Roles for B[a]P and FICZ in subchondral bone metabolism and experimental temporomandibular joint osteoarthritis via the AhR/Cyp1a1 signaling axis

Bone loss due to smoking represents a major risk factor for fractures and bone osteoporosis. Signaling through the aryl hydrocarbon receptor (AhR) and its ligands contributes to both bone homeostasis and inflammatory diseases. It remains unclear whether the same AhR signaling axis affects the temporomandibular joint (TMJ). The aim of this study was to investigate possible mechanisms which mediate bone loss in the TMJ due to smoking. In particular, whether benzo[a]pyrene (B[a]P), a carcinogen of tobacco smoke, induces expression of the AhR target gene, Cyp1a1, in mandibular condyles. Possible functions of an endogenous ligand of FICZ, were also investigated in a TMJ-osteoarthritis (OA) mouse model. B[a]P was administered orally to wild-type and AhR−/− mice and bone metabolism was subsequently examined. TMJ-OA was induced in wild-type mice with forceful opening of the mouth. Therapeutic functions of FICZ were detected with μCT and histology. Exposure to B[a]P accelerated bone loss in the mandibular subchondral bone. This bone loss manifested with osteoclastic bone resorption and upregulated expression of Cyp1a1 in an AhR-dependent manner. In a mouse model of TMJ-OA, FICZ exhibited a dose-dependent rescue of mandibular subchondral bone loss by repressing osteoclast activity. Meanwhile, in vitro, pre-treatment with FICZ reduced RANKL-mediated osteoclastogenesis. B[a]P regulates mandibular subchondral bone metabolism via the Cyp1a1. The AhR ligand, FICZ, can prevent TMJ-OA by regulating osteoclast differentiation.

A long-term smoking habit can have many consequences, one is the development of osteoporosis. This condition is recognized as a critical determinant of fracture risk. For those with a history of smoking, there is more than 85% high risk factor of hip-fracture and up to a 40% higher overall risk factor of fracture 1,2 . The mechanistic details mediating these effects are unknown, especially in relation to metabolism of the mandibular subchondral bone. Subchondral bone and articular cartilage act as a unit to maintain the structural and functional integrity of joint 3,4 . Subchondral bone provides the mechanical support and nutrition supply for overlying articular cartilage 5 . The presence of more than 4000 compounds in cigarette smoke also represents a daunting number of candidates to consider.
Signaling through the aryl hydrocarbon receptor (AhR) and its ligands has been shown to contribute to both bone homeostasis and inflammatory diseases. The effects of halogenated and polycyclic aromatic hydrocarbons (PAH) in the environment, including benzo[a]pyrene (B[a]P) containing in cigarette smoke, are also mediated through the AhR. It has been established that binding of agonist B[a]P to the receptor AhR induces translocation of the complex to the nucleus where it forms a heterodimer with AhR nuclear translocator (ARNT). As a Treatments and TMJ-OA model. Both WT and AhR −/− mice were administered B[a]P (120 mg/kg; Tokyo Chemical Industry, Tokyo, Japan) or corn oil (control) daily via oral gavage in six doses for 6 days.
WT mice were subjected to forced mouth opening-induced TMJ-OA 15 and received a tail vein injection of FICZ twice a week. Two different concentrations of FICZ were administered (FICZ low: 100 μg/kg, FICZ high: 100 mg/kg). FICZ (Focus Biomolecules #10-1463, Plymouth Meeting, PA, USA) was dissolved in dimethyl sulphoxide (DMSO) (0.5 mg/mL) for these injections. Experimental TMJ-OA was established according to a forced mouth opening method as previously described 15 . In brief following an intraperitoneal injection of 50 mg/kg somnopentyl, adverse mechanical stress was applied to the TMJ of mice with a consistent and repetitive mouthopening protocol. A custom-made spring was used to deliver a force of 2N at maximal mouth opening (measured to be 14 mm, passively, in 8-week-old C57BL/6 WT mice). The TMJ of the mice in the loaded group was subjected to mechanical loading by forceful opening of the mouth for 3 h/days for 5 days. Individual spring forces were measured with a mechanical test system (autograph AG-X 1 kN, SHIMADZU, Kyoto, Japan). This method was approved by the Animal Care and Ethical & Use Committee of Okayama University (Permit No. OKU-2020861).

Isolation of macrophage and primary mandibular chondrocytes. Bone marrow macrophages
(BMMs) were differentiated into multinucleated mature osteoclasts as previously described 20 . Briefly, isolated BMMs were cultured with macrophage colony stimulating factor (M-CSF, 20 ng/mL) and receptor activator of nuclear factor kappa-B ligand (RANKL, 100 ng/mL) for 6 days. TRAP activity was subsequently detected with staining (Kit 387-A; Sigma-Aldrich, St. Louis, MO, USA). For applications in vitro, FICZ was dissolved in DMSO and prepared at concentrations of 10, 100, or 200 ng/mL. Primary mandibular chondrocyte cells were isolated from the condyles according to a previously published procedure 10 . In brief, TMJ condylar cartilage tissues were dissected from 6 to 10-week-old mice. Pieces of cartilage were minced with a scalpel and then were digested with 3 mg/mL collagenase (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 4 mg/mL dispase (Gibco, Grand Island, NY) in PBS at 37 ℃ with shaking. After 3 h, enzymatic digestion was stopped. The resulting cell suspension was filtered through a nylon mesh (70-μm pore size; BD Falcon, Franklin Lakes, NJ) to eliminate cell-matrix residues, then was centrifuged for 10 min at 250×g. The chondrocytes obtained were washed thrice with α-MEM and cultured in 5% CO 2 at 37 ℃ in basal medium that consisted of α-MEM supplemented with 20% FBS, 2 nmol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco), and 100 mmol/L 2-mercaptoethanol (Gibco). After 4-6 days, the adherent cells were detached with trypsin-EDTA (Gibco) and were passaged.
A mouse chondroprogenitor cell line, ATDC5 (RIKEN BioResource Center Cell Bank, Tsukuba, Japan), was cultured as a monolayer in high-glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich) with 5% FBS. www.nature.com/scientificreports/ μ-Computed tomography (μCT). Mandibular condyles were resected from wild-type (WT) and AhR −/− mice treated with B[a]P. After soft tissues were removed, the bones were incubated in 70% ethanol overnight. Image acquisition was performed the next day with a high-resolution microcomputed tomography scanner (SkyScan 1176 Scanner; Bruker, Billerica, MA, USA) at 200 μA and 50 kV. To prevent dehydration and movement, samples were tightly covered with plastic wrap during image acquisition. Thresholding was applied to distinguish background noise from bone images. A posterior region in the midsagittal section of the mandibular condyle was established as the region of interest (ROI). At a resolution of 9 μm/pixel, microstructural parameters of each ROI were analyzed. These parameters included: trabecular separation (Tb.Sp), trabecular thickness (Tb. Th), and bone volume to trabecular bone volume ratio (BV/TV).
Tissue preparation, histologic staining, and analysis. Resected TMJ tissues were fixed in freshly prepared 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 2 days and decalcified in the EDTA for 20 days and then embedded into paraffin. After preparing serial sagittal sections using a microtome (Carl Zeiss HM360, Jena, Germany), sections were stained with TRAP in order to detect multinucleated-osteoclasts, while staining with Hematoxylin-Eosin (HE) and Safranin-O provided scoring for features of cartilage disease (e.g., structural abnormalities and changes in cellularity). Glycosaminoglycan loss and distribution were also examined. In brief, area of proteoglycan staining of the cartilage of the central and posterior thirds of the mandibular condyles were quantified using Image J software (NIH, Bethesda, MD, USA) 21  Detection of apoptotic cell death by TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining. A TUNEL method was used to assay the distribution of apoptotic chondrocyte cells.
After the 3′-hydroxyl terminus of DNA strand breaks were labeled, apoptotic cells were detected using an In Situ Apotosis Detection Kit (FUJIFILM Wako Pure Chemical), according to the attached company's protocol. Microscopy was used to observe TUNEL + apoptotic cells (KEYENCE).

Isolation of RNA.
Frozen condyles were homogenized with Precellys Evolution lysis and homogenization system (Bertin Instruments, Montigny-le-Bretonneux, France). A PureLink RNA isolation mini kit (Thermo Fisher Scientific, Waltham, MA, USA) was subsequently used in order to isolate mRNA, followed by the manufacturer's instructions. The NanoDrop One spectrophotometer (Thermo Fisher Scientific) was utilized to estimate RNA concentrations.
Quantitative PCR (q-PCR). Total RNA was extracted using PureLink spin column kit (Thermo Fisher Scientific), and a ReverTra Ace qPCR RT kits (TOYOBO, Osaka, Japan) were used to reverse transcribe mRNA into cDNA, according to the company's instructions. Each cDNA samples were then prepared in a 10-μl total volume qPCR reaction containing real-time PCR Master Mix SYBR Green (TOYOBO) and 1 μM primer pairs. Levels of Trap, Cathepsin K, Integrin β 3 , Cyp1a1, aggrecan, Col2a1, and Sox9 mRNA were analyzed with a LightCycler 96 System (Roche Diagnostic, Mannheim, Germany). These levels were corrected by the glyceraldehyde 3-phosphate dehydrogenase, GAPDH, as an endogenous control and then evaluated according to the comparative cycle threshold method (ΔΔCt). The primer sequences used in amplification are shown in Supplemental Table S1. www.nature.com/scientificreports/

Staining of actin ring and bone resorption pits.
BMMs were plated on dentin slices and cultured with M-CSF and RANKL for 6 days to generate mature osteoclasts. Dentin slices were fixed with 4% PFA in PBS for 20 min and permeabilized in 0.1% Triton X-100. F-actin was stained with Alexa Fluor 488-phalloidin. The number of osteoclasts with actin rings were counted per dentin slice under a conventional microscope equipped with a charge-coupled device camera (Olympus, Tokyo, Japan). The cells were then removed from dentin slices with mechanical agitation. Dentin slices were incubated with peroxidase-conjugated wheat germ agglutinin (Sigma Aldrich) for 1 h and stained with 3,3′-diaminobenzidine (Sigma Aldrich) for 30 min. Bone resorption pits were analyzed using a light microscope (KEYENCE) and pits area per field were quantified using Image J software (NIH) 21 .
Analysis of osteoblast mineralization and differentiation. MC3T3 E1 cells were grown up to 21 days in osteogenic media including 20 mM β-glycerophosphates and 50 mM ascorbic acids. After fixation cells were appropriately stained using 0.2% alizarin-red S and alkaline phosphatase staining kit (AK20; Cosmo Bio, Sapporo, Japan). Osteoblastic marker genes such as Alpl, Osteocalcin, and Col1a1 were detected by qPCR. Osteoblastic activities were analyzed in tetracycline and calcein double-labeled sections in vivo.

Statistical analysis.
Each experiment was independently repeated more than three times; and for the all sets of each condition, experiments were performed at least in duplicate or triplicate. Statistical data analyses were respectively performed by applying one-way analysis of variance (ANOVA) accompanied with post-hoc Tukey's honest significant differences test or Student t-test, as appropriate for each case. p values less than 0.01 or 0.05 were considered as statistically significant.

AhR ligand B[a]P increases osteoclastic activity.
To examine whether osteoclastogenic activity is affected by B[a]P in vivo, TRAP staining was performed. Compared with corn oil-treated control WT mice, osteoclast activity was significantly accelerated in B[a]P-administered WT mice ( Fig. 2A,B). It was previously demonstrated that the femoral metaphysial trabecular bone of AhR −/− mice exhibited an increased bone mass compared with WT controls 23 . Consistent with this report, we also observed an increase in bone mass in the mandibular condylar subchondral regions in AhR −/− mice. Meanwhile, TRAP activity in the same bone was

Cyp1a1 and caspase expression is necessary for B[a]P-induced mandibular bone resorption.
A number of genes which regulate metabolism are activated by the AhR, including genes encoding Cyp1 enzymes. In B[a]P-administered WT mice, higher levels of Cyp1a1 mRNA and Cyp1a1 protein were detected (Fig. 3A,B). Immunohistochemical analysis further showed that B[a]P treatment of AhR −/− mice did not affect condyle expression of Cyp1a1 (Fig. 3C). Thus, exposure to Ahr agonist B[a]P only significantly increased condyle levels of Cyp1a1, AhR and caspase-3 in WT mice (Fig. 3C). The active forms of caspase-3 and caspase-9 were found to be markedly upregulated after B[a]P treatment in chondrocyte progenitor cell line ATDC5 by immune blot analysis in a time-dependent manner (Fig. 3D).  www.nature.com/scientificreports/ In control mice treated with corn oil, an abundance of proteoglycans was observed in the hypertrophic layer of the extracellular matrix (Fig. 4A,B). In contrast, the extent of proteoglycan staining in cartilage from B[a]P-treated WT mice was reduced. Moreover, fibrocartilage at the surface layer of the mandibular condylar cartilage was www.nature.com/scientificreports/ intact despite degradation of the mandibular condylar cartilage (Fig. 4A,B). For the B[a]P-treated AhR −/− samples, Safranin O staining showed no obvious histomorphological differences (Fig. 4A,B). Meanwhile, IHC assays of articular cartilage resected from B[a]P-administered WT mice showed loss of aggrecan, Col2a1 and Sox9 protein expression. In AhR −/− mice, there were no obvious histomorphological changes in articular cartilage after B[a]P treatment (Fig. 4A,C-E). Assays of mRNA levels of aggrecan, Col2a1, and Sox 9 were also lower in mandibular condyles of B[a]P-administered WT mice (Fig. 4F-H). In contrast, these levels were unaffected in mandibular condyles of the AhR −/− mice (Fig. 4F-H).

Protective role of FICZ on mandibular cartilage damage and subchondral bone loss in experimental murine TMJ-OA.
To establish a murine model of TMJ-OA, mechanical stress were applied to the TMJs of WT C57BL/6 mice. Briefly, a jaw maximal opening device was loaded to the interincisal-teeth holding the mandible in a maximal open posture (Fig. 5A) 15 . The TMJ-OA model was established after severe destruction of the subchondral bone was achieved. The mice then received an intravenous injection of FICZ dissolved in DMSO, or DMSO alone, twice a week (Fig. 5B). When three-dimensional (3D) reconstructions were generated of the subchondral bones in both treatment groups, severe destruction was observed in the TMJ-OA mice receiving DMSO alone (Fig. 5C-F). In contrast, destruction was prevented in a dose-dependent manner with FICZ administration (Fig. 5C-F). When TMJ sections from control WT mice were stained with HE, the articular cartilage exhibited a smooth surface and normal cellularity. In contrast, staining of the joints from the TMJ-OA mice revealed OA-like degenerated lesions, including irregularities of chondrocyte alignment in the condylar cartilage layers and subchondral bone loss. However, these lesions were attenuated in a FICZ dose-dependent www.nature.com/scientificreports/ manner (Fig. 6A). TRAP staining showed osteoclasts around the subchondral bone in the vehicle-treated TMJ-OA group. Meanwhile, the reduction in the numbers/activities of osteoclasts was observed a dose-dependently in two FICZ treatment group (Fig. 6B). When expression of Cyp1a1 was detected, the level was significantly higher in mandibular condyles from the vehicle-treated TMJ-OA group, while lower levels Cyp1a1 were observed dose dependently in the mandibular condyles from both groups of FICZ-treated TMJ-OA mice (Fig. 6C). To analyze whether normal or abnormal chondrocytes undergoing apoptotic cells were induced in degraded zones such as cartilage and subchondral bone layer, TUNEL staining was performed. Significant increase in the numbers of TUNEL + cell were detected in the vehicle-treated TMJ-OA group compared to fewer TUNEL + cells in the FICZ-treated TMJ-OA groups. The latter was associated with a protective effect against osteoclast-mediated subchondral bone resorption and cartilage degradation (Fig. 6D-J). Further characterization of condyles from the FICZ-treated TMJ-OA mice showed that mRNA expression levels of Cathepsin K, Trap, and Integrin β 3 were markedly downregulated, as well as Cyp1a1 expression (Fig. 6K-N). To further analyze and confirm the accelerated apoptosis that were observed in some layers of mandibular condyles in TMJ-OA, cleavage of caspase-3 was analyzed after the FICZ treatment. FICZ treatment dramatically repress the cleaved caspase-3 expression in the isolated mandibular chondrocytes in a dose dependent manner while active form of caspase-3 was upregulated in the vehicle-treated TMJ-OA group (Fig. 6O).

FICZ disrupts actin rings to inhibit bone-resorption activity of osteoclasts. To further investigate
the effect of the relationship between FICZ and AhR axis on osteoclasts, WT BMMs were treated with FICZ, an endogenous AhR ligand. Formation of TRAP + cells in cultures of WT BMMs was attenuated in a FICZ dosedependent manner (Fig. 7A,B). In contrast, treatment with RANKL dramatically increased Cyp1a1 expression in DMSO-treated WT BMMs, yet not in FICZ-treated cells (Fig. 7C). Protein levels of Cyp1a1, Cathepsin K, Integrin β 3 , and NFATc1 expressions were also decreased following FICZ treatment by immunoblot analysis (Fig. 7D). Cytoskeletal reorganizations, representatively actin ring formation, is closely related to the boneresorbing activities of activated osteoclasts 24 . When RANKL-stimulated pits-formation assays were performed, a dose-dependent effect of FICZ treatment was observed. For example, the bone slices-resorbing activities of activated osteoclasts was in part inhibited in 100 ng/mL FICZ, or was nearly inhibited in 200 ng/mL FICZ (Fig. 7E,F). Correspondingly, actin rings were essentially disrupted within 10 h of FICZ addition (Fig. 7G,H). Taken together, these results might indicate that FICZ-mediated suppression of the osteoclastic bone resorption activity may involve disruption of actin rings. Fig. S1A,B). Furthermore, the key osteobastic gene expressions of Alpl, Osteocalcin, and Col1a1 mRNAs were upregulated in the FICZ-stimulated MC3T3 E1 cells (Supplemental Fig. S1C). These results are largely supported by the findings of in vivo imaging analyses of the bones and minerals apposition rates determined by tetracyclines and calcein-double fluorescence labelling, which also showed significant acceleration in minerals apposition or bone formations rate in FICZ-treated mice (Supplemental Fig. S1D-F).

Discussion
Skeletal healing and bone mass in individuals with a history of smoking have been found to be adversely compromised, independent of gender. In particular, nicotine has been found to inhibit distraction osteogenesis 25 , spinal fusion 26 , and fracture healing in rabbits 27 . Traditionally, smoke-induced bone fractures or osteoporosis have been attributed to impaired the osteoblastic bones and minerals formation. However, conflicting reports regarding the role of tobacco-smoke carcinogens on bone resorption have been published 28,29 . Dioxins are a component of cigarette smoke and exhibit a capacity to inhibit osteoblast formation and differentiation [30][31][32] . Moreover, it has been reported that the low dose of the AhR agonist concentrations such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or B[a]P accelerates osteoclastogenesis and bone mineral resorptions, while low rates of osteoclastogenesis are observed in AhR −/− mice 23 . In our own recent study, we demonstrated that the AhR-c-Fos signaling axis in osteoclasts helps regulate fracture healing 8 . These findings are consistent with the hyperresorption that is observed in smokers 28,29 , and the ~ 60% increase in bone resorption that characterizes mice overexpressing constitutively activated AhR 33 . In mice exposed to smoke, delays in chondrogenesis have been associated with tibial fractures 34 . Consequently, we hypothesize that B[a]P, one of the major components of cigarette-smoke, impairs bone metabolism in mandibular condyles via AhR ligands. It has been reported that AhR ligands mediate toxic effects on various types of tissues, including hard palate tissue and tooth 35,36 . However, the effects of the AhR pathway on mandibular condyle tissue have remained unclear. In particular, the effects of B[a]P and FICZ on mandibular bone metabolism or TMJ-OA have not been investigated. Therefore, to the best of our knowledge, our present research is the first and novel to report B[a]P-mediated effects on mandibular subchondral bone, which is a site of tissue healing distinct from the lung. To evaluate biological effects of cigarette smoke on cultured cells, B[a]P containing in cigarette smoke have been generally used. However, use of B[a]P or smoke extracts may not completely mimic in vivo smoking situations. Overall, we observed that the AhR-targeted gene, Cyp1a1, is upregulated in those from mandibles of mice following exposure to B[a]P, which implies those activation in AhR signaling may be involved. Previously, it was demonstrated that α-naphthoflavone inhibits Cyp1a1 activity and significantly reduces B[a]P-induced apoptosis 37 . In contrast, 2,3′,4,5′-tetramethoxystilbene and pyrene, specific inhibitors of Cyp1b1, do not suppress B[a]P-induced apoptosis. Kim et al. also reported that Cyp1a1, and not Cyp1b1, is involved in mediating B[a]P-induced activation of cleaved caspase-3 in human RL95-2 endometrial cancer cells and Hepa1c1c7 cells 38 . In our present studies, a significant increase of Cyp1a1 mRNA was detected in the mandibular www.nature.com/scientificreports/  www.nature.com/scientificreports/ dependent. Correspondingly, several studies have demonstrated that inhibitory crosstalk takes place between these two signaling pathways [42][43][44] . Thus, it is possible that this same cross talk affects osteoclast and chondrocyte differentiation as well. Nicotine, an exogenous stimulator of the cholinergic system, is also important component of cigarette smoke. Several studies reported the role of cholinergic system in the bone homeostasis in the context of osteoporosis and osteoarthritis 45,46 . Acetylcholine or exogenous activation via nicotine or muscarine, stimulates osteoblast proliferation and osteoclast apoptosis 45 . Acetylcholinesterase (AChE), an enzyme catalyzing the degradation of acetylcholine, plays an important role in the cholinergic regulation by terminating the action of acetylcholine. It has recently been reported that the expression of AChE can also be found in non-neuronal tissues like the immune system and bone tissue 46 . After induction of bone loss in mice using RANKL, the AChE inhibitor donepezil could resucue the RANKL-induced bone loss. During osteoclastogenesis of bone marrow macrophages an upregulation of the AChE expression has been observed. Moreover, genetic knockdown of AChE via siRNA suppressed RANKL-induced osteoclast differentiation 46,47 . These results suggest that the signaling interaction between AhR and AChE might be a potential therapeutic target for bone disease like osteoporosis and TMJ-OA. The TMJ-OA experimental model that was established in the present study with mechanical stress applied to the mandibular condyle is consistent with that used in other studies 15,[48][49][50][51][52] . This model is characterized by chondrocyte alignment irregularities in the layers of mandibular condylar cartilage, marked depletion of proteoglycans, and subchondral bone destructions, and OA-like degenerated lesions. It has been recognized that OA is not only a problem of cartilage damage but also a whole joint disorder 3,4 . In particular, articular cartilage and subchondral bone function as a unit to maintain the structural and functional integrity of the joint 4,9 . The animal models to recapitulate the disturbance of subchondral bone as well as cartilage degeneration are needed to better understand the cartilage bone unit in the pathogenesis of TMJ-OA. The results obtained in the present study with this model are also consistent with those reported for models of early TMJ-OA induced with surgical joint manipulations 53,54 , local deliveries or applications of chemicals 55,56 , and gene deletions or modification 57,58 . The origin of TMJ-OA is primarily non inflammatory, unlike synovitis and rheumatoid arthritis. The pathology of TMJ-OA involves abrasion and deterioration of the mandibular articular cartilage, as well as local turnover and thickening of the underlying bone remodeling. Moreover, those changes are often involved in secondary inflammatory reactions. In the TMJ, excessive mechanical stress is one of the key factors in the induction of degradating cartilages in the mandibular condylar. Consequently, use of forced mouth opening is successful in establishing an experimental TMJ-OA model mouse to evaluate the initialization and progression toward TMJ-OA.
In these current studies, administration of the AhR endogenous ligand, FICZ, significantly attenuated bone loss in the mandibular subchondral bone and the inflammatory response in our TMJ-OA model. Subchondral bone remodelling occurred with angiogenesis prior to microscopic changes in articular cartilage on the posterior tibia in the anterior cruciate ligament transection (ACLT) mouse model 59 . Our result consistent with the previous reports that the subchondral remodeling reflects the early stage of OA 9 . In addition to routine bone parameters, evaluation of the porosity in the subchondral bone might be an emerging indicator in the early stage of TMJ-OA. The in-depth understanding of subchondral bone disturbance in TMJ-OA will lead to a paradigm shift in therapies for TMJ-OA. Similar results also have been obtained in other studies of FICZ. For example, in a murine model about ligature-induced periodontitis, FICZ treatment reduced the inflammatory response 60 . Similarly, FICZ downregulated epithelial-derived IL-7 expressions and repressed inflammation in the gastro-intestinal tract of mice with DSS-induced colitis 61 , and has inhibited imiquimod-induced skin inflammation 62 . However, it has been observed that AhR ligands are not universally applicable for controlling inflammation. For example, while TCDD (also known as another family of smoke contaminants as dioxin) exhibits immunomodulatory properties, it is the most toxic among the AhR ligands 63 . Inhibition of osteoclast formation by 3-methylcholanthrene (3MC) containing in cigarette smoke is evident at very low concentrations (10 -9 M), whereas inhibition of osteoblast formation occurs at higher concentrations (10 -7 M), suggesting that 3MC has a stronger inhibitory effect on osteoclastogenesis than osteoblast formation 64,65 . Further research also suggests the preferential targeting of osteoclasts by 3MC and other AhR ligands by examining the downstream effects of the AhR signaling. Another AhR-ligands, kynurenines, are secreted from the human tumors to promote production of regulatory T cells (Tregs) in order to mediate potential autoimmune issues 66 . The present results indicate that FICZ is able to mitigate TMJ-OA. To better elucidate the mechanisms mediating regulation of inflammation by AhR signaling, we performed in vitro cell culture assays with BMMs. Following treatment with FICZ, RANKL-induced formation of osteoclasts was found to be inhibited. FICZ treatment also reduced RANKL-induced expression of Cyp1a1, as well as the osteoclastic markers, integrin β 3 , NFATc1, and cathepsin K. Huai et al. 67 previously demonstrated that pre-treatment with increasing concentrations of FICZ reduced expression of NLRP3 in mouse macrophages stimulated with lipopolysaccharide and inflammasomes. When TMJs were compared between mice with and without FICZ treatment, the former exhibited a dose-dependent attenuation of cartilage degradation within the hypertrophic layer of the condylar cartilage. Further research is essential to better understand the role of these changes in relation to chondroprotection and homeostasis of the ECM in cartilage as well as the molecular mechanism of osteoblastogenesis. Survival of OA chondrocytes also requires further study.
In conclusion, we present data supporting AhR expression in mandibular condyles and activation of AhR signaling upon binding of B[a]P. Furthermore, in the presence of B[a]P, osteoclast activity as well as the expression of aggrecan, Col2a1 and Sox9 are altered in the subchondral bone and mandibular chondrocytes. It is possible that overactivation of the AhR pathway induces disordered bone remodeling and inappropriate bone turnover in mandibular condyles. Based on the data we obtained from qPCR analysis, radiographs of mandibular condyles, and histological assays, the AhR endogenous ligand, FICZ, provides protection from inflammatory responses and prevents bone loss in mandibular subchondral bone in TMJ-OA. It is anticipated that these findings will facilitate the development of a new therapeutic approach for AhR-initiated inflammatory diseases, in combination with further evidences of a mechanistic details of AhR signaling in mandibular condyles.