Periodontitis is a common inflammatory disorder affecting the tissues surrounding the teeth, which can lead to the destruction of periodontal tissue and tooth loss. Resveratrol, a natural phytoalexin, exerts multiple biological effects. For example, its anti-inflammatory activity has been widely studied for the treatment of inflammatory bowel disease for a number of years. However, its effect on bone repair and new bone formation in an inflammatory microenvironment is not well understood. Accordingly, the effect of resveratrol on inflammation-affected human periodontal ligament stem cells (hPDLSCs) requires further investigation. In the present study, the effect of tumor necrosis factor-α (TNF-α), resveratrol, or the combination of both on the osteogenic differentiation of hPDLSCs, as well as the underlying mechanisms involved, were investigated. Cell Counting Kit-8 assay, alkaline phosphatase staining, Alizarin red staining, Oil Red O staining, reverse transcription-quantitative PCR and western blotting were used in the present study. It was demonstrated that resveratrol enhanced hPDLSC osteogenesis and reversed the inhibitory effects of TNF-α on this process. Further mechanistic studies indicated that resveratrol exerted anti-inflammatory activity by activating the ERK1/2 pathway, decreasing the secretion of interleukin (IL)-6 and IL-8 induced by TNF-α, and enhancing hPDLSCs osteogenesis. The present study suggested that resveratrol may be a novel and promising therapeutic choice for periodontitis.
Conventional therapies have succeeded in controlling periodontal inflammation but cannot restore the damage to periodontal tissues (
Decreased estrogen levels are the primary cause of osteoporosis in menopausal women. Accordingly, estrogen can be used to prevent postmenopausal osteoporosis (
Furthermore, the bone protective activities of resveratrol have attracted extensive interest (
Tumor necrosis factor-α (TNF-α) plays a critical role in inflammation, which can be rapidly secreted by macrophages and T lymphocytes (
In the present study, the effect of resveratrol and TNF-α on hPDLSC proliferation and osteoblast differentiation was investigated.
hPDLSCs were collected from wisdom teeth and premolars extracted from 20 individuals aged 12–20 years. Volunteers included 15 males and five females with a mean age of 14 years (age, 12–20 years). The recruitment was carried out between June and September 2017. All procedures were performed at the Department of Oral & Maxillofacial Surgery (School of Stomatology, Shandong University), and written informed consent was obtained from each participant and the legal guardians of all children. The extracted teeth were placed in α-Minimal Essential Medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with penicillin (400 U/ml) and streptomycin (400 mg/ml) on ice and were delivered to the Shandong Provincial Key Laboratory of Oral Tissue Regeneration immediately. Periodontal membranes were scraped from the middle third of the healthy non-carious root surface and cut into 1.0 mm3 fragments with an aseptic scalpel, as previously described (
The effect of resveratrol on hPDLSC viability was investigated using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.), according to the manufacturer's instructions. hPDLSCs were seeded into 96-well plates at a density of 3×103 per well. Cells were then cultured with complete medium (α-MEM containing 10% FBS) for 24 h at 37°C. After the cells attached to the flask, the culture medium was changed to complete medium containing TNF-α (1,5 and 10 ng/ml; PeproTech) and/or Resveratrol (0.1,1 and 10 µM; Sigma-Aldrich; Merck KGaA). The medium was subsequently changed every three days. On day one, three and five, the culture medium in each well was substituted for 10% CCK-8 reagent (100 µl). Next, the 96-well plate was incubated for 2 h in an incubator at 37°C. Absorbance was recorded at a wavelength of 450 nm using a SPECTROstar Nano ultraviolet spectrophotometer (Spectro Analytical Instruments GmbH).
To analyze cell surface marker proteins, hPDLSCs at passage 3 were collected using 0.25% trypsin and resuspended in PBS. In brief, 1×106 cells in suspension were incubated with fluorescent dye-conjugated monoclonal antibodies at 4°C for 20 min in the dark, washed three times using PBS (Corning, Inc.), and then analyzed using a flow cytometer and FlowJo® software (version 10.5.2; BD Biosciences) was used to analyze the data. The antibodies used for these experiments were CD34-PE (cat. no. 12-0349-41; 5 µl/test; eBioscience; Thermo Fisher Scientific, Inc.), CD44-PE (cat. no. 12-0441-81; 0.125 µg/test; Affymetrix; Thermo Fisher Scientific, Inc.), CD90Thy-1-PE (cat. no. 15-0909-42; 5 µl/test; Affymetrix; Thermo Fisher Scientific, Inc.), CD105-APC (cat. no. 17-1057-42; 5 µl/test; Affymetrix; Thermo Fisher Scientific, Inc.) and CD45-PE (cat. no. 12-0451-82; 0.125 µg/test; eBioscience; Thermo Fisher Scientific, Inc.).
ALP is an osteogenic marker expressed at the early stage of osteogenic differentiation (
hPDLSCs were seeded in 6-well plates at 1.5×105 cells per well and cultured for 21 days in osteogenic medium with or without resveratrol treatment, as indicated in the experiments (Resveratrol 1 µM; TNF-α 10 ng/ml). After treatment, the cells were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. After three washes with deionized water, hPDLSCs were stained with 1% alizarin red (pH 4.2, Beijing Solarbio Science & Technology Co., Ltd.) for 10 min at room temperature. Then, 600 µl of 10% (w/v) cetylpyridinium chloride (Sigma-Aldrich; Merck KGaA) was added to the stained wells and the absorbance of the extracted dye was assayed at 562 nm using a spectrophotometer (SPECTROstar Nano).
For adipogenic induction, hPDLSCs (1×104/well) were seeded in a 24-well plate. Then, the cells were exposed to adipogenic induction medium that included α-MEM containing 10% FBS, 0.2 mmol/l indomethacin (Sigma-Aldrich; Merck KGaA), 2 µM dexamethasone (Beijing Solarbio Science & Technology Co., Ltd.), 0.01 g/l insulin (Sigma-Aldrich; Merck KGaA) and 0.5 mmol/l isobutyl-meth-ylxanthine (Sigma-Aldrich; Merck KGaA). Following 4 weeks of induction, the lipid droplets were stained with Oil Red O (cyagen Biosciences, Suzhou, china) for 30 min at room temperature, and then observed with an inverted fluorescent microscope (Olympus Corporation) at a magnification of ×400.
hPDLSCs (2×105/well) were seeded in 6-well plates. Total RNA was isolated after seven, 14 and 21 days of culture using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized from 1.0 µg of total RNA using RevertAid First Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was performed utilizing the Roche Light Cycler 480 with a 10 µl reaction volume. The reaction system contained 5 µl SYBR® Premix Ex Taq™ (Takara Bio, Inc.), 1 µl cDNA, 0.2 µl of each primer and 3.6 µl RNase-free H2O, following the manufacturer's protocol. The thermal cycling conditions were as follows: Incubation at 95°C for 15 min, 45 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 35 sec, and a final extension at 72°C for 3 min. The relative expression levels of
To measure protein levels, hPDLSCs were cultured in a 6-well plate at a density of 2×105 per well for further treatment. Cells were cultured with osteogenic induction medium, osteogenic induction medium + Resveratrol 1 µmol/l, osteogenic induction medium + TNF-α 10 ng/ml or osteogenic induction medium + Resveratrol 1 µmol/l + TNF-α 10 ng/ml, respectively. After 3 weeks of osteogenic induction, cells were rinsed three times with PBS before being collected with RIPA buffer (Beyotime Institute of Biotechnology) for 30 min on ice. Then, the protein concentrations were measured using a bicinchoninic acid assay (Beijing Solarbio Science & Technology co., Ltd.). Next, 10 µg protein samples were separated using 10% SDS-PAGE gels and then transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, incubated with primary antibodies overnight at 4°C, and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (cat. no. SA00001-2 and SA00011; both at 1:10,000 dilution; Proteintech Group, Inc.) for 1 h at room temperature. The bands were detected using the chemiluminescent HRP substrate (EMD Millipore) and scanned utilizing an Amersham Imager 600 (GE Healthcare Life Sciences). Semi-quantitative analysis of western blotting was performed with the Image J software (version 1.47; National Institutes of Health). GAPDH is used as reference protein in the study. The primary antibodies and dilution ratios were as follows: Rabbit anti-human ALP (cat. no. ab108337; 1:20,000; Abcam), rabbit anti-human GAPDH (cat. no. cst2118; 1:1,000; Cell Signaling Technology, Ltd.), rabbit anti-human Runx2 (cat. no. cst12556s; 1:1,000: Cell Signaling Technology, Ltd.), rabbit anti-human ERK1/2 (cat. no. cst4695; 1:1,000; Cell Signaling Technology, Ltd.), and rabbit anti-phosphorylated (p)-ERK1/2 (cat. no. cst4370; 1:1,000; Cell Signaling Technology, Ltd.).
To activate the ERK1/2 pathway, hPDLSCs (2×105 per well) were seeded in a 6-well plate. The cells were cultured with α-MEM containing 10% FBS at 37°C for 24 h. Then, the cells were maintained in α-MEM containing 0.5% FBS for 48 h to synchronize the cells and reduce basal ERK1/2 activity (
To further clarify the effect of resveratrol (1 µmol/l) or resveratrol (1 µmol/l) plus PD98059 (10−5 mol/l) on TNF-α-induced inflammatory cytokine production, after TNF-α treatment, the levels of secreted IL-6 and IL-8 within the conditioned medium were measured using an ELISA kit (IL-6 cat. no. 430507 and IL-8 cat. no. 431507; BioLegend, Inc.) according to the manufacturer's instructions. The optical density values were measured using a microplate reader at 450 and 570 nm, and values at 570 nm were subtracted from the absorbance at 450 nm for subsequent data analysis.
All experiments were performed in triplicate. The significance of differences among the groups was assessed by one-way ANOVA followed by Tukey's post-hoc test. The significance of differences among multiple groups at different time points was assessed by two-way ANOVA followed by Tukey's post-hoc test. Data were analyzed using GraphPad Prism (version 6; GraphPad Software, Inc.). All data are presented as the mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.
The characteristics of hPDLSCs were confirmed by examining their morphology, multi-potency, and expression of cell surface markers. After seven days of incubation, hPDLSCs displayed a typical spindle-shaped morphology (
Resveratrol (10−8 to 10−5 M) was reported to increase human bone marrow-derived mesenchymal stem cell (BMSC) growth dose-dependently (
To examine the effect of resveratrol and TNF-α on osteogenesis, cells were cultured for 14 days with resveratrol (0.1, 1 and 10 µmol/l) or TNF-α (1, 5 and 10 ng/ml). After 21 days of culture, alizarin red staining was carried out (
Furthermore, the mRNA expression of
hPDLSCs were treated with resveratrol (1 µmol/l) and/or TNF-α (10 ng/ml) during the three weeks of osteogenic induction. Western blot analysis showed that protein levels of ALP and Runx2 in the TNF-α-treated group were significantly decreased compared with those in the control group (
To assess cytokine secretion in response to resveratrol (1 µmol/l) and TNF-α (10 ng/ml), hPDLSCs were treated with either TNF-α alone or with both TNF-α and resveratrol for 24 h. Data from RT-qPCR showed that the mRNA expression of
The MAPK pathway, comprised of serine/threonine protein kinases, plays an important role in regulating cell migration, proliferation and differentiation (
Plaque bacteria are the initiating factor of periodontitis, and the host reaction stimulated by these microorganisms is the main cause of periodontal tissue destruction (
In the present study, a comprehensive assessment of the effect of TNF-α and resveratrol on the proliferation and osteogenic differentiation of hPDLSCs was carried out. Results showed that TNF-α could significantly decrease hPDLSC viability and osteogenic differentiation at a high concentration (10 ng/ml), whereas co-treatment with TNF-α and resveratrol rescued such TNF-α-induced inhibitory effects.
TNF-α is a 17-kDa cytokine produced by mononuclear macrophages and other immune cells (
In addition, Feng
To the best of our knowledge, the effect of TNF-α on the osteogenic differentiation of periodontal ligament stem cells has not been previously reported. In the present study, the effect of different TNF-α concentrations on the proliferation and osteogenic differentiation of hPDLSCs was assessed. Data showed that TNF-α (10 ng/ml) could significantly inhibit both processes in hPDLSCs. Furthermore, the present study found that co-treatment of hPDLSCs with resveratrol and TNF-α could prevent this inhibitory effect. Similar protective effects of resveratrol in response to TNF-α have been were reported in cardiac stem cells (
Previous studies have also shown that resveratrol possesses potent bone-protective properties. Specifically, Song
Tseng
The present study was the first to find that Resveratrol can attenuate the secretion of inflammatory cytokines mediated by TNF-α. Upon further exploring the underlying mechanisms, it was found that the mRNA expression of
In conclusion, the present study is the first to demonstrate that resveratrol not only enhances the osteogenesis of hPDLSCs by activating the ERK1/2 pathway, but also exerts anti-inflammatory effects by activating this pathway. Ultimately, resveratrol decreases the TNF-α-mediated secretion of inflammatory cytokines and enhances osteogenesis in hPDLSCs. Therefore, resveratrol may be a good candidate for an anti-inflammatory agent that can induce bone remodeling in response to an inflammatory microenvironment. However, further
The authors would like to thank the Director of Shandong Provincial Key Laboratory of Oral Tissue Regeneration for providing technical support.
This work was supported by a grant from the National Natural Science Foundation of China (grant. no. 81371180).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JZ designed the experiments. JY and XW performed the experiments. JZ, XW, DM, HG and DZ analyzed the data. JY wrote the manuscript. All authors read and approved the final manuscript.
This study was approved by the Medical Ethical Committee of the School of Stomatology, Shandong University. Each participant and the legal guardian of all children provided written informed consent in accordance with The Declaration of Helsinki.
Not applicable.
The authors declare that they have no competing interests.
alkaline phosphatase
bone marrow-derived mesenchymal stem cells
human periodontal ligament stem cells
mesenchymal stem cells
tumor necrosis factor-α
Identification and characteristics of hPDLSCs. Morphological characterization of hPDLSCs in (A) primary culture and (B) culture at passage 3. A periodontal membrane fragment is present and shown in A. Flow cytometry analysis of surface markers expressed on hPDLSCs, showing that they were positive for (D) 0CD44, (F) CD90 and (G) CD105, and negative for (C) CD34 and (E) CD45. (H) Following 3 weeks of culture in osteogenic induction medium, the cells were stained with alizarin red. Mineralized nodules are shown. (I) Following 3 weeks of culture in adipogenic induction medium, the cells were stained with Oil Red O. Lipid globules are shown (black arrow). hPDLSCs, human periodontal ligament stem cells; PE, phycoerythrin.
Cell proliferation of hPDLSCs upon resveratrol and TNF-α treatment. Cell proliferation of hPDLSCs treated for 1, 3 or 5 days with (A) resveratrol (0.1, 1 and 10 µmol/l), (B) TNF-α (1, 5, and 10 ng/ml) or (C) resveratrol (1 µmol/l) and TNF-α (10 ng/ml), measured by the Cell Counting Kit-8 assay. All experiments were performed in triplicate. *P<0.05 and **P<0.01 vs. the control group. hPDLSCs, human periodontal ligament stem cells; TNF-α, tumor necrosis factor-α.
Effects of resveratrol and TNF-α at different doses on osteogenesis of hPDLSCs. (A) Representative image of hPDLSCs cultured for 14 days in osteogenic medium, osteogenic medium + TNF-α 10 ng/ml or osteogenic medium + resveratrol 1 µmol/l were used for ALP. Magnification, ×10. (B) Representative image of hPDLSCs were cultured for 21 days in osteogenic medium, osteogenic medium + TNF-α 10 ng/ml or osteogenic medium + resveratrol 1 µmol/l for alizarin red staining. Magnification, ×10). (C) Relative ALP activity. (D) Mineralized nodule analysis. Relative mRNA expression of (E)
Resveratrol rescues TNF-α-induced inhibition of osteogenesis in hPDLSCs and attenuates the secretion of inflammatory cytokines induced by TNF-α. hPDLSCs were treated with TNF-α (10 ng/ml), resveratrol (1 µmol/l) or both (A) Western blot analysis of ALP and Runx2 levels in hPDLSCs cultured for 21 days. Semi-quantitative analysis of (B) ALP and (C) Runx2 protein levels in hPDLSCs cultured for 21 days. Relative expression of (D)
ERK1/2 pathway is required for the inhibitory effects of resveratrol on TNF-α-induced inflammation and enhanced osteogenesis in hPDLSCs. Levels of p-ERK/total ERK were (A) determined by western blot analysis and (B) quantified in hPDLSCs treated with resveratrol (1 µmol/l) for the indicated times (0, 5, 15, 30 and 60 min). Levels of p-ERK/total ERK were (C) determined by western blot analysis and (D) quantified in hPDLSCs treated with resveratrol (1 µmol/l) or resveratrol (1 µmol/l) plus PD98059 (10−5 mol/l). hPDLSCs treated with TNF-α (10 ng/ml) and resveratrol (1 µmol/l) or TNF-α (10 ng/ml), resveratrol (1 µmol/l) and PD98059 (10−5 mol/l), then cultured for 24 h. (E) Relative mRNA expression levels of IL-6 and IL-8. Protein concentrations of (F) IL-6 and (G) IL-8. *P<0.05 and **P<0.01 vs. control. $P<0.05 vs. IL-6 (TNF-α + resveratrol) group. &P<0.05 vs. IL-8 (TNF-α + resveratrol) group. TNF-α, tumor necrosis factor-α; hPDLSCs, human periodontal ligament stem cells; p, phosphorylated; IL, interleukin.