The proliferation, migration and differentiation capacities of human periodontal ligament fibroblasts (HPDLCs) are important for the treatment of periodontal diseases. The aim of the present study was to investigate whether icariin could promote these abilities in HPDLCs, and explore the cellular mechanisms therein. The results indicated that icarrin markedly blocked apoptosis, and increased the viability and migration of HPDLCs, particularly at the concentrations of 20 and 50 µM. In addition, icariin significantly promoted HPDLCs to synthesize extracellular matrix, which was reflected by the decreased expression of matrix matalloproteinase-1 and increased expression of tissue inhibitor of metalloproteinase-1. Furthermore, the levels of bone morphogenetic protein 2, collagen I, osteoprotegerin and alkaline phosphatase were markedly elevated by icariin, indicating that icariin was able to promote the osteogenic differentiation capability of HPDLCs. Icariin also inactivated the Toll-like receptor 4 (TLR)-4/nuclear factor (NF)-κB signaling pathway by suppressing the expression levels of TLR-4 and phosphorylated p65, and by blocking p65 nuclear translocation. These results suggested that icarrin increased the survival, migration and osteoblastic differentiation of HPDLCs by inhibiting the TLR-4/NF-κB signaling pathway.
Human periodontal ligament, a band of fibrous connective tissue, has shock absorption ability and prevents tooth and alveolar bone injury during chewing (
Icariin (2-(4′-methoxyphenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(3′-methyl-2-butylenyl)-4-chromanone, the major active ingredient of epimedii, possesses a broad spectrum of pharmacological applications, such as the protection of bone, cartilage, neuro and cardiovascular system (
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade components of ECM (
In the present study, HPDLCs were isolated and cultured in the absence or presence of icariin at different concentrations to explore the molecular mechanisms of icarrin in HPDLCs. These results strongly demonstrated that icariin promote HPDLCs to differentiate into osteoblasts and stimulate ECM synthesis via suppressing TLR-4 and phosphorylation of NF-κB.
HPDLCs were isolated from the molar of a female patient (25 years old) at Shanghai Stomatological Hospital. Briefly, periodontal ligament tissue dissected from patients were chopped into pieces and digested with 0.25% Trypsin/EDTA (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C for 1 h. Then, cells were centrifugated at 1,000 × g for 10 min and subsequently cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. A complete clinical examination was performed in all participants, clinical data were recorded and written consent was obtained from each subject. The experimental protocols were approved by the Ethics Committee of Shanghai Stomatological Hospital (Shanghai, China).
Icariin was obtained from the Chinese National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Cell viability was measured using Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, HPDLCs were planted into a 96-well plate at the concentration of 5×103/well. After adherence, cells were treated with icariin at different concentrations for different times. Then, CCK-8 solution was added into cells, and cells were incubated for another 4 h. The average value of optic density was detected using a Microplate Spectrophotometer (Thermo Fisher Scientific, Inc.) at a wavelength of 450 nm.
HPDLCs were cultured in the in the absence and presence of various concentrations of icariin for 24 h. Then, cells packed by centrifugation at 1,000 × g. After washing with PBS for 3 times, cells were stained with Hoechst 33258 at 37°C for 5 min in the dark. Images (magnificaion, ×400) were captured under a confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).
2×104 cells in each group were seeded into serum-free medium in the insert coated with Matrigel (BD Biosciences, San Jose, CA, USA), and the lower chamber was filled with DMEM containing 10% FBS. After incubation for 24 h, the invaded cells were stained with crystal violet. Cells were counted in at least 6 randomly selected fields under a light microscope (Olympus, Tokyo, Japan).
Proteins were isolated from HPDLCs using a Total Protein Extraction kit (Applygen Technologies, Inc., Beijing, China). Then, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% BSA, membranes were incubated with primary antibodies specific for matrix matalloproteinase-1 (MMP-1), bone morphogenetic protein 2 (BMP2), collagen I (Col I), osteoprotegerin (OPG), toll-like receptor 4 (TLR-4), nuclear factor-κB (NF-κB), GAPDH (Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated IgG at room temperature for 1.5 h. Blots were observed using enhanced chemiluminescence.
HPDLCs were planted into a 96-well plate at the concentration of 2×105 cells/ml. After different treatment, the cells were incubated for another 24 h. Then, culture supernatants were collected and ALP level was assessed using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA). The experiment performed strictly according to the manufacturer's instructions.
HPDLCs were cultured in chamber slides, followed by different treatment. Then, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Trion and blocked with 5% BSA in PBS for 1 h at room temperature. Then, cells were incubated with NF-κB p65 (Abcam) at 4°C overnight, followed by incubation with second antibody for 30 min. After washing, cells were counterstained with DAPI. Cells were observed under a confocal laser scanning microscope (Carl Zeiss Group, Germany).
Cell apoptosis was measured using Annexin V Apoptosis Detection kit (BD Biosciences). Briefly, HPDLCs were treated with Icariin at different concentrations for 24 h. Then, cells were stained with Annexin V-FITC and PI for 15 min. Cell apoptosis was detected using FACS Calibur flow cytometer (BD Biosciences).
All statistical analysis was carried out using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Analysis was performed using one-way analysis of variance followed by a Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.
The structure of icariin was shown in
To investigate whether icariin was related to cell apoptosis, the morphological changes of icariin-treated HPDLCs were measured by Hoechst 33258 staining and flow cytometry. Results suggested that the icariin treatment decreased the cell apoptosis in a dose-dependent manner (
To explore the effect of icariin on the motility of HPDLCs, cell motility was measured using Transwell. As illustrated in
To determine whether icariin could affect the osteogenic differentiation of HPDLCs, the level of related proteins were measured using western blotting. Results suggested that, BMP2, Col I and OPG expressions were slightly elevated in icariin (20 µM) group and remarkably increased in icariin (50 µM) group compared with control group (
Recently, various researches have suggested that the activation of TLR-4/NF-κB pathway could inhibit osteogenic differentiation, cell viability and motility. Thus, TLR-4/NF-κB pathway was examined. It is observed that icarrin decreased TLR-4 expression and p65 phosphorylation in a dose-dependent manner (
HPDLCs is one of the most important cells in periodontal ligament. Due to the stem cell-like property, HPDLCs have potent proliferation, differentiation and migration abilities (
Both proliferation and migration of HPDLCs are essential for repair and regeneration. The results from current studies have demonstrated that icariin could promote the proliferation and migration of various cells. For example, icariin was found to tremendously enhance the osteoblast proliferation and Col I level using membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry (
Previous studies have demonstrated that ECM degradation is closely related to MMPs and TIMPs, for TIMPs help to regenerate ECM via binding to MMPs to decrease their activity (
Apoptosis, a common cellular behavior, has irreplaceable functions in multicellular organisms. Accumulated studies have indicated that HPDLCs apoptosis is closely associated with the development of periodontitis (
BMP2, Col I, OPG and ALP have potent capacity of promoting osteoblast differentiation and inducing osteogenesis (
It has been suggested and supported by studies that the inactivation of TLR-4 and NF-κB was involved in ECM synthesis and osteogenic differentiation (
In conclusion, the current study has illustrated that icarrin treatment decreased the apoptosis and increased the viability and migration of HPDLCs. However, HPDLCs have stem cell-like characteristics and the osteogenic differentiation and ECM synthesis abilities could be remarkably enhanced by icariin treatment via inactivating of TLR-4/NF-κB pathway. Our study is valuable for unraveling the underlying mechanism of icarrin as a candidate drug for periodontal diseases.
The authors would like to thank Shanghai Stomatological Hospital (Shanghai, China) for providing advice and technical support during the present study.
No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HJL analyzed and interpreted the main data regarding the cell function study and immunofluorescence. XYL was responsible for study design and the drafting of the manuscript. DBJ conducted the statistical analysis. All authors read and approved the final manuscript.
The experimental protocols were approved by the Institute Research Medical Ethics Committee of Shanghai Stomatological Hospital (Shanghai, China); written informed consent was obtained from all participants.
Written informed consent was obtained from all participants.
The authors declare that they have no competing interests.
extracellular matrix
matrix matalloproteinase-1
tissue inhibitor of metalloproteinase-1
bone morphogenetic protein 2
collagen I
osteoprotegerin
Toll-like receptor 4
nuclear factor-κB
Effect of icariin on HPDLC proliferation. (A) The structure of icariin. (B) HPDLC viability was detected by CCK-8 assay. Cells were treated with icariin at different concentrations (0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, 50, 100, 200, 300 and 400 µM) for 24 h. (C) Cell viability was measured using CCK-8 in HPDLC cells treated with icariin at different concentrations (0, 10, 20 and 50 µM) and different times (1, 2, 3 and 4 days). The experiments were repeated at least 3 times with similar results, and data are presented as the mean ± standard deviation. *P<0.05 vs. HPDLC. CCK, Cell Counting Kit; HPDLC, human periodontal ligament fibroblast.
Icariin suppresses the apoptosis of HPDLCs. HPDLCs were administered icariin at different concentrations (0, 10, 20 and 50 µM) for 24 h. (A) Cell apoptosis was determined using immunofluorescence assay (magnification, ×400). (B) Histogram representing the statistical analysis of the rate of cell apoptosis. The experiments were repeated at least 3 times with similar results, and data are presented as the mean ± standard deviation. *P<0.05 and ***P<0.001 vs. HPDLCs. HPDLC, human periodontal ligament fibroblast; PI, propidium iodide.
Icariin induces HPDLC motility and fibrosis. HPDLCs were administered icariin at different concentrations (0, 10, 20 and 50 µM) for 24 h. (A) A Transwell assay was performed to assess the migration ability of HPDLCs (magnification, ×400). (B) The expression levels of MMP-1 and TIMP-1 were evaluated by western blotting. The experiments were repeated at least 3 times with similar results, and data are presented as the mean ± standard deviation. *P<0.05 vs. HPDLCs. HPDLC, human periodontal ligament fibroblast; MMP-1, matrix matalloproteinase-1; TIMP-1, tissue inhibitor of metalloproteinase-1.
Icariin enhances HPDLC osteogenic differentiation ability. HPDLCs were administered icariin at different concentrations (0, 10, 20 and 50 µM) for 24 h. (A) The levels of BMP2, Col I and OPG were detected by western blotting. (B) ELISA was employed to evaluate the ALP levels. The experiments were repeated at least 3 times with similar results, and data are presented as the mean ± standard deviation. *P<0.05 vs. HPDLCs. HPDLC, human periodontal ligament fibroblast; BMP2, bone morphogenetic protein 2; Col I, collagen I; OPG, osteoprotegerin; ALP, alkaline phosphatase.
Icariin inactivates the TLR-4/NF-κB signaling pathway. HPDLCs were administered icariin at different concentrations (0, 10, 20 and 50 µM) for 24 h. (A) Western blot analysis was performed to detect the expression of TLR-4, p65 and p-p65. (B) The nuclear translocation of p65 was observed using an immunofluorescence assay (magnification, ×400). The experiments were repeated at least 3 times with similar results, and data are presented as the mean ± standard deviation. *P<0.05 vs. HPDLCs. HPDLC, human periodontal ligament fibroblast; TLR-4, Toll-like receptor 4; NF-κB, nuclear factor-κB; p-, phosphorylated.