented as mean ± SD, n = 6. * represents p < 0.05 vs. control; # represents p < 0.05 vs. Periodontitis control. a-e represents a significant difference (p < 0.05) between mean values of different experimental groups as determined by one-way ANOVA followed by DMRT analysis.

Export to PPT

3.2 Cucurbitacin B reduces inflammatory cell influx and improves histology

Histopathologic analysis of the excised maxillae from rats that were induced experimental periodontitis exhibited severe inflammatory cell infiltration, alveolar bone loss, severely damaged cementum, presenting scores between 2 and 3. The periodontium of rats administered with cucurbitacin B showed considerably preserved cementum, alveolar process, and reduced cellular influx (Fig. 2a and b). 50 mg/kg cucurbitacin B treated animals revealed almost near normal histology with negligible infiltration of inflammatory cells and well-preserved collagen and cementum.

Close

Fig. 2

Cucurbitacin B reduced inflammation following ligature-induced periodontitis (a) and histopathological analysis (b) Values are represented as mean ± SD, n = 6. * represents p < 0.05 vs. control; # represents p < 0.05 vs. Periodontitis control. a-e represents a significant difference (p < 0.05) between mean values of different experimental groups as determined by one-way ANOVA followed by DMRT analysis [a-Control; b-Periodontitis + Cucurbitacin B (50 mg/Kg)].

Export to PPT

3.3 Cucurbitacin B regulates the expressions of RANK/RANKL and OPG following induction of periodontitis

RANK and RANKL expression in the periodontal tissues of rats with ligature-induced periodontitis was elevated following when related to control group (Fig. 3a and b). OPG expression, however, was reduced (p < 0.05) to 63.11% in rats presenting periodontitis vs. control rats. Cucurbitacin B treatment significantly (p < 0.05) reduced RANK and RANKL expression while enhancing OPG levels to 94.01% with 50 mg/kg dose. The expression of RANK reduced from 169.10% to 103%, and RANKL levels reduced from 177.09% to 107.02% upon administration of 50 mg/kg cucurbitacin B.

Close

Fig. 3

Cucurbitacin B down-regulated RANK/RANKL and OPG expressions (a) Cucurbitacin B down-regulated RANK/RANKL and OPG expressions and (b) Immunohistochemical analysis Values are represented as mean ± SD, n = 6. * represents p < 0.05 vs. control; # represents p < 0.05 vs. Periodontitis control. a-e represents a significant difference (p < 0.05) between mean values of different experimental groups as determined by one-way ANOVA followed by DMRT analysis. [a-Control; b-Periodontitis + Cucurbitacin B (12.5 mg/Kg); c-Periodontitis + Cucurbitacin B (50 mg/Kg)].

Export to PPT

3.4 Cucurbitacin B reduces the levels of inflammatory mediators

Cytokines are well documented to play a crucial role in periodontitis. The levels of inflammatory cytokines (TNF-αand IL-1β) were found to be higher in animals with ligature-induced periodontitis vs. normal control (Fig. 4a). MPO activity was also observed to be markedly (p < 0.05) increased in ligature-induced rats in comparison to control (Fig. 4b). MPO activity reduced from 21.75 nmol/g tissue to 5.10 nmol/g tissue on treatment with 50 mg cucurbitacin B. However, the levels of IL-10 were observed to be slightly raised in animals that were induced periodontitis. The increase, however, was non-significant. The levels were brought down to almost near normal from 270 pg/mL to 265 pg/mL, 263 pg/mL and 259 pg/mL on treatment with 12.5 mg/kg, 25 mg/kg and 50 mg/kg respectively. Cucurbitacin B treatment at 12.5 mg, 25 mg/kg and 50 mg/kg doses were found to reduce the levels of TNF- α and IL-1β in the gingival tissues. Interestingly, IL-10 levels were found to be near normal and not much altered on cucurbitacin B administration.

Close

Fig. 4

Effect of Cucurbitacin B on inflammatory mediators (a) Cucurbitacin B reduced the levels of inflammatory cytokines-IL-10, IL-1β and TNF-α (b) MPO activity (c-d) Relative expressions of COX-2 expression (d) Immunohistochemical analysis were significantly downregulated by cucurbitacin B. Values are represented as mean ± SD, n = 6. * represents p < 0.05 vs. control; # represents p < 0.05 vs. Periodontitis control. a-e represents a significant difference (p < 0.05) between mean values of different experimental groups as determined by one-way ANOVA followed by DMRT analysis [a-Control; b-Periodontitis + Cucurbitacin B (12.5 mg/Kg); c-Periodontitis + Cucurbitacin B (50 mg/Kg)].

Export to PPT

Close

Fig. 4

Effect of Cucurbitacin B on inflammatory mediators (a) Cucurbitacin B reduced the levels of inflammatory cytokines-IL-10, IL-1β and TNF-α (b) MPO activity (c-d) Relative expressions of COX-2 expression (d) Immunohistochemical analysis were significantly downregulated by cucurbitacin B. Values are represented as mean ± SD, n = 6. * represents p < 0.05 vs. control; # represents p < 0.05 vs. Periodontitis control. a-e represents a significant difference (p < 0.05) between mean values of different experimental groups as determined by one-way ANOVA followed by DMRT analysis [a-Control; b-Periodontitis + Cucurbitacin B (12.5 mg/Kg); c-Periodontitis + Cucurbitacin B (50 mg/Kg)].

Export to PPT

Immunohistochemical analysis reveals a significant increase (p < 0.05) in the expression levels of the COX-2 enzyme in the animals following ligature (Fig. 4 c-d). The expressions were found to increase from 2.1 folds to 1.05 Cucurbitacin B administration prior and after ligature resulted in a significant reduction in tissue COX-2 level compared to ligature-induced animals. 50 mg/kg cucurbitacin B exhibited the highest anti-inflammatory effect as compared to were doses of 12.5/kg and 25 mg/kg.