Local delivery of simvastatin maintains tooth anchorage during mechanical tooth moving via anti‐inflammation property and AMPK/MAPK/NF‐kB inhibition

Abstract Simvastatin (SMV) could increase tooth anchorage during orthodontic tooth movement (OTM). However, previous studies on its bone‐specific anabolic and anti‐inflammation properties were based on static in vitro and in vivo conditions. AMPK is a stress‐activated kinase that protects tissue against serious damage from overloading inflammation. Rat periodontal ligament cells (PDLCs) were subjected to a serial of SMV concentrations to investigate the optimization that promoted osteogenic differentiation. The PDLCs in static and/or tensile culturing conditions then received the proper concentration SMV. Related factors expression was measured by the protein array, real‐time PCR and Western blot. The 0.05UM SMV triggered osteogenic differentiation of PDLCs. The inhibition of AMPK activation through a pharmacological approach (Compound C) caused dramatic decrease in osteogenic/angiogenic gene expression and significant increase in inflammatory NF‐κB phosphorylation. In contrast, pharmacological activation of AMPK by AICAR significantly inhibited inflammatory factors expression and activated ERK1/2, P38 MAPK phosphorylation. Moreover, AMPK activation induced by SMV delivery significantly attenuated the osteoclastogenesis and decreased the expression of pro‐inflammatory TNF‐α and NF‐κB in a rodent model of OTM. The current studies suggested that SMV could intrigue intrinsic activation of AMPK in PDLCs that promote attenuate the inflammation which occurred under tensile irritation through AMPK/MAPK/NF‐kB Inhibition.

mechanically induced physical movement raises great attention for shortening the treatment cycle and reducing the patients' incompliance caused by pain and discomfort, by either accelerating local movement rate or precisely increasing tooth anchorage in the alveolar. 3 Existing biological therapy includes chemical methods (cytokines, hormone, drugs, growth factors and other biological substances) and gene therapy, among which the most potential translational approach is the locally administrated meditations. 4 Simvastatin (SMV), as the HMG-CoA reductase inhibitor, is a cholesterol-lowering statin drug. 5 Recently, its protective bone anabolic and anti-resorptive properties have been unveiled and lead to a wide application in large-size bone defect regeneration, therapeutic management of osteoporosis and periodontitis correction. [6][7][8] According to clinical trials observation, SMV intake was beneficial for chronic periodontitis control for its function in inflammation limitation and periodontal tissues repair. [9][10][11] Animal studies have shown that SMV when administered systemically resulted in increased tooth anchorage and reduced root resorption. 3,12 To be mentioned, the pocket injection of 1.2% SMV gel decreased space re-opening after orthodontic space closure in human anterior teeth. 13 We thus assume a bone anabolic property of SMV on periodontal tissue under a biomechanically induced inflammatory condition.
Adenylate-activated protein kinase (AMPK) is a silk and threonine-protein kinase which mainly coordinates the metabolism of energy. 14,15 Lately, researchers discovered its roles in autophagy inducement, inflammation control, cancer metastasis and autoimmune inhibition. [16][17][18][19][20][21][22] Our group also found that AMPK-α1 knockout (AMPK-α1 -/-) mice presented larger inflammatory periodontal bone defects and expressed higher levels of inflammatory cytokines than wild-type (WT) mice. 23 Interestingly, OTM is a biomechanical process accompanied by tension side bone regeneration and pressure side bone resorption, which indicates the AMPK activation in the inflammation-mediated osteogenesis under biomechanical irritation. 24 Current in vitro observations grounded on the static-culturing periodontal ligament cells (PDLCs), which led to big inconsistency from the actual stress state of the PDLCs during OTM. In this study, we focused on the anti-inflammation effect of SMV on tensile side of the orthodontic moving tooth in a rodent model. An in vitro dynamic culturing system was also applied to mimic the stressing condition in OTM to explore the potential mechanism concerning the AMPK regulation.

| Animal for OTM model
Twelve-week-old male Sprague Dawley (SD) rats of 250 g ± 15 g were obtained from the Tongji Medical College Animal Center.
All animal procedures were approved by the Animal Care and Experiment Committee of Tongji Hospital, Tongji medical college, Huazhong University of Science and Technology. Animals were housed in specific pathogen-free (SPF) condition at an ambient 24-26°C temperature, 55%-60% humidity under a 12:12-hour light/dark cycle. Adequate measures were implemented to minimize pain or discomfort of animals during all procedures. The study compliance with the ARRIVE guidelines.

| Acquisition and cultivation of human PDLCs
Ten human healthy pre-molars were collected from four patients ( 25 The explants were kept in a 5% CO 2 incubator at 37°C. Cells at passage 3-5 were adopted for the following tests. 26

| The optimization of SMV concentration
Periodontal ligament cells were seeded into 24-well plates at a density of 4.0 × 10 4 cells/mL. One day later, six concentrations of SMV (0, 0.01, 0.05, 0.1, 0.5 and 1UM) (Sigma-Aldrich) in supplemented DMEM were offered. The cell numbers in each hole were evaluated on days 1, 4, 7 and 10 (Multisizer, Beckman Coulter Inc). PDLCs were seeded in 6-well plates at a density of 5 × 10 4 cells/well and cultured in the six concentrations prepared in osteogenic culture medium (supplemented DMEM with 50 mg/mL ascorbic acid, 10 mmol/L b-glycerophosphate and 10 −8 mol/L dexamethasone, Sigma). After 7-and 14-day inducement, ALP staining (Beyotime Biotech, Jiangsu, China) and semi-quantitative analysis (Sigma) were performed. The total protein content was determined using a protein assay kit (Bio-Rad). The osteogenic gene expression of RUNX2, VEGF, OPN and OCN was measured by RT-PCR on days 1 and 7. Finally, after 4 weeks inducement, fixed PDLCs were marked by the Alizarin Red staining 27 and the Von Kossa staining. 28 Osteogenic medium (0UM SMV) was used as control. All experiments were performed in triplicate.

| Protein array
The human protein membrane array (QAH-PDD-1, Ray Biotech) simultaneously profiles 20 proteins were in quadruplicate, and the supernatant of group i, ii, iii and iv was pulled together for measurement. 30 The cytokine array membranes were blocked for 30 minutes and for 2 hours in the collected supernatants. After washing, the membranes were incubated with biotin-conjugated antibodies (1:250 dilution, 1 mL per array membrane) at room temperature for 2 hours and washed again. The horseradish peroxidase-conjugated streptavidin solution (1:1000, 2 mL) was added and incubated for 2 hours followed by a third wash step. Proteins were detected by the enhanced chemiluminescence, and the membranes were exposed to X-ray films.

| Real-time Quantitative PCR
The total cellular RNA was extracted using the TRIzol onestep method (Invitrogen Life Technologies) and reverse-transcribed to cDNA using a cDNA Synthesis kit (TaKaRa Bio, Inc, Otsu, Shiga, Japan). Primers were synthesized commercially . The genes expression was evaluated using a real-time PCR kit (TaKaRa Bio). GAPDH was used as the housekeeping gene. The 2 −∆∆CT method was used to quantify the folded expression level. All tests were repeated in triplicate.

| Western blotting analysis
Total proteins of the cells were extracted (RIPA lysis buffer, Sigma) and measured (BCA Protein Assay Kit, Pierce Biotechnology).
Protein homogenates were run on SDS-PAGE gel (Beyotime) and transferred onto a PVDF membrane (Millipore). After 5% milk blocking, the membranes were incubated overnight in primary an-

| Pre-treatment of drug injection and Orthodontic model establishment
Four groups of pre-conditioning were randomly given to all animals: group, 10 minutes after the Compound C administration, AICAR (30 mg/kg) was given. A total of 3 times dosing were completed before orthodontic appliance fixation. 31 The orthodontic wire was a titanium-nickel alloy measuring 0.228 mm in diameter and 14 mm in length. 32 After intraperitoneal injection of 3.5 mg/100 g pentobarbital, the buccopalatal grooves of the M1 and M2 were enlarged with a diamond bur (NO. 145, Shofu) on the occlusal surfaces to deep enough to fit the spring wire. The site was dried, etched with 65% phosphoric acid (Shofu) for 20 seconds, rinsed with water and dried. The tips were brought together and maintained at a distance of 3 mm by a circular frame to deliver an initial force of 30 g. The springs were seated into the occlusal grooves and bonded with dental resin (Shofu). The frame was removed to activate the spring after bonding. The rats were then allowed to recover from anaesthesia and returned to their cages.
At the end of 1 week active tooth movement, the orthodontic appliances were removed. Animals were euthanasia and perfused with 10% buffered formalin. The maxillae were carefully isolated and trimmed into single blocks together with three right upper molars before storage in 4% neutral formaldehyde solution at 4°C overnight. 33

| Micro-CT measurement
The alveolar samples were assessed using a micro-CT system (μCT-80, Scanco Medical, Bassersdorf, Switzerland) as previously described. The microfocus of the X-ray source had a spot size 7 mm and maximum voltage 36 kV. 34

| Histological and histomorphometric assays
All samples were decalcified in 10% EDTA before embedding.
The specimens were cut into 5 μm sections and prepared for H&E staining. The histomorphometric analysis was performed under the bright-field setting on a light microscope (Leica-microsystems) on six samples per group. 37 On three randomly selected images  38 Finally, the sections were treated with a mixture of a tartaric acid solution and acid phosphatase substrates (Sigma). 28 The TRAP labelled cells were calculated in the corresponding region of H&Estained sections.

| Statistical analysis
The experimental data were presented as the mean ± standard derivation. Statistical analysis in this study was performed using  Figure 1D). Finally, we tested the calcified nodule formation in control and 0.05UM SMV groups. Both Alizarin Red and Von Kossa staining showed that the 0.05UM SMV induced more calcified nodules in larger size than the control group ( Figure 1E).

| SMV promoted osteogenesis on the tensile side of periodontium during OTM via antiinflammatory effect
SMV shows protective bone anabolic and anti-resorptive properties. 6,11 We here focused on identifying the intrinsic factor(s) responsible for the therapeutic effects of SMV in increasing tooth anchorage.
We took advantage of protein array technology and scanned 20 pro-and anti-inflammatory factors in the co-stimuli of tension and SMV-sensitized PDLCs. We found that IL-6 was the most pronounced cytokine induced by PDLCs under the co-stimuli (Figure 2A,B). To detect the potential effect of AMPK during SMV-induced anti-inflammation, we subsequently measured the expression levels of p-AMPK/ AMPK and p-P65/P65 (NF-κB) in the cells. Our data demonstrated that increased AMPK was negatively connected with IL-6 secretion ( Figure 2C). Also, we confirmed that IL-6 mRNA were correspondingly reduced after indicated treatment and AMPK mRNA levels were correspondingly elevated by the co-stimuli ( Figure 2D,E).
The intervention of AMPK signalling was analysed by the admin-  Figure 3D). For NF-κB, the inhibition of AMPK significantly elevated its expression in all the time-points (P < .01). This change was corrected after the administration of AMPK activator. However, the tensile culture condition did not show statistic influence on RUNX2 expression ( Figure 3E). On the other hand, the expression level of AMPK significantly increased in tensile condition at 12 hours and 1 day. And SMV collaboratively increased the expression level at 6 hours (*, P < .05) and 12 hours (**, P < .01). This alteration could be inhibited by Compound C ( Figure 3F). Meanwhile, there was no significant difference in the expression of AMPK-α (P > .05).

| SMV promoted osteogenesis on the tensile side of periodontium during OTM via AMPK/MAPK/ NF-ΚB inhibition
To

| Surgical procedures and gross observation
In general, a total of 24 SD rats received orthodontic appliance

| Micro-CT assessment
The maxillae samples were examined by micro-CT before the following process. Massive bone resorption was noted around the distal root of the first maxillary molar in the control group, while the height of the bones around the distal roots in the SMV group was basically maintained at the bifurcation position ( Figure 6A).
The moved distance of first maxillary molar in control group was 497.13 ± 63.28 μm, which was significantly higher than the SMV group 27.56 ± 31.15 μm and AICAR group 172.45 ± 55.05 μm (**, P < .01). There was no statistical difference between the control group and the Compound C group 417.91 ± 50.36 μm (P > .05; Figure 6B). The BV/TV percentage was consistent with 3D reconstruction observation. The control group 63.84 ± 7.36% was the lowest, while the SMV group 79.49 ± 3.38% the maximum (**, P < .01). The treatment of Compound C reduced the BV/TV to 60.55 ± 4.56%, and the addition of AICAR significantly increased the ratio to 72.8 ± 6.08% (**, P < .01; Figure 6C).

| Histological observation
Rest specimens were decalcified for paraffin sections preparing.
H&E staining showed that the pressure side of the periodontium was narrower than the tension side ( Figure 7A). Observing the periodontal ligament on the tension side of the distal root (within the yellow square), we found that the thickness of periodontal ligament was larger in the control group, their arrangement was more chaotic, and the nucleus appeared to be significantly longer than the other the AICAR group (1.5 ± 0.55) (*, P < .05). There was also a statistic difference between the control group and the AICAR group (*, P < .05; Figure 7E). In terms of cell size and cell amount, the control group was obviously larger than the other three groups (**, P < .01; Figure 7F).

| DISCUSS IONS
Control of stabilization during orthodontic treatment is considered of significant importance since it helps avoid undesirable tooth movements that may occur as a consequence of the reaction forces applied to move teeth. 39 1 and 3). This was consistent with previous findings that SMV could lift the expression of BMP-2 and VEGF mRNA in osteoblasts after local application of SMV. 50 Since bone formation is a coupling process involving osteogenesis and angiogenesis, this result proves the bone anabolic property of SMV on PDLCs in the tensile condition. Interestingly, there were significant differences among the gene expression levels of BMP-2, VEGF, TNF-ɑ and AMPK on most time-points between the tension and the static groups, which highlighted the importance of tension force system application.
NF-κB is a well-known transcription factor associated with inflammation and bone resorption. 51 Many studies reported orthodontic mechanical stress could trigger the NF-κB pathway activation, which mediated the expression of pro-inflammatory genes such as TNF-α and IL-6, as proved in our histological findings. 52 Therefore, inhibition of NF-κB activation is an important process in increasing tooth anchorage. On the other hand, ERK and p38 MAPK belong to the MAPKs signalling pathway, which played a central role involved in inflammatory response and cell survival. 53,54 For certain, recent studies indicated the phosphorylation of p38 and ERK1/2 MAPKS was activated in stress-stimulated PDLCs. 55 We then verified the effect of SMV on the MAPKs signalling pathway, and our results revealed SMV markedly inhibited stress-stimulated ERK and p38 MAPK phosphorylation in PDLCs, and these manifestations had significant changes after treatment with Compound C, the inhibitor of AMPK. The pre-condition of AICAR significantly reversed the inhibition trend and increased the pro-inflammatory factors expression level. Furthermore, we tested the expression level of TNF-α and IL-1.
Similar changes were identified under the Compound C and AICAR treatment. AMPK is an energy sensor that is activated by several types of stresses and has been described as a negative regulator of inflammatory response to IL-1, IL-6 and TNF-α. 56 Here, we detected the inhibition of NF-κB phosphorylation and the most significant reduction in IL-6 levels among the 20 cytokine in the SMV stimulated PDLCs ( Figure 2). Thus, a relationship was figured between AMPK activation and SMV-induced anti-inflammation. Previous studies reported that the activation of AMPK can reduce the inflammatory level caused by overload exercise on muscle cells and reverse harmful inflammation activity to a state of regenerative inflammation that induces muscle regeneration. 57 Studies also reported that AMPK signalling is a negative regulator of NF-κB-TNFα inflammatory axis in the treatment of hepatic inflammation and fibrosis. 58 Our work confirmed with previously studies and first identified that the anti-inflammatory and bone protection effect of SMV were associated with suppressing MAPKs phosphorylation and blocking NF-κB activation via AMPK.
However, this study mainly discussed the tension irritation which could not represent the integral stressing status of the tooth under orthodontic force. Also, a complete understanding of mechanic bone formation requires the identification and analysis of osteoclast and its responses. After that, this study recognized the inhibition of AMPK/MAKP/NF-κB pathway mediating SMV bone-protective effect on the enhancement of tooth anchorage during OTM.

ACK N OWLED G EM ENTS
Statement of sources of funding for the study: This work was supported by the National Natural Science Foundation of China (No. 81800981 and No. 81700940).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no known conflicts of interest associated with this publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.