TPCA-1 negatively regulates inflammation mediated by NF-κB pathway in mouse chronic periodontitis model

Bo Wang1,2 | Shizhu Bai3 | Jiang Wang4 | Nan Ren3 | Rui Xie3 | Geng Cheng3 | Yan Yu1
1 School of Public Health, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, China
2 Department of Stomatology, Xi’an People’s Hospital, Xi’an, Shaanxi, China
3 Digital Center, The Hospital of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, China
4 Department of General Dentistry & Emergency, The Hospital of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, China


The dysregulation of immune system plays a crucial function in periodontitis devel- opment. Pro-inflammatory cytokines are thought to be critical for the generation and development of periodontitis. The enhanced activity of osteoclasts contributes to periodontitis pathogenesis. Nuclear factor-κB (NF-κB) signaling pathway directly enhances osteoclast differentiation and maturation. 2-[(aminocarbonyl)amino]-5-(4- fluorophenyl)-3-thiophenecarboxamide (TPCA-1) is a IκB kinases (IKK) inhibitor. This research aimed to investigate whether TPCA-1 had influence on the pathogenesis of chronic periodontitis. Mouse chronic periodontitis was induced by an in vivo ligature- induced periodontitis model. TPCA-1 was intravenously injected into mice after chronic periodontitis induction. Bone marrow-derived macrophages were cultured in macrophage colony-stimulating factor (M-CSF)-conditioned media with receptor activator of nuclear factor-kappa B ligand (RANKL) induce in vitro osteoclast dif- ferentiation. Western blot was used to analyze protein levels and mRNA levels were analyzed through qRT-PCR. TPCA-1 promoted osteoclastogenesis and osteoclast- related gene expression in vitro. The production of pro-inflammatory cytokines in osteoclasts induced by lipopolysaccharides was inhibited by TPCA-1 in vitro. In vitro TPCA-1 treatment inhibited Aggregatibacter actinomycetemcomitans (A.a)-induced expression of pro-inflammatory cytokines and NF-κB signal activation in osteoclasts. The induction of chronic periodontitis was inhibited by the absence of IKKb in mice. This research demonstrates that the treatment of TPCA-1 negatively regulates in- flammation response and inhibits the osteoclastogenesis through the inactivation of NF-κB pathway in mouse chronic periodontitis model.

chronic periodontitis, NF-κB, osteoclast, TPCA-1


As a complex infectious disease, periodontitis is caused by sev- eral different factors (Van Dyke, 2017). Periodontitis patients are typically found to suffer by more than one risk factors. Two of the most important pathogens during the generation of periodontitis are Aggregatibacter actinomycetemcomitans (A.a) and Porphyromonas gingivalis (Michalowicz et al., 2000; Teles et al., 2013). Periodontitis is diagnosed through pathologic loss of periodontal ligament and al- veolar bone (Slots, 2017).
The immunity during the pathogenesis of periodontitis is com- posed of innate and adaptive cellular and humoral responses and aims at inhibiting pathogens invasion (Ebersole et al., 2017). The dys- regulation of immune system plays a crucial function in periodontitis development. Pro-inflammatory cytokines are thought to be critical for the generation and development of periodontitis (Shibata, 2018). During periodontitis development, pro-inflammatory cytokines in periodontal tissues triggers inflammatory mediator production, osteoclasts formation, matrix metalloproteinase expression, and matrix-producing cell death, resulting in periodontal alveolar bone and connective tissue loss (Graves & Cochran, 2003).
Osteoclasts are derived from monocyte/macrophage lineage and are capable to resorb mineralized bone matrix (Fu & Shi, 2020; Gu et al., 2015). The enhanced activity of osteoclasts alters bone remodel- ing and contributes to periodontitis pathogenesis (Li etal., 2018). Factors in nuclear factor-κB (NF-κB) signal are crucial regulators of osteoclast differentiation and NF-κB signal directly enhances osteoclast differ- entiation and maturation (Lacey et al., 1998; Yamashita et al., 2007). Meanwhile, the inhibition of Janus kinase 2/signal transducer and acti- vator of transcription 3 (JAK2/STAT3) pathway shows effect in the sup- pression of osteoclast generation (Li et al., 2013). 2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) is a IκB kinases (IKK) inhibitor. It is reported that the administration of TPCA-1 not only inhibits NF-κB signaling pathway but also blocks STAT3 re- cruitment to upstream kinases and suppresses the activity of STAT3 signaling pathway (Nan et al., 2014). Other research showed that IKK-2 inhibitor TPCA-1 represses nasal epithelial inflammation in vitro (Sachse et al., 2011). TPCA-1 reduces pro-inflammatory cytokines and antigen-induced T-cell proliferation in murine collagen-induced arthri- tis (Podolin et al., 2005). Thus, the administration of TPCA-1 may influ- ence osteoclast differentiation and maturation and contributes to the therapy of periodontitis.


2.1 | Animals

Eight-week-old C57BL/6 background wild-type mice were ob- tained from Animal Model Research Center of Nanjing University. B6 background IKKbfl/fl mice (EM:01921) were purchased from EMMA. IKKbfl/fl mice were crossed with C57BL/6 background Lyz2- Cre mice to generate myeloid cell conditional IKKb knockout mice (IKKbfl/fl; Lyz2Cre/+, IKKbMKO). B6/129 background STAT3fl/fl mice (016923) were obtained from Jackson Laboratory. STAT3fl/fl mice were crossed with Lyz2-Cre mice to generate myeloid cell condi- tional STAT3 knockout mice (STAT3fl/fl; Lyz2Cre/+, STAT3MKO). All ani- mal experiments were approved by the Ethics Committee of Xi’an Jiaotong University Health Science Center.
The chronic periodontitis mouse model was established in this re- search. Four mL/kg 10% chloralhydrate was intraperitoneal injected into mice to perform anesthesia. On one side of the maxilla bone, the first and second molars subgingival were ligated with silk sutures (3/0) (Johnson & Johnson Medical) through continuous “∞” method. Before the ligature induction, sutures were pretreated by Pg solution. During the 4 weeks induction, mice were supplied with high-sugar drinking water. Ligation was observed every 2 weeks and replaced when the ligation was loosened or displaced. After 4 weeks induction, the liga- tion was removed. The distance from the cement–enamel junction to the alveolar bone crest (CEJ-ABC) was defined as periodontal bone height and measured by micro-CT (Gully et al., 2014; Ihn et al., 2017). Briefly, specimens were scanned using a cone-beam micro-CT system (Skyscan1176; Skyscan) with x-ray generator (accelerated potential: 50 kV, beam current: 500 μA, shutter speed: 900 ms). Scans were re- constructed and 3D digitized images were generated using the sup- porting analyzing software (CT Analyser Version 1.10; Skyscan).
TPCA-1 (Tocris Bioscience) was suspension in phosphate- buffered saline (PBS) solution. Two-hundred μg/kg TPCA-1 was intravenous injected into mice after chronic periodontitis induction once. After inducing the chronic periodontitis mouse model, the mice were fed with high-sugar drinking water for 4 weeks. During this period, the ligation was checked twice weekly and was replaced if had been displaced or loosened. Four weeks later, the ligation was removed, and SRT1720 (Selleck Biochem) was administered via oral gavage for 4 weeks according to different dosages. Blood, gingival crevicular fluid (GCF), and gingival tissue samples were collected for further investigation. In this research, mice were divided into no- treatment (NT) group with no chronic periodontitis, PBS treatment group with chronic periodontitis, and TPCA-1 treatment group with chronic periodontitis. Each group had 10 mice.

2.2 | Cells

In vitro osteoclastogenesis was performed in this research. The femurs and tibia were collected from relative mice and the bone marrow cells were flushed out. Bone marrow cells were cultured in DMEM medium (Sigma-Aldrich) supplemented with 20% fetal bovine serum (Sigma), 100 U/ml penicillin-streptomycin (Sigma), and 30% macrophage col- ony stimulating factor (M-CSF)-containing conditioned medium for 4–6 days to get bone marrow-derived macrophages. Bone marrow- derived macrophages were cultured in M-CSF-conditioned media for 4 days and treated by 25–100 ng/ml receptor activator of nuclear factor-κB ligand (RANKL) (R&D Systems, Minneapolis, MN) for another 3 days to induce in vitro osteoclast differentiation.

2.3 | Reagents

Lipopolysaccharides (LPS, Escherichia coli 055:B5; L6529) were pur- chased from Sigma-Aldrich. TNF were purchased from Peptech. Recombinant murine RANKL were purchased from R&D Systems.

2.4 | Bacteria culture

In this research, because of its high prevalence and pathogenicity in periodontitis, Aggregatibacter actinomycetemcomitans (A.a.) strain VT1729, serotype b strain, was employed. A.a. was plated and cul- tured on 3% Trypticase soy broth and 0.6% yeast extract (TSBYE) 1.5% agar plate with 100 μg/ml ampicillin for 2 days. The A.a. was cultured in TSBYE broth with 100 μg/ml ampicillin on a shaker at 37°C overnight.

2.5 | qRT-PCR

Total RNA was extracted by Nucleospin RNA II kit (Macherey-Nagel). Reverse transcription was performed by First Strand cDNA Synthesis Kit (Sigma). Real-time PCR was executed by SYBR Green Real-Time PCR Master Mixes (ThermoFisher) following manufacturer’s instruc- tion. Primers used in this experiment were listed in Table 1.

2.6 | Western blot

Cells were lysed by RIPA buffer (Beyotime) and centrifuged to get protein sample. Western blot was performed by the standard method. Antibodies used in this experiment were shown here: anti- IKBα (C-21), anti-STAT3 (C-20), anti-IKKα (H-744), anti-p65 (C-20, sc-372), anti-phospho-IκBα (Ser32, 14D4) (Cell Signaling Technology), anti-phospho-STAT3 (#9131) (Cell Signaling Technology), anti- phospho-NF-kB p65 (#3033) (Cell Signaling Technology), anti- phospho-IKKα/β (Ser176/180,16A6) (Cell Signaling Technology).

2.7 | Flow cytometry

For flow cytometry, fluorescently labeled antibodies to PB-conjugated anti-CD4, anti-CD11c, PE-conjugated anti-B220, anti-F4/80, PerCP5. 5-conjugated anti-Gr-1 (Ly6G), APC-CY7-conjugated anti-CD11b, and anti-CD8 from eBioscience were used at 1:100 dilution.

2.8 | ELISA

The concentrations of IL-6, TNF-α, and IL-1b were evaluated by Mouse Ready-SET-Go! ELISA kits (eBioscience) based on manufac- turer’s instruction.

2.9 | TRAP staining

For TRAP staining, cell medium was removed and cells were fixed by 1% glutaraldehyde for 15 min. Then, the cells were stained using TRAP staining kit (Cosmo Bio) based on the manufacturer’s instruction.

2.10 | Statistical analysis

Statistical analysis was performed by GraphPad PRISM 6.0 soft- ware. Data were presented as mean ± SEM. Student t test or one- way analysis of variance (ANOVA) followed by the Tukey’s post hoc test was used to calculate the differences between each group.


3.1 | TPCA-1 treatment has no effect on the development and survival of immune cells in mice

To investigate the effect of TPCA-1 on the immune system, mice were treated with TPCA-1 through intravenous injection. The mo- lecular structure of TPCA-1 was shown in Figure 1A. The proportion of relative immune cell in spleen of TPCA-1-treated mice was ana- lyzed by flow cytometry. As shown in Figure 1B, B cells, T cells, CD4+ T cells, and CD8+ T cells in spleen were not influenced by TPCA-1. We also detected the influence of TPCA-1 on the bone marrow of mice. Flow cytometry results demonstrated that the amount of mac- rophage and neutrophil shown no difference between PBS-treated mice and TPCA-1-treated mice (Figure 1C). Thus, the administration of TPCA-1 had no influence on the development and survival of im- mune cells in mice bone marrow and spleen.

3.2 | TPCA-1 promotes in vitro osteoclastogenesis and osteoclast-related gene expression

The influence of TPCA-1 on osteoclastogenesis was detected. Bone marrow-derived macrophages were cultured in M-CSF-conditioned media with 50 ng/ml RANKL for 3 days to induce in vitro osteoclast differentiation. During this process, different dosages of TPCA-1 were added to the medium. qRT-PCR results illustrated that the administration of TPCA-1 significantly elevated the mRNA levels of osteoclast genes Cstk, Acp5, and Calcr (Figure 2A). The expressions of osteoclast genes were enhanced by TPCA-1. Furthermore, TRAP staining was employed to investigate the differentiation of osteoclast. Figure 2B shows that osteoclastogenesis was dramatically enhanced by TPCA-1 in vitro. The effect of TPCA-1 on the viability of osteoclast was also detected in this research. The 24 hr treatment of various concentrations of TPCA-1 had no influence on the osteoclast viabil- ity in vitro (Figure 2C). The in vitro osteoclastogenesis was enhanced through TPCA-1, while the osteoclast viability was not influenced.

3.3 | TPCA-1 depresses LPS-induced pro- inflammatory cytokine production in osteoclasts

Then, we detected whether TPCA-1 influenced pro-inflammatory cytokine production in osteoclasts. Primary osteoclasts were pre- treated with 30 nM TPCA-1 for 1 hr and pro-inflammatory cytokines was induced by 100 ng/ml LPS. qRT-PCR results proved that the expressions of LPS-induced Il6, Tnf, and Il1b were significantly sup- pressed by the administration of TPCA-1 (Figure 3A). LPS-induced pro-inflammatory cytokines levels in the supernatants of osteo- clasts analyzed through ELISA. Il-6, Tnf-α, and Il-1b levels in TPCA- 1-treated osteoclasts supernatants were significantly lower than in PBS-treated osteoclasts after LPS induction (Figure 3B). So, the pro- duction of pro-inflammatory cytokines in osteoclasts was inhibited by TPCA-1.

3.4 | TPCA-1 inhibits A.a-induced production of pro-inflammatory cytokines in osteoclasts via suppressing NF-κB activity

Pro-inflammatory cytokine production in osteoclasts was also in- duced by A.a. Primary osteoclasts were pretreated with 30 nM TPCA-1 for 1 hr and the production of pro-inflammatory cytokines was induced by 10 CFU of A.a per cell. As shown by Figure 4A, the expressions of Il6 and Tnf in osteoclasts induced by A.a were inhib- ited by the administration of TPCA-1. Meanwhile, the activities of NF-κB signaling pathway and STAT3 signaling pathway were inves- tigated by Western blot. Figure 4B indicated that IKKα/β, IκBα, p65, and STAT3 phosphorylation were all depressed by TPCA-1 treat- ment. Thus, the activity of NF-κB pathway and STAT3 pathway in osteoclasts was both inhibited by TPCA-1.

3.5 | TPCA-1 ameliorates inflammatory response in chronic periodontitis mouse model

To further confirm these results in vivo, chronic periodontitis was induced in STAT3MKO or IKKbMKO mice. Through micro-CT, the CEJ- ABC in chronic periodontitis mouse model was analyzed. The in- duction of chronic periodontitis elevated the CEJ-ABC in wild-type mice, while the treatment of TPCA-1 reduced the elevated CEJ-ABC in mice with chronic periodontitis (Figure 5A and B). In STAT3MKO mice, the change in CEJ-ABC during chronic periodontitis induction and TPCA-1 treatment was not influenced (Figure 5A). However, in IKKbMKO mice, chronic periodontitis induction had no significant in- fluence on CEJ-ABC with or without TPCA-1 treatment (Figure 5B). These data demonstrated that the absence of STAT3 in osteoclasts had no influence on the pathogenesis of chronic periodontitis, but the absence of IKKb inhibited chronic periodontitis generation in mice. Through qRT-PCR, the expression of Il6, Tnf, and Il1b in gin- gival tissues was examined. Figure 5C shown that elevated Il6, Tnf, and Il1b mRNA levels in mice with chronic periodontitis were dra- matically down-regulated by the administration of TPCA-1. So, the inflammatory response in gingival tissues of mice with chronic peri- odontitis was ameliorated by TPCA-1.


During the pathogenesis of many immunoinflammatory diseases of bone, one of the basic physiologic processes is bone resorption (Hienz et al., 2015). In recent decades, plenty of researches have fo- cused on the investigation of the cellular and molecular mechanism of bone resorption during periodontal disease development in human (Gruber, 2015; McCauley & Nohutcu, 2002; Wiebe et al., 1996). The discovery of unknown biological mechanisms which participates in the regulation of bone resorption and remodeling processes is im- portant for understanding bone cell function and the pathophysi- ology of accelerated bone loss in periodontal disease. The newer advances in the molecular mechanism of periodontitis pathogenesis shed light on the development of novel therapeutic strategies.
NF-κB family is composed of several different transcription factors, including NFκB1, NFκB2, p65 (RelA), RelB, and cRel (Du et al., 2012). Evidences have demonstrated that NF-κB sig- naling pathway is involved in the regulation of cell proliferation, cell survival, innate immunity, and inflammation response (Beg & Baltimore, 1996; Hoesel & Schmid, 2013; Wang et al., 1996). Under normal circumstances, NF-κB transcription factors bind with IκB inhibitory proteins and stay at an inactive state in the cytoplasm of cells (Hayden & Ghosh, 2008). In the canonical NF-κB pathway, various stimuli trigger the activation of the IKKγ/IKKβ/IKKα com- plex and then promote the dissociation of the cytosolic inactive NF-κB/IκB complexes (Yu et al., 2020). The typical stimulating molecules in canonical NF-κB pathway include LPS, IL-1β, and TNF-α (Schmid & Birbach, 2008). These stimulations are mediated by tumor necrosis factor receptor (TNFR), interleukin-1 receptor (IL-1R), and Toll-like receptor (TLR) (Perkins & Gilmore, 2006). The NF-κB pathway activation enhances IκB phosphorylation, ubiquiti- nation, and degradation, which in turn promotes the DNA binding of p50-containing dimers and enhances relative gene expression (Mitchell & Carmody, 2018). NF-κB activation is a part of the im- mune defense. The pro-inflammation cytokines such as TNFα, IL-1, IL-6, and IL-8 and adhesion molecules are induced by NF-κB sig- naling pathway and, thus, promote the recruitment of leukocytes to inflammation site (Fan et al., 2013). TPCA-1 is an IKK inhibitor which can suppress the activity of NF-κB signaling pathway both in vivo and in vitro (Du et al., 2012; Gaddipati et al., 2015). The administration of TPCA-1 may regulate the inflammation response through its function in the inhibition of NF-κB signaling pathway. In human glioma cells, TPCA-1 inhibits the activity of NF-κB sig- naling pathway and type I interferon-mediated antiviral action (Du et al., 2012). TPCA-1 reduces pro-inflammatory cytokines and antigen-induced T-cell proliferation in murine collagen-induced ar- thritis (Podolin et al., 2005). In this research, the mice were treated by 200 μg/kg of TPCA-1. The immune cells in spleen and bone marrow were analyzed through flow cytometry. The proportions of B cell and T cell in spleen and macrophage and neutrophil in bone marrow were not influenced by the treatment of TPCA-1. So, the development and survival of immune cells were not altered by TPCA-1.
Osteoclasts are differentiated from hematopoietic stem cells and play crucial function in bone resorption (Bruzzaniti & Baron, 2006). Based on its high migration ability and mineralized bone degradation ability, osteoclast is critical for several processes in bones, includ- ing bone growth, bone integrity maintenance, bone remodeling, and the keep of bone homeostasis (Bar-Shavit, 2007; Teitelbaum, 2007). Since osteoclast is the principal bone resorptive cell, the local stim- ulation of its activity is the major procedure of alveolar bone loss (Saffar et al., 1997). Hematopoietic precursors differentiate into macrophages and monocytes, and eventually fuse into mature multicellular osteoclasts (Zou & Bar-Shavit, 2002). This differen- tiation process is promoted by both the contact with osteocalcin- containing mineralized bone particles and the key factors, including osteoclast differentiation factors (RANKL), tumor necrosis factors, interleukins, and M-CSF/CSF-1 (Nagasawa et al., 2007; Tanaka et al., 1993; Yasuda et al., 1998). RANKL is essential for osteoclast differentiation. RANKL binds with its receptor RANK to stimulate the activation of NF-κB and MAPKs and further the activation of nuclear factor of activated T-cells c1 (NFATc1) (Boyle et al., 2003; Kim & Kim, 2016). NFATc1 is the principal osteoclastogenesis regu- lator which induces the expression of osteoclast-specific genes and further regulates the differentiation and functions of osteoclasts (Kim & Kim, 2014; Kim et al., 2005). These facts indicate that the inhibited NF-κB signal through TPCA-1 may interfere the normal de- velopment of osteoclasts.
Through the treatment of M-CSF and RANKL, the bone marrow-derived macrophages were induced to differentiate into osteo- clasts. During this process, the expressions of osteoclast genes Cstk, Acp5, and Calcr were all enhanced by the administration of TPCA-1. Meanwhile, the number of osteoclasts differentiated from macro- phages was also elevated by TPCA-1 treatment. The administration of TPCA-1 promoted the expression of osteoclast-related genes in bone marrow-derived macrophages and enhanced osteoclastogen- esis. However, the cell viability of osteoclasts was not influenced by TPCA-1.
LPS is crucial component of Gram-negative bacteria cell wall. LPS has been proved to be the receptor activator of RANKL- mediated osteoclastogenesis and enhance osteoclastogenesis both in vivo and in vitro (Orcel et al., 1993). If pre-osteoclasts are ex- posed to LPS after being treated by RANKL, the osteoclastogen- esis will be promoted. If pre-osteoclasts are treated by RANKL and LPS simultaneously, the osteoclastogenesis will be inhibited (Park et al., 2015; Takami et al., 2002). The existence of LPS trig- gers the production of pro-inflammatory cytokines in relative tis- sues (Yucel et al., 2017). The LPS-induced osteoclastogenesis and bone destruction have association with the enhanced production of pro-inflammatory cytokines (Chiang et al., 1999; Zou & Bar- Shavit, 2002). In this research, primary osteoclasts were pretreated with TPCA-1 for 1 hr and then treated by LPS to induce the produc- tion of cytokines. Both LPS-enhanced productions of IL-6, TNF- α, and IL-1b in primary osteoclasts were all depressed by TPCA-1 treatment.
A.a is a critical pathogen which participates in the pathogene- sis of periodontitis. The periodontal infection of A.a will cause local and systemic inflammation response through the invasion of gingival epithelium and the release of endotoxins and exotoxins (Henderson et al., 2010). Endotoxins are widely expressed in all Gram-negative bacteria and induce pro-inflammatory response in host. However, A.a has two unique kinds of exotoxins, leukotoxin (LtxA), and cy- tolethal distending toxin (Cdt) (Melgar-Rodriguez et al., 2016). Cdt released by A.a is able to enhance the expression of RANKL and promote the osteoclastogenesis (Belibasakis et al., 2005). Primary osteoclasts were pretreated with TPCA-1 for 1 hr and then stimu- lated with A.a to investigate whether TPCA-1 also influenced the response of osteoclasts to the infection of A.a. The enhanced ex- pressions of Il6 and Tnf in osteoclasts induced by A.a were inhibited by TPCA-1. Meanwhile, the activated NF-κB signal and STAT3 sig- nal of A.a-stimulated osteoclasts were both suppressed by TPCA-1 treatment. Thus, the enhanced osteoclastogenesis by the infection of A.a was inhibited through TPCA-1. In vivo experiments were employed to further confirm these results. In STAT3MKO mice, the absence of STAT3 had no influence on the induction of chronic peri- odontitis and the function of TPCA-1. However, in IKKbMKO mice, the absence of IKKb significantly inhibited the generation of chronic periodontitis in mice. The NF-κB signaling pathway played the domi- nant regulation function in the generation of chronic periodontitis in mice. The administration of TPCA-1 also inhibited the expression of pro-inflammatory cytokines in gingival tissues of mice with chronic periodontitis.
There were some limitations in this research. The other IKK in- hibitors have been reported to simultaneously inhibit osteoclasto- genesis and inflammatory bone loss. While in this research, in vitro experiments proved that TPCA-1 inhibited the production of pro- inflammatory cytokines in osteoclasts and promoted osteoclasto- genesis and osteoclast-related gene expression. The paradox that existed with the effects of TPCA-1 on the promotion of osteoclasto- genesis and the inhibition of inflammation response was not be fully explained. The administration of TPCA-1 not only inhibited NF-κB signaling pathway but also blocked STAT3 recruitment to upstream kinases and suppresses the activity of STAT3 signaling pathway. Meanwhile, the inhibition of JAK2/STAT3 pathway showed effect in the suppression of osteoclast generation. Due to the multiple targets of TPCA-1, we proposed that osteoclast differentiation- regulated TPCA-1 was mainly depending on the STAT3 signal, but the IKK-mediated NF-kB was indispensable for the inductions of inflammatory cytokines. Therefore, TPCA-1 showed opposite func- tions on distinct signal pathways. Thus, TPCA-1 treatment pro- motes osteoclastogenesis and osteoclast-related gene expression. In chronic periodontitis mouse model, the administration of TPCA-1 also ameliorated inflammatory response. Thus, the result of TPCA-1 in bone marrow-derived osteoclasts might not have the similar ef- fect in mouse model, since the other effectors might also participate in the regulation of osteoclastogenesis. Further experiments should be performed to explore the effect of TPCA-1 on osteoclastogenesis in mouse model. The other limitation in this research was that the connection between the in vitro and in vivo findings should be filled. In in vitro experiments, pro-inflammatory cytokine production in os- teoclasts was induced by A.a. In in vivo experiments, the chronic periodontitis mouse model was established by ligature. To further confirm the conclusion in this research, the induction of chronic periodontitis mouse model should be performed by ligatures soaked with A.a.


In conclusion, the treatment of TPCA-1 negatively regulates in- flammation response but promotes the osteoclastogenesis through the inactivation of NF-κB pathway in mouse chronic peri- odontitis model.


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