Rosuvastatin Regulates Odontoblast Differentiation by Suppressing NF-κB Activation in an Inflammatory Environment

Abstract

Rosuvastatin is a synthetic statin of 3-hydroxy-methyl-3-glutamyl coenzyme A reductase inhibitor. It has pleiotropic characteristics including hepatic selectivity, minimal metabolism, inhibition of inflammation, and induction of osteoblast differentiation. In this study, dental pulp stem cells (DPSCs) were treated with lipopolysaccharide alone or with rosuvastatin. Then, we examined the accelerative effects of rosuvastatin on odontoblast differentiation and mineralized nodule formation by real-time polymerase chain reaction (RT-PCR), western blot, alizarin red S staining, and alkaline phosphatase staining. The extent of anti-inflammation was determined by RT-PCR and analysis of the expression of tumor necrosis factor α, interleukin 1β (IL-1β), and IL-6. Furthermore, the activation of nuclear factor kappa B (NF-κB) was determined by western blot. This study demonstrates that rosuvastatin may speed up odontoblast differentiation and rescue inflammatory reaction by suppressing the NF-κB signaling pathway. It is believed that our findings provide novel perceptions on odontogenic differentiation of DPSCs.

Introduction

Dental caries is a communicable disease in which the host, diet, and microbial communities interact after a period of time in this way to stimulate demineralization of the dental enamel with resultant caries formation (Caufield et al., 2005). Dental caries is still one of the most common diseases in the world (Ferreira-Nobilo et al., 2014). The incidence of dental caries is polyinfection of gram-positive bacterium and gram-negative bacterium. Streptococcus mutans is the acknowledged pathogenic organism of dental caries and plays important functions in cariogenic biofilm formation (Loesche, 1986).

Numerous studies have suggested that S. mutans stimulates production of proinflammatory cytokines (Benabdelmoumene et al., 1991; Russell and Mansson-Rahemtulla, 1989; Soell et al., 1994). Cytokines are associated with the beginning and advancement of dental caries, and their levels elevate in caries-active saliva (Evans et al., 1990; Gornowicz et al., 2012; Kim et al., 2002). Therefore, the understanding of the mechanisms of restraining inflammatory cytokines release may provide new therapeutic strategies for dental caries.

Rosuvastatin, an inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is widely used as a cholesterol-lowering reagent to prevent cardiovascular disease (Fletcher et al., 2005; Wang et al., 2010). Moreover, rosuvastatin has recently been suggested to have pleiotropic effects in various systems, including cardiovascular system, immune system, and skeletal system (Hileman et al., 2017; Monjo et al., 2010; Qiu et al., 2017).

Interestingly, growing evidence shows that statin therapy has local effects that significantly reduce the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), IL-6, and so on in gingival crevicular fluid (Subramanian et al., 2013; Suresh et al., 2013). Indeed, rosuvastatin is a next-generation statin with significantly anti-inflammation effect and has the ability to inhibit c-Jun terminal kinase and nuclear factor kappa B (NF-κB) activation and reduce the expression of IL-8, IL-6, monocyte chemoattractant protein 1, intercellular adhesion molecule 1, and cyclooxygenase 2 (Qian et al., 2015). Based on above-mentioned reports of the anti-inflammatory effects of rosuvastatin, we explore whether it is involved in regulating the inflammatory response in experimental cellular model of dental caries in vitro.

It is well known that NF-κB plays a crucial role in inflammation and cell survival (Imran et al., 2015). NF-κB is present in the cytoplasm as a heterotrimer composed of IκBα, p50, and p65 subunits. Upon activation of the complex, NF-κB is freed to enter the nucleus by activation-initiated degradation of IκBα. In the nucleus, NF-κB binds to specific genes that have DNA-binding sites for NF-κB to regulate gene transcription (Moynagh, 2005). The control of proinflammatory progress in dental caries may also be associated with the activation of NF-κB pathway that contributes to aberrant inflammatory cytokines secretion during early dental caries events.

Recently, in view of the fact that inhibition of NF-κB may decrease the release of inflammatory cytokines and enhance differentiation of odontoblasts and formation of collagen matrix (Hozhabri et al., 2015), we investigated whether rosuvastatin could control odontoblast differentiation of dental pulp stem cells (DPSCs) and whether the function was associated with the inhibition of the NF-κB pathway.

Materials and Methods

Cell culture

All samples were acquired from generally healthy patients (18–25 years of age) after obtaining informed consents that were approved by the Ethics Committee of the Affiliated Hospital of Nantong University. Healthy pulp tissues (n = 10) were isolated from the caries-free teeth of patients without oral infection undergoing extraction of fully erupted third molars. Inflamed pulp tissues (n = 10) were obtained from molars with deep caries. We isolated DPSCs from healthy pulp (N-DPSCs) and inflamed pulp (I-DPSCs) by cleaning the tooth surface, cutting around the cementoenamel junction using sterilized dental fissure burs, and then opening to reveal the pulp chamber. The pulp was digested in a solution of 3 mg/mL collagenase type I for 1 hour at 37°C.

Single-cell suspensions were obtained by passing the digested tissues through a 70 μm cell strainer (BD Falcon). Cell suspensions of dental pulp were seeded into 25 cm² culture dishes and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂ (Ledesma-Martinez et al., 2016). The medium was changed every 3 days. Approximately 7–10 days after seeding, the cells became nearly confluent. Cells were passaged at the ratio of 1:3 when they reached 85%–90% confluence. The cell populations were characterized by positive staining with anti-CD34, STRO-1, and c-kit, and the absence of CD45 (Feng et al., 2013; Wang et al., 2018). Cells from the third to fifth passages were used in all experiments.

Odontoblast differentiation

DPSCs (2 × 10⁴ cells/dish) were cultured in 35 mm culture dishes (Costar, Cambridge, MA) in odontogenic differentiation (OD) medium containing a-minimum essential medium (Invitrogen, Carlsbad, CA), 15% FBS (Gibco-BRL Life Technologies, Inc., Gaithersburg, MD), 10 mmol/L β-glycerophosphate, 50 mg/mL α-ascorbic acid, 10 nmol/L dexamethasone (Sigma-Aldrich, St. Louis, MO), 0.292 mg/mL glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin G for 0, 3, 7, 14, and 21 days, replacing the medium every 2 days. Then, the cells were collected for determination of the odontoblast differentiation by evaluating mRNA expression of odontogenic markers, including dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP).

The experimental treatment groups consisted of differentiation media with Escherichia coli lipopolysaccharide (LPS) serotype 0111: B4 (Sigma) and/or rosuvastatin (AstraZeneca, United Kingdom) or BMS-345541 (Sigma). After being induced for 0, 3, 7, 14, and 21 days, cells were prepared for alizarin red S staining, alkaline phosphatase (ALP) staining, and immunofluorescence. Protein and RNA were extracted for western blot analysis and real-time polymerase chain reaction (RT-PCR).

Alizarin red S and ALP staining

DPSCs were fixed with 4% paraformaldehyde (PFA) for 1 hour and washed with phosphate-buffered saline (PBS). Cells were then stained with 40 mmol/L alizarin red S (pH = 4.2) for 10 minutes under conditions of gentle agitation. Absorbance of the extracted alizarin red S stain was measured at 570 nm. DPSCs were subjected to ALP staining using the ALP assay kit (JianCheng, Nanjing, China) according to the manufacturer's instructions.

Western blot analysis

Total extracts of cells were lysed in buffer consisting of 150 mM NaCl, 50 mM TRIS, 2% sodium dodecyl sulfate (SDS), and a protease inhibitor mixture and centrifuged at 12,000 rpm for 12 minutes at 4°C. The proteins were subjected to SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes at 350 mA for 2.5 hours in a blotting apparatus (Bio-Rad, CA). After blocking in Tris-buffered saline with Tween with 5% nonfat dry milk, the membranes were incubated overnight at 4°C with primary antibodies. Then, the membranes were reacted with corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. β-actin and β-tubulin were used as the internal control for the cytoplasmic and nuclear proteins.

The following primary antibodies were used: β-actin (anti-mouse; Santa Cruz), β-tubulin (anti-mouse; Sigma), TNF-α (anti-mouse; Santa Cruz), IL-1β (anti-mouse; Santa Cruz), IL-6 (anti-mouse; Santa Cruz), p65 (anti-mouse; Santa Cruz), p-p65 (anti-mouse; Santa Cruz), and IκBα (anti-rabbit, Cell Signaling).

Immunofluorescent staining

DPSCs were seeded into the 24-well plates, fixed with 4% PFA for 1 hour, rinsed with PBS containing 0.1% Triton X-100 (PBST), and blocked for 30 minutes in PBST supplemented with 10% FBS. Cells were incubated with a primary antibody against p65 (1:100) (anti-mouse; Santa Cruz) in the same solution overnight at 4°C. Then cells were rinsed and incubated with secondary antibodies for 2 hours at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) (1:1000; Santa Cruz). Finally, the cells were examined using a Leica fluorescence microscope (Germany).

Real-time PCR

Total cellular RNA was extracted from the cells and reverse transcribed using conventional protocols. PCR amplification was performed using the following primer sets: GAPDH, forward: 5′-TCCATGACAACTTTGGTATCG-3′ and reverse: 5′-TGTAGCCAAATTCGTTGTCA-3′; DSPP, forward: 5′-GGAGACAAGACCTCCAAGAGTA-3′ and reverse: 5′-TGCTGGGACCCTTGATTTCTA-3′; DMP1: 5′-TGGGGATTATCCTGTGCTCT-3′ and reverse: 5′-GCTGTCACTGGGGTCTTCA T-3′; ALP, forward: 5′-TGAAATATGCCCTGGAGC-3′ and reverse: 5′-TCACGTTGTTCCTGTTTAG-3′; BMP-2, forward: 5′-CTAGACCTGTATCGCAGGCA-3′ and reverse: 5′-TTTCCCACTCGTTTCTGGTA-3′; TNF-α, forward: 5′-GGAAGACCCCTCCCAGATAG-3′ and reverse: 5′-CCCCAGGGACCTCTCTCTAA-3′; IL-1β: forward: 5′-ATAAGCCCACTCTACAGCT-3′ and reverse: 5′-ATTGGCCCTGAAAGGAGAGA-3′; IL-6: forward: 5′-GTACCCCCAGGAGAAGATTC-3′ and reverse: 5′-CAAACTGCATAGCCACTTTC-3′.

All the primer sequences were determined using established GenBank sequences. The primers were used to amplify the duplicate PCRs. Each sample was calculated from three independent experiments and GAPDH was used as an internal control.

MTT colorimetric assay

DPSCs were seeded at a cell density of 3 × 10³ cells/well into 96-well plates and treated with LPS (20 μg/mL) and/or rosuvastatin (10 μM). After incubation for 1, 3, 5, and 7 days, the proliferation rate of the cells was assessed by MTT (3-dimethylthiazol-2, 5-diphenyltetrazolium bromide; Sigma) test. The culture medium was added with 50 μL of MTT (50 mg/mL; Sigma) to each well and the cells were cultured for 4 hours at 37°C under 5% CO₂. The precipitate was extracted with dimethyl sulfoxide and the plates were read on a microplate reader at a wavelength of 540 nm and repeated in triplicate.

Statistical analysis

All experiments were repeated at least in triplicate, and values were presented as mean ± standard deviation. The significance of differences between the experimental groups and controls was analyzed using analysis of variance. Statistical significance was evaluated by the independent samples t-test using SPSS v17.0 software. Differences in which p was <0.05 were considered to be statistically significant.

Results

Effect of rosuvastatin on odontoblast differentiation in DPSCs in noninflammatory microenvironment

To assess odontoblast differentiation of different concentrations of rosuvastatin on DPSCs, we measured the mRNA levels of odontogenic markers, including DMP1, DSPP, ALP, and bone morphogenetic protein-2 (BMP-2) in the odontogenic medium after incubation periods of 0, 7, 14, and 21 days with rosuvastatin. As given in Figure 1A–D, the mRNA levels of DMP1, DSPP, ALP, and BMP-2 significantly increased after 7 days in the rosuvastatin-treated group (10 μM) compared with untreated control group. The data also showed that rosuvastatin upregulated odontoblast differentiation of DPSCs by alizarin red S staining and ALP staining (Fig. 1E). Therefore, 10 μM was selected as the optimal concentration for subsequent experiments.

FIG. 1.

Rosuvastatin promotes odontoblast differentiation of DPSCs in noninflammatory microenvironment. (A–D) DPSCs were cultured in OD medium (Normal) and added rosuvastatin (0.1, 1, 10, and 100 μM) for 0, 7, 14, and 21 days. The levels of DMP1, DSPP, ALP, and BMP-2 mRNA significantly increased after 7 days in the rosuvastatin-treated group (10 μM) compared with untreated Normal group (*p < 0.05). (E) DPSCs were cultured in OD medium (Normal) and added rosuvastatin (0.1, 1, 10, and 100 μM) for 21 days. Rosuvastatin (10 μM) upregulated odontoblast differentiation of DPSCs by alizarin S staining and ALP staining. ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; DMP1, dentin matrix protein-1; DPSCs, dental pulp stem cells; DSPP, dentin sialophosphoprotein.

Effect of rosuvastatin on odontoblast differentiation in DPSCs in inflammatory microenvironment

We isolated and characterized DPSCs from pulp tissues of teeth that had been clinically diagnosed as deep caries and compared them with those from healthy teeth. Alizarin red S staining and ALP activity decreased in I-DPSCs compared with N-DPSCs (Fig. 2A), suggesting impaired odontoblast differentiation in I-DPSCs. Furthermore, LPS was used to stimulate DPSCs to imitate the microenvironment of deep caries induced by S. mutans (a gram-positive bacterium and the most important pathogen in the development of caries) to examine the effect of rosuvastatin on odontoblast differentiation in DPSCs.

FIG. 2.

Rosuvastatin promotes odontoblast differentiation of DPSCs in inflammatory microenvironment. (A) N-DPSCs and I-DPSCs were cultured in OD medium and added LPS (20 ng/mL) and/or rosuvastatin (10 μM) for 21 days. Mineralized matrix was strongly decreased in I-DPSCs by alizarin S staining and ALP staining. (B) DPSCs were cultured in completed medium (Normal) and OD medium and added LPS (20 ng/mL) and/or rosuvastatin (10 μM) for 21 days. The OD group formed more mineralized nodules and LPS resulted in significant decrease in mineralized nodules formation compared with the OD group, but rosuvastatin easily inhibited the effect of LPS by alizarin S staining and ALP staining. I-DPSCs, DPSCs from inflamed pulp; LPS, lipopolysaccharide; N-DPSCs, DPSCs from healthy pulp; OD, odontogenic differentiation.

After 3 weeks of culture, compared with the normal group, the OD group with odontogenic-inducing medium formed more mineralized nodules (Fig. 2B). It indicated that odontogenic-inducing condition played a crucial role in the odontoblast differentiation of DPSCs. In the meantime, LPS resulted in significant decrease in mineralized nodules formation compared with the OD group, but rosuvastatin easily inhibited the effect of LPS (Fig. 2B).

Effect of rosuvastatin on DPSCs proliferation

To understand the effect of rosuvastatin on cell proliferation, the MTT assay was performed with 10 μM of rosuvastatin for 7 days. Results showed that rosuvastatin treatment had no significant effects on proliferation of DPSCs. However, suppressing the proliferative capacity of DPSCs with LPS could be reversed by 10 μM rosuvastatin (Fig. 3A, B).

FIG. 3.

The proliferative ability of DPSCs. (A) Cells were plated in 12-well plates and proliferation was determined by counting cell numbers at the indicated daily intervals. Growth curves after stimulations of LPS (20 ng/mL) and/or rosuvastatin (10 μM). Rosuvastatin treatment had no significant effects on proliferation of DPSCs, but rosuvastatin easily reversed the effect of LPS suppressing the proliferative capacity of DPSCs. (B) The cell proliferation of DPSCs was determined by the MTT assay. Triplicate reactions and three separate experiments were performed. All results are presented as mean ± standard error of mean (*p < 0.05).

Effect of rosuvastatin on LPS-induced inflammatory cytokines expression

To test the effect of rosuvastatin on regulating TNF-α, IL-1β, and IL-6 expressions in DPSCs, western blot was conducted to examine the protein expression levels. Upon stimulation with LPS and rosuvastatin, compared with the control group, enhanced expressions of TNF-α, IL-1β, and IL-6 by LPS were reduced by rosuvastatin pretreatment (Fig. 4A). The mRNA expression levels were detected by RT-PCR for TNF-α, IL-1β, and IL-6 (Fig. 4B–D). Rosuvastatin (10 μM) statistically significantly attenuated these mRNA expressions.

FIG. 4.

Rosuvastatin inhibits LPS-induced inflammatory cytokines expression. (A) DPSCs were cultured in completed medium (Normal) and added rosuvastatin (10 μM) and/or LPS (20 ng/mL) for 6 hours. Western blot analysis showed that the levels of proinflammatory cytokine production (TNF-α, IL-1β, and IL-6) were significantly increased in the LPS-challenged cells and rosuvastatin was a potent inhibitor of this effect for both TNF-α, IL-1β, and IL-6. (B–D) The mRNA expression levels were detected by RT-PCR for TNF-α, IL-1β, and IL-6 (*p < 0.05). IL, interleukin; RT-PCR, real-time polymerase chain reaction; TNF-α, tumor necrosis factor α.

NF-κB signaling plays a crux role in regulating the odontoblast differentiation of DPSCs

NF-κB is a noted transcription factor concerned in the process of inflammation. Rosuvastatin was checked to see whether it exerted an inhibiting effect on this process. LPS improved the phosphonation of p65, the degradation of IκBα, and the nuclear migration of p65, whereas rosuvastatin inhibited all these processes (Fig. 5A). Immunofluorescence results showed that p65 translocated to the nucleus after exposure to LPS, but rosuvastatin effectively restrained LPS-induced p65 nuclear translocation (Fig. 5B). BMS-345541, an NF-κB pathway inhibitor, exhibited similar effect to rosuvastatin (Fig. 5A, B).

FIG. 5.

Rosuvastatin promotes odontoblast differentiation by inactivating NF-κB pathway. (A) DPSCs were cultured in OD medium and added LPS (20 ng/mL), and/or rosuvastatin (10 μM) or BMS-345541 for 21 days. Western blot analysis showed that LPS improved the phosphonation of p65, the degradation of IκBα, and the nuclear migration of p65, whereas rosuvastatin and BMS-345541 inhibited all these processes. β-actin was used as the internal control for total proteins, whereas β-tubulin was used as the internal control for nuclear protein. (B) The expression of p65 in the cytoplasm and nucleus of DPSCs treated with LPS (20 ng/mL), and/or rosuvastatin (10 μM) or BMS-345541 was examined by immunofluorescent staining. Nuclei are stained with DAPI. Scale bar 100 μm. All experiments were performed three times independently. (C) Schematic of rosuvastatin regulates odontoblast differentiation through suppression of NF-κB activation. NF-κB can be activated in DPSC added LPS. The activation of NF-κB can inhibit the capacity of odontoblast differentiation in inflammatory microenvironment. Rosuvastatin decreases p-p65, degrading the nucleus translocation of p65 and enhancing odontoblast differentiation capacity. DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; NF-κB, nuclear factor kappa B.

Discussion

The aim of this study was to extend our previous review and to include the potential character of rosuvastatin in the progress of odontoblast differentiation. This study work revealed the following: (1) rosuvastatin can accelerate odontoblast differentiation of DPSCs in both noninflammatory microenvironment and inflammatory microenvironment; (2) rosuvastatin is able to significantly reduce LPS-induced TNF-α, IL-1β, and IL-6 production from human cultured DPSCs; and (3) rosuvastatin may enhance odontoblast differentiation capacity by inhibiting NF-κB signaling.

Rosuvastatin has a long half-life and is a potent HMG-CoA reductase inhibitor (McTaggart et al., 2001). Earlier studies have shown that rosuvastatin promotes osteoblast differentiation in vitro as demonstrated by increased BMP-2 gene expression and secretion, and ALP activity in bone mesenchymal stem cells (BMSCs) (Monjo et al., 2010). DPSCs and BMSCs share many biological properties like marker expression, immunomodulatory activity, gene repertoire, and differentiation potential. Whereupon, we hypothesize that the effect of different concentrations of rosuvastatin on odontoblast differentiation in DPSCs in vitro and the molecular mechanisms of this effect are similar to BMSCs.

Generally, LPS activates and leads to the production of proinflammatory cytokines through NF-κB in DPSCs. We examined the effect of LPS-induced activation of NF-κB signaling. It is well established that NF-κB plays a crucial role in the regulation of expression of inflammatory cytokines like TNF-α, IL-1β, and IL-6, thus exhibiting the anti-inflammatory effect that should require a capacity to reduce the activation of this pathway.

The NF-κB family of transcription factors is involved in controlling many of these proinflammatory cell signals and is activated both in the early stages and in advanced stages of lesions (Hajra et al., 2000). Normally, NF-κB inactivation is associated with inhibitors of IκB that exist in the cytoplasm. After the phosphorylation and degradation of IκBα, the NF-κB p65 subunit is released and translocated to the nucleus to regulate downstream proinflammatory genes (Fujioka et al., 2004; Liu et al., 2011). Fortunately, in our study, as we expected, LPS increased the phosphorylation of p65 and accelerated NF-κB activity.

In addition, several evidences indicate that chronic inflammation impedes DPSC differentiation and dentine reparative processes (He et al., 2005; Smith et al., 2005). Of note, the cytokines are essentially expressed by odontoblasts, likely in anticipated disease events, and their levels can be significantly upregulated by both bacterial elements, LPS and TNF-α (Reing et al., 2009). Both the protein and RNA levels of proinflammatory mediators are enhanced in diseased pulpal tissue in dental caries compared with that in healthy ones (Cooper et al., 2010; McLachlan et al., 2004). In our study we observed that the anti-inflammatory mechanism of rosuvastatin in activated DPSCs subsequently suppressed the NF-κB signaling pathway. Interestingly, except for its anti-inflammatory activity, rosuvastatin is highlighted by its odontogenic capacity.

The connections between rosuvastatin and dentin function remain unclear. Rosuvastatin may directly activate the expression of odontoblast genes to control odontoblast differentiation. It may also indirectly control the production of proinflammatory cytokines. There are also some limitations in this study. What is the effect of rosuvastatin in OD in vivo? Does rosuvastatin control odontoblast differentiation also through other signaling pathways? Does rosuvastatin have an effect in the crosstalk network between the destruction of the dental tissues and the formation of reparative dentinogenesis? Further researches are warranted to address these questions.

Footnotes

Acknowledgments

The study was partly supported by Graduate Student Innovation of Science and Technology Projects funded by Jiangsu Province (No. SJLX16_0567) and Nantong University (No. YKC16092), Nantong Science and Technology Project (MS32015030 and MS22015091), and a Project funded by Affiliated Hospital of Nantong University (S11954).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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