Periodontal diseases are chronic inflammatory diseases that develop due to microbial dental plaque and its products, as well as the immune-inflammatory response initiated by the host against these products and various risk factors, resulting in the destruction of the supporting tissues of the teeth [1, 2]. According to studies conducted to explain the pathogenesis of periodontal disease, it is reported that the destruction in the periodontal disease process is affected by the long-term release of reactive oxygen species as well as the activation of the immune system [2,3,4].

Antioxidants are known to protect tissues from the destructive effects of inflammation and regulate the oxidative imbalance caused by inflammation [5]. Therefore, the homeostatic imbalance between reactive oxygen species and antioxidant defense systems plays a crucial role in the pathogenesis of periodontal diseases [6]. In recent years, the regulation of this balance by antioxidant molecules and their effects on various cell types have been subjects of research. For this reason, studies have intensified on the impact of antioxidants, which play a very important role in preventing oxidative damage, in the treatment of inflammatory diseases.

For example, Xiong et al. investigated the effects of quercetin, a natural antioxidant from the polyphenol group, on human gingival fibroblast cells stimulated with P. gingivalis lipopolysaccharide, and found that quercetin exhibited a dose-dependent anti-inflammatory effect [7]. In the study conducted by Li et al., the effects of resveratrol, another natural antioxidant, on gingival fibroblast cells stimulated with P. gingivalis LPS were investigated and according to the results of the study, strong anti-inflammatory and antioxidant effects were obtained [8]. In the study examining the effects of antioxidant ascorbic acid on the wound healing model created with gingival fibroblast cells, it was observed that ascorbic acid increased the expression of biomarkers related to wound healing activity [9]. In another in vitro study conducted by Nizam et al., it was determined that dose-dependent application of α-tocopherol, which has known antioxidant properties, accelerated the proliferation rate and wound healing process in periodontal ligament and gingival fibroblast cells [10].

There are studies in the literature examining the effects of various antioxidants on periodontal cells. However, the limited number of studies on the use of antioxidant natural molecules in the treatment of periodontal diseases, their healing effects on periodontal tissues, and their roles on periodontal cells indicate that further research on these antioxidants is needed. In line with this information, our study evaluated the effects of pterostilbene, a known potent antioxidant that has not previously been assessed for its effects in the field of periodontology, on gingival fibroblast cells.

Pterostilbene is a dimethylated analog of resveratrol, the most popular and widely studied of the stilbene group of antioxidants [11]. It acts as a phytoalexin, a compound that mainly functions to protect the plant against pathogens and other environmental stresses, and exhibits a range of pharmacological properties, notably anti-inflammatory, and antioxidant effects [12,13,14].

To the best of our knowledge, no studies have been encountered that investigate the effect of pterostilbene on periodontal diseases. Additionally, there is insufficient data regarding the potential cytotoxic and proliferative effects of pterostilbene on gingival fibroblasts. Therefore, we aimed to evaluate the anti-inflammatory, antioxidant, and wound-healing effects of various concentrations of pterostilbene on gingival fibroblast cells stimulated by lipopolysaccharide secreted by Porphyromonas gingivalis (P. gingivalis). With this purpose, a wound model created on gingival fibroblast cultures was used to determine these effects of PTS and we aimed to analyze the levels of IL-1β, TNF-α, IL-6, TGF-β, SOD, GSH-Px, bFGF, and collagen type I.

The present study was performed at the Gazi University Faculty of Medicine Immunology Laboratory. The study protocol was approved by Clinical Investigation Ethics Committee of Gazi University’s Faculty of Dentistry (ID E-21071282–050.99–462,173).

Human primary gingival fibroblast cells were purchased from ATCC (ATCC® PCS-201–018, Virginia, USA). The cells were thawed at 37 °C. The cells were centrifuged fibroblast growth kit (ATCC® PCS-201–041, Virginia, USA) and precipitated by centrifugation at 1200 rpm for 5 min. The supernatant was then discarded and the cell pellet was resuspended with the recommended fibroblast growth kit in 25 cm2 culture flask. The cultures were incubated in a humidified incubator with 5% carbon dioxide at a temperature of 37 °C. Upon reaching 80% to 90% confluence, the cells were dissociated using trypsin–EDTA solution (%0.05 trypsin–EDTA with phenol red, Gibco) at 37 °C for 10 min, followed by the establishment of subcultures in culture plates. The amplification of a sufficient number of cells used for MTT, wound model, and ELISA stages was accomplished by repeating these procedural steps.

LPS derived from P. gingivalis (InvivoGen US, LPS-PG Standard, San Diego, California) was purchased to induce an inflammatory response in gingival fibroblast cells. In our study, the most appropriate P. gingivalis LPS concentration was determined to be 1 μg/mL and LPS was prepared at a concentration of 1 μg/mL under the literatüre [7, 15,16,17]. 10 mg of pterostilbene (Phyproof® Reference Substances, trans-Pterostilbene, Vestenbergsgreuth, Germany) was prepared in 0.3 mL of dimethyl sulfoxide solution. Subsequently, to obtain a solution at the desired concentrations from the prepared stock solution, dilution was performed using the serial dilution method. Stock solution was diluted at 0.1 μM, 1 μM, 10 μM, 25 μM, 50 μM, 100 μM, 200 μM.

Pterostilbene concentrations prepared at different doses were applied to the wells containing the cells. 96 cell plate was placed in the incubator for 1 day. Then, 10 μL of MTT solution (Carl Roth GmbH & Co. Kg, Thiazolyl Blue, Karlsruhe, Deutschland) was pipetted into control and cell wells, and the cells were further incubated at 37 °C for 4 h. After removal from the incubator, 100 μL of sodium dodecyl sulfate solution was added to the cells, and the 96-well plate was left to incubate again overnight. Gingival fibroblast cells proliferation and viability were performed at 24 h. Absorbance was measured at 570 nm in an ELISA reader.

A wound healing kit (Ibidi® Culture-Insert 2 Well in μ-Dish 35 mm, Gräfelfing, Germany) consisting of three parts designed for cell adhesion and proliferation was used. Silicone inserts, which included two rectangular wells separated by a barrier measuring 500 mm in width, were placed in cell culture dishes with a diameter of 35 mm. 70 μL of cell suspension, containing 104 cells, was applied to each well of the silicone insert. The wound healing kit was then incubated at 37 °C in an environment with 5% CO2. Once the cells completely covered the wells, the silicone inserts were gently removed using sterile tweezers, creating a 500 μm wide cell-free area. The cell culture dishes were washed with phosphate-buffered saline and then filled with fresh medium for both control and test groups (control group, 1 μg/mL LPS, 1 μg/mL LPS + 1 μM PTS, 1 μg/mL LPS + 10 μM PTS). Images of the wound area were taken at 0, 24, 48, and 72 h under a fluorescence microscope (Leica DM4000) (Fig. 1). A transparent counting grid was used to count cells in standardized images. The cells in the wound area were counted and recorded, and the numerical data obtained were compared and evaluated as a percentage.

In vitro wound healing model images of human gingival fibroblast cells after 24, 48, and 72 h of incubation

The concentrations of IL-1β, TNF-α, IL-6, TGF-β, SOD, GSH-Px, bFGF and Collagen Type I in supernatants were measured with enzyme-linked immunosorbent assay (ELISA) using human IL-1β (Elabscience® Human IL-1β ELISA kit, Houston, Texas, USA), TNF-α (Elabscience® Human TNF-α ELISA kit, Houston, Texas, USA), IL-6 (Elabscience® Human IL-6 ELISA kit, Houston, Texas, USA), TGF-β ((Elabscience® Human TGF-β ELISA kit, Houston, Texas, USA), SOD (Cayman Chemical Superoxide Dismutase Assay Kit, Ann Arbor, Michigan, USA), GSH-Px (Cayman Chemical Glutathione Peroxidase Assay Kit, Ann Arbor, Michigan, USA), bFGF (Elabscience® Human bFGF/FGF2 ELISA kit, Houston, Texas, USA) and Collagen Type I (Elabscience® Human COL1α1 ELISA kit, Houston, Texas, USA) ELISA kits.

The statistical analysis was performed using the GraphPad Prism 9.5.1 software (GraphPad Prism Software Inc., San Diego, CA) program. Group comparisons were conducted using a one-way ANOVA, and differences between the two groups were assessed using Student's t-test. Statistical significance was determined when P < 0.05.