Therapeutic potential of PPARα agonist in ligature-induced experimental periodontitis

Abstract Inflammation-related immune responses and bone metabolism lead to extensive tooth loss in periodontitis. Objective: This study aims to investigate the effect of peroxisome proliferator-activated receptor (PPAR) alpha agonist anti-inflammatory treatment in vitro and in ligature-induced experimental periodontitis in vivo . Methodology: Splenocytes were isolated from C57BL/6J mice and cultured for 48 hours under the following conditions: control, P. gingivalis lipopolysaccharide (LPS) (1 µg/ml); experimental, LPS (1 µg/ml) + PPARα agonist (fenofibrate) at 1, 10, 50, 100 µM. MRNA and secreted protein levels of TNF-α expression were detected by RT-qPCR and ELISA, respectively. Silk ligatures (7-0) were tied around maxillary second molars of C57BL/6J mice for two weeks. Optimized doses of fenofibrate (50 µM) and vehicle control were injected into the contralateral side of the palatal gingiva on days three, six, and nine. At day 14, bone resorption, osteoclastogenesis, and gingival mRNA expression levels of TNF-α, IL-1β, IL-6, and RANKL/OPG were measured by micro-computed tomography, Tartrate-resistant acid phosphatase (TRAP) staining, and Real-time quantitative PCR, respectively. Results: TNF-α expression in cultured spleen cells were significantly increased in the presence of LPS, when compared with the control group, and significantly reduced by fenofibrate treatment in a dose-dependent manner from 1-100 µM (p<0.05). Gingival mRNA levels of TNF-α, IL-1β, IL-6, and the ratio of RANKL/OPG, were significantly decreased after injection of fenofibrate, when compared to the control side (p<0.05). Periodontal bone loss and TRAP positive cell formation were significantly decreased on the side with an injection of fenofibrate, as compared to the control side (p<0.05). Conclusions: An anti-inflammatory treatment, PPARα agonist, inhibited inflammation and periodontal bone loss in ligature-induced experimental periodontitis.


Introduction
The periodontium consists of the gingiva, periodontal ligament, cementum, and the alveolar bone proper.
These teeth-surrounding tissues support teeth in the maxillary and mandibular bones and maintain teeth functioning. 1 Periodontal disease is a progressive inflammatory process affecting teeth-surrounding tissues, destroying bone-supporting tissues, inducing bone resorption, and constituting the leading cause of bone loss in teeth. 2 A various range of inflammatory cytokines and signaling pathways have been shown to mediate the pathological process of periodontitis. 2 Among these, inflammatory factors, tumor necrosis factor-alpha (TNF-α), and interleukin-I (IL-1) play the causative pathological roles of destroying periodontal tissues. 3 The excessive production of IL-1 and TNF is believed to be an overreaction of the host's immune response to periodontal pathogens. 4 IL-1 induces adhesion molecules and a number of inflammatory factors to attack the periodontal tissues, resulting in the loss of connective tissue attachment, osteoclast formation, bone resorption, and loss of alveolar bone. 5 On the other hand, TNF-α mediates the loss of fibroblasts in the pathogenic process of periodontal infections. [5][6][7] Antagonizing IL-1 and TNF-α in experimental periodontitis has shown the beneficial effects of inhibiting the activity of pro-inflammatory cytokines and the further spread of the inflammation in the periodontal tissues. 7 PPARs are members of the nuclear hormone receptor and function as transcription factors regulating the expression of genes in the metabolism and inflammation. 8 Binding with selective ligands activates PPAR and results in heterodimerization with the retinoid X receptor (RXR), which regulates gene expression 8 . A previous study has shown that PPARα activation upregulates the overexpression of the IL-1 receptor antagonist (IL-1ra). 9 It has also been shown that activated PPARα binds to c-Jun and to the p65 subunit of NF-κB, 10 and that PPARα expression is higher in periodontitis and peri-implantitis groups than in healthy patients, whereas RXR shows a reverse pattern to PPARα, higher in healthy individuals than in periodontitis and periimplantitis patients. 14 In this study, we evaluate the anti-inflammatory effect of fenofibrate, a PPARα agonist, in an experimental ligature-induced periodontitis model. Our study focuses on key inflammation events, including the effect of fenofibrate on the production of IL-1 and TNF-α, and bone loss.

Methodology
Animal: Wild-type (WT) C57BL/6 mice (Jackson Laboratory) aged eight to 10 weeks were used for experiments. Half of the mice used were males and half, females. A total of 24 mice (six groups, four per group) was used in an in vitro study and a total of 24 mice (two groups, 12 per group) was used in an in vivo study. The animal experiments were approved by the Institutional Animal Care and Use Committee.
Cell preparation and culture: Mice spleens were collected and carefully ground into an IMDM medium (Gibco) through a 2 cm X 2 cm metal mesh. After going through a 100 μm cell strainer, the ACK lysis buffer (Life Technologies) was applied to lysis erythrocytes. After, a 40-μm cell strainer was used. was used to analyze these skulls. The Seg3D software was used to establish the quantitative 3-D images, followed by measurements of bone resorption volume, as previously described, 15 with blinding of groups.
Briefly, the volume of interest (VOI) of a cylinder with a diameter of 1.0 mm and a height of 1.0 mm was defined from the cement-enamel junction plane. The empty space volume (ESV) surrounding the teeth was measured by the total VOI volume minus bone volume.
Then, the bone loss of each sample was calculated by its ESV minus the average ESV in the no ligature control group.
Tissue histological analysis: The maxillary part of skulls was harvested and put in 4% formaldehyde for fixation. Then, skulls were decalcified in 10% EDTA for two weeks at 4ºC, with sloshing, and embedded in paraffin. Five μm tissue sections were cut in parallel with the long axis of the molars. These sections were stained with an acid phosphatase kit (378A, Sigma) and counterstained with hematoxylin for tartrateresistant acid phosphatase (TRAP) analysis. The number of multinucleated TRAP-positive cells which were considered osteoclasts were counted (n=6 animals/group) as previously described. 15

Statistical analysis
Quantitative data were expressed as means ± SD.
Statistical analysis was performed using unpaired Student's t-test for comparisons of any two groups of data sets. Statistical significance was set at p<0.05.

Results
Effects of PPARα agonist treatment on TNF-α mRNA level of splenocytes induced by LPS.
TNF-α is a proinflammatory cytokine released by macrophages which plays a substantial pathologic role in periodontitis. 3 TNF-α specially mediates the loss of fibroblasts and is associated with bone loss in periodontitis. We, therefore, tested the ability of fenofibrate to reduce the production of TNF-α. The splenocytes collected from mice were incubated with lipopolysaccharide (LPS) (10 ng/ml), one of the most potent stimuli for macrophages to produce large quantities of proinflammatory cytokines, including TNF-α. As Figure 2 shows, LPS induced a four-fold increase in the expression of TNF-α at a transcriptional level, which is significantly reduced by fenofibrate, in a dose-dependent manner, from 1 to 100 µM.
Effects of PPARα agonist treatment on TNF-α protein expression level of splenocytes induced by LPS. PPARα agonist inhibited bone loss in a ligatureinduced experimental periodontitis mouse model.
To explore the protective potential of fenofibrate on periodontitis in vivo, we first tested the effect of fenofibrate on bone loss in a ligature-induced experimental periodontitis mouse model ( Figure   4A). We observed that the application of ligatures

Discussion
In our ligature-induced experimental periodontitis model, the levels of TNF-α and IL-1 were elevated, which is in line with the findings in patients with periodontal diseases. 5 In patients with chronic periodontitis, aggressive periodontitis, and periimplantitis, the levels of TNF-α and IL-1 were elevated and contributed to patients' susceptibility to the disease. 16- 18 Graves' study showed that, when TNF-α and IL-1 were antagonized with antibodies, a causeand-effect relationship between inflammation degree and periodontitis severity was verified. 5 Our study showed that increases in TNF-α, IL-1, Fenofibrate is an agonist of PPARα, a nuclear transcriptional factor. 8 Schaefer's study showed that LPS increases TNF-α in both wild-type and PPARα-null mice. Treatment with PPARα agonist reduces TNF-α in wild-type mice but not in PPARα-knockout mice, 19 suggesting that PPARα is essential for fenofibrate lowering TNF-α. Mouse splenocytes were separated from C57/BL6 wild-type (WT) mice aged eight to 10 weeks and treated with LPS (1 µg/ml), LPS (1 µg/ ml) + Fenofibrate (1 μM, 10 μM, 50 μM, and 100 μM) for 48 hours. The protein expression levels of TNF-α were measured by ELISA kits and analyzed. (mean ± SD, n=4, compared with LPS group, *p<0.05, **p<0.01, N.S., no significance) suggesting that PPARα is a potentially therapeutic target for periodontitis. In this study, we have been unable to determine how TNF-α and IL-6 are regulated Silk ligatures were tied around maxillary second molars on both sides of C57/BL6 mice mouths on day 0; fenofibrate (50 µM, 2 µl) or Vehicle (PBS, 2 µl) was injected on days three, six, and nine (A). Maxilla were collected on day 14, measured by 3D micro-CT (B), and analyzed as bone resorption (volume/mm3¬) (C). (mean ± SD, n=6 mice per group, **p<0.01).  Ligature duration (14 days), on our experimental periodontitis mice model, is one of the limitations of our study. Although our previous studies showed that 14-day ligatures were enough to generate significant bone loss and changes in inflammatory cytokines, 15,30 it is very important for us to study these changes in shorter (7 days Inflammation plays a crucial pathogenic role in the process of periodontitis. This study, investigating the effect of fenofibrate, a PPARα agonist, on inflammatory response and bone protection in a ligature-induced experimental periodontitis model, aimed to explore a new therapeutic target as an alternative strategy for periodontitis. In summary, our study has provided Silk ligatures were tied around maxillary second molars of both sides of C57/BL6 mice mouths on day 0; fenofibrate (50 µM, 2 µl) or Vehicle (PBS, 2 µl) was injected on days three, six, and nine. Gingival tissue mRNA levels of TNF-α, IL-1, IL-6, and ratio of RANKL/OPG were measured and analyzed. (mean ± SD, n=6, *p<0.05, **p<0.01). first-hand evidence that fenofibrate has a potentially therapeutic benefit for treating periodontitis.