Dose-dependent green tea effect on decrease of inflammation in human oral gingival epithelial keratinocytes: in vitro study

This in vitro study aimed to analyze the anti-inflammatory and wound healing potential of green tea extract (GTE) in human gingival epithelial keratinocytes (HGEK) treated with lipopolysaccharides (LPS). A cell viability assay was conducted using MTT to determine nontoxic levels of GTE on immortalized HGEK. Cells were concomitantly treated with LPS (1 μg/ml) and GTE (1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) to assess inflammation. Gene expression levels of inflammatory markers IL-β1, IL-6, and TNFα were measured by RT-PCR and their protein production was assessed by ELISA. The scratch wound healing assay was used to investigate the effects of different concentrations of GTE on cell migration. We also explored the effect of GTE on the induction of the Nrf2/HO-1 pathway in the cells with or without LPS. GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml significantly enhanced cell viability (p < 0.05). And IL-β1, IL-6, and TNFα gene expression presented up to 10-fold decrease compared with LPS-treated cells, which was also similarly found on the protein levels. At the same concentrations, cell migration increased. The mechanism results showed that GTE produced the anti-inflammatory response by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and increasing the level of anti-oxidant protein heme oxygenase-1 (HO-1). GTE may be potentially used as oral rinse anti-inflammatory drug for treatment and prevention of oral inflammatory diseases, which is shown here by the ability to reduce the inflammation and increase in cell migration in a dose-dependent manner.


Introduction
Growing interest in natural remedies has brought about an increase in scientific reports on substances such as GTE (Camellia sinensis). Green tea is a worldwide popular drink that has many compounds containing large amounts of important antimutagenic and anticarcinogenic polyphenol nutrients used medicinally in Asia for 5000 years [1]. Green tea consumption has been associated with health promotion as it is a natural product without side effects [2]. Components of GTE are basically known as polyphenols, such as epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG) [3][4][5]. Among the green tea polyphenol components, EGCG was found to be the most abundant constituent with over 30% of total catechins [6], and it was shown to reduce dentin surface loss under erosive/ abrasive conditions in oral mouth rinsing [7][8][9]. EGCG has also been suggested to influence the cell anti-inflammatory activity by suppressing the synthesis and inhibiting action of many pro-inflammatory mediators, nitric oxide synthase, peroxynitrite, reactive oxygen/nitrogen species, and cyclooxygenase-2 [10][11][12][13][14][15][16]. Oral tissue inflammation is primarily initiated by epithelial gingival cells and macrophages when exposed to pro-inflammatory cytokines, interferon-γ, or oral bacterial LPS (lipopolysaccharides) [17]. Activation of macrophage may stimulate the release of inflammatory mediators also known as pro-inflammatory cytokines (interleukin-β1, interleukin-6, tumor necrosis factor α) [17,18]. The production of inflammatory mediators and cytokines in prolonged oral tissue inflammation (i.e., periodontitis) can cause extensive cellular and tissue destruction.
To better prevent or avoid prolonged side effect of oral tissue inflammation, development of more antiinflammatory agents is still needed as to be employed in treatment of oral diseases. In recent years, the inflammatory-inhibitory potential of naturally derived GTE substances has gained increasing attention. In comparison with steroidal or non-steroidal chemical drugs for treating inflammation, naturally derived substances for preventing prolonged inflammation have limited side effects and fewer intolerance issues, and could be available at lower costs than synthetic drugs [10]. Previous studies have shown green tea polyphenols, such as EGCG, to inh i b i t m a t r i x -m e t a l l o p r o t e i n a s e -2 a n d m a t r i xmetalloproteinase-9 key proteases involved in destructive periodontal diseases [19]. However, the exact role of these EGCGs in mediating inflammation on oral tissues is still unknown since most of previous research have been focusing on the use of green tea EGCC on reducing oral hard tissue erosion-abrasion [7,9]. As previous studies have focused on the use of green tea extract polyphenols on inflammation in other cell line types and in animal models, it seems expedient to study the application of green tea phenols for their potential to block cytokine-involved inflammatory cascades in oral cells. Further, more knowledge is needed on how these EGCG polyphenols would improve oral tissue wound healing. In addition, other pathways were pointed to assist on progression of inflammation and one of these pathways is the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway [20]. The activation of Nrf2 triggered by medication has increasingly gained attention on its possible use in therapies for inflammatory disorders [21,22] and proved that Nrf2 pathway indeed may play a critical role in inflammation. However, there have been only few Nrf2 activators found to be employed to support oral therapies. Thus, the development of new activating clinical drugs of Nrf2 pathway should be an important goal in the pharmaceutical industry.
Accordingly, the purpose of the present study was to analyze the distinct anti-inflammatory activity and wound healing potential, given the ease of application, of green tea extract topically on the oral epithelium via oral mouth rinse hygiene products. To this end, we have demonstrated the antiinflammatory potential of green tea extract in human gingival epithelial keratinocytes treated with periodontopathogenic bacterial endotoxin (LPS) by assessing the positive effects on cell viability, wound healing, and downregulation of important inflammatory markers. Also, our findings suggest that GTE could be a strong potential Nrf2 activator for the treatment and prevention of oral inflammatory diseases.
Gingival epithelial keratinocytes (0.1 × 10 6 cells/ml) were recultured in 12-well plates supplemented with fresh medium containing different concentrations of GTE (1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml). After an exposure time of 24 h, the solutions were aspirated and the cells washed with PBS 1× before culture medium was newly added. At 24 h after exposure to the respective GTE solution, 500 ml of MTT was added to each well and incubated for 4 h at 37°C in the dark. In the next step, MTT was removed by aspiration from the wells and isopropanol was added (200 ml, 1 N HCl) to solubilize the MTT-formazan crystals formed. The test absorbance at 570 nm and reference absorbance at 630 nm were measured using a spectrophotometer plate reader (Corning Costar, Corning, NY, USA). Experiments were performed in triplicate.

Scratch wound healing migration assay
To determine the effect of GTE concentrations on wound healing, a scratch-wounded monolayer model was used. The cells were seeded at a density of 0.1 × 10 6 cells/ml and cultured into each well of a 12-well plate and incubated for 24 h at 37°C until confluent. Prior to the scratch assay, the cells were exposed to 10 μg/ml of mitomycin C (Sigma-Aldrich) in serum-free media for 2 h, which inhibited mitosis of the cells. The wound was produced by scratching with a 10-μl pipette tip (700-900 μm in diameter). Following PBS 1× washes to remove cell debris, the remaining adherent cells were divided into 6 treatment groups: (1) negative control (PBX 1×); (2) positive control (1 μg/ml LPS from P. gingivalis, Sigma-Aldrich); (3) HGEK treated with 1 μg/ml LPS and 1 mg/ml GTE; (4) HGEK treated with 1 μg/ml LPS and 2.5 mg/ml GTE; (5) HGEK treated with 1 μg/ml LPS and 5 mg/ml GTE; (6) HGEK treated with 1 μg/ml LPS and 10 mg/ml GTE. Digital images were captured using a camera-equipped, inverted microscope (Carl Zeiss, Inc., Thorwood, NY, USA) and wound width measurements were subtracted from wound width at time 0 to obtain the net wound closure. The distance between edges of injured monolayers was measured using the ImageJ software (National Institutes of Health, USA) in pixels and wound closure was expressed as the difference in widths at 0 h and 24 h after wound simulation.

Western blot analysis
Western blotting was performed to determine the protein expression of Nrf2 and HO-1 proteins after cell treatment with LPS and GTE. The cells (0.1 × 10 6 cells/ml) were seeded in a 12-well plate and incubated for 24 h at 37°C. After incubation, the cells were treated with various concentrations of GTE (1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) and exposed to LPS (1 μg/ml) for 24 h. Protein was extracted with radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail and centrifuged at 12,000×g for 15 min at 4°C. The supernatant protein was quantified using a bicinchoninic acid assay (BCA, Thermo Fisher Scientific, Rockford, USA) and stored at − 80°C. Total lysates were resolved in SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane and incubated with primary antibodies and the corresponding secondary antibodies. Immune complexes were visualized by the use of an enhanced chemiluminescence western blotting system (BioRad, Richmond, CA).

Statistical analysis
The mean values and standard deviations were computed for the cell viability test and multiple comparisons were conducted by analysis of variance (ANOVA), followed when appropriate by Bonferroni post hoc tests with a global significance level of 5% to assess the statistical significance of the differences between the experimental groups (SPSS version 22.0, Munich, Germany). All the in vitro experiments were performed in triplicate and from three independent experiments unless otherwise mentioned. Differences were considered significant at p < 0.05.

Effect of GTE on HGEK cell viability
The cell viability, as a preliminary study, was shown to be significantly enhanced after GTE exposure at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml compared with the cells cultured without treatment (p < 0.05 at 24 h) and with LPS treatment by performing the cell viability assay. Based on these results and previous studies, the green tea extract concentrations of 1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml were selected for further experiments. GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml showed significant increase a maximum of 1.5-fold compared with untreated control, while 1 mg/ml did not increase viability (Fig. 1).

Green tea extract downregulation of markers for inflammation
To investigate whether GTE could inhibit LPS-induced inflammatory gene expression of IL-β1, IL-6, and TNFα, cells were simultaneously pretreated with GTE (1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) and LPS (1 μg/ml) for 24 h. The results showed that LPS (1 μg/ml) treatment remarkedly increased by almost 10-fold the expression of IL-β1, IL-6, and TNFα compared with untreated cells (Fig.  2a). Thus, LPS-treated keratinocytes showed the highest level of all pro-inflammatory cytokines and modulators tested in this study. Further, the positive control LPS (1 μg/ml) was used as a standard to obtain GTE inflammatory inhibition activity toward IL-β1, IL-6, and TNFα expression. GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml decreased all pro-inflammatory cytokines tested in this study compared with each positive control (8-fold change decrease) (LPS 1 μg/ml) (Fig. 2a). The protein levels of IL-β1, IL-6, and TNFα by ELISA analysis have also shown significant decrease on GTE test cell groups (GTE at 2.5 mg/ml, 5 mg/ml, and 10 mg/ml and LPS at 1 μg/ml) compared with the positive control (LPS 1 μg/ml) (p < 0.05), confirming the RT-PCR results (Fig. 2b).
Scratch wound healing assay (cell migration) HEGK cells exposed to GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml similarly elicited increase in cell migration at 24 h compared with untreated negative control and positive control (LPS 1 μg/ml) (up to 40%). Wound closure was observed to be almost complete after 24 h in the presence of GTE (2.5 mg/ml, 5 mg/ml, and 10 mg/ml). Positive and negative controls showed around 80% incomplete healing patterns at 24 h (Fig. 3).

Mechanism analysis via Nrf2 signaling pathway
In order to understand whether GTE could exert its antiinflammatory effect through activating the Nrf2 pathway, the downstream protein HO-1 of the Nrf2 pathway was investigated in this study. Compared with LPS-stimulated HEGK cells, GTE (10 mg/ml) increased Nrf2 and HO-1 gene expression and protein production (p < 0.05, Fig. 4a, b).

Discussion
The purpose of this study was to ascertain the antiinflammatory and wound healing stimulating effects of green tea extract on human gingival epithelial keratinocyte cells challenged with LPS. Our findings sustain the hypothesis that GTE is a powerful anti-inflammatory agent and in a dose-  reported biological response in other cell types, such as antioxidant, antimicrobial, and anti-inflammation effects of EGCG which is one of green tea's compounds [10][11][12][13][14][24][25][26]. Green tea also has shown antimicrobial activity against most oral bacteria. Additionally, it may enhance oral peroxidase activity and prevent the establishment and progression of periodontitis [27][28][29]. Based on its anti-inflammatory and antioxidant effects, green tea EGCG could be considered for treatment of innumerous diseases, including neurological diseases, diabetes, and hypertension [30][31][32]. GTE compounds can suppress nuclear factor Bkappa-light-chain-enhancer^of activated B cells and downstream signaling inflammatory mediators (nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, prostaglandine-E2) in vitro and could be responsible for its significant anti-cancer or chemopreventive activity [33]. Our results (Fig. 1) suggested that GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml has a positive effect on oral gingival keratinocyte cell viability by almost duplicating viability compared with the non-treated control. However, it was shown that green tea EGCG could provoke increased cytotoxicity in cells when applied at higher concentrations. An EGCG concentration higher than 100 μM was reported to cause production increase of H 2 O 2 and oxidative DNA damage, while concentrations of more than 200 μM EGCG could even affect cell cycle progression [34,35]. Consequently, the concentration of EGCG in the test green tea solution should be carefully controlled for in in vitro assay applications. In this study, green tea solutions containing a maximum of ≈ 50 μM EGCG concentration were observed to be safe based on the cell viability test (Fig. 1). GTE solutions at 2.5 mg/ml, 5 mg/ml, and 10 mg/ml concentrations downregulated the expression of inflammation markers IL-β1, IL-6, and TNFα at 24 h (Fig. 2a) and reduced protein production of the same analyzed genes (Fig.  2b). IL-β1, IL-6, and TNFα decreased in gene expression compared with those in the control, and the combination with GTE (at 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) and LPS (1 μg/ml) still showed the positive effects of GTE by reducing inflammatory status. LPS is an important component of the outer membrane of Gram-negative bacteria, which promotes cellular signal transduction through toll-like receptors and secretion of proinflammatory interleukins, eicosanoids, and nitric oxide [36]. As seen in cells treated only with LPS ( Fig. 2a, b, darker columns), the gingival keratinocytes treated with lipopolysaccharide here resulted in a significant increase of IL-β1, IL-6, and TNFα gene expression and protein production compared with the negative control. This is a predicted biological reaction to LPS bacterial endotoxin, especially from the highly oral pathogenic bacteria P. gingivalis. A decrease in inflammation was also observed in previous in vitro studies using different cell lines, which showed GTE's potential for use as antiinflammatory drugs, providing its ability to reduce the production of nitric oxide, cyclooxygenase-2, interleukin-6, interleukin-1β, and tumor necrosis factor α in active macrophages [37][38][39]. Tumor necrosis factor α is responsible for increase in levels of inflammation by upregulating other proinflammatory cytokines (e.g., interleukin-6, interleukin-1) and consequently inducing angiogenesis and nuclear factor Bkappalight-chain-enhancer^of activated B cell transcription, and stimulating nitric oxide production [40]. Tumor necrosis factor α has been targeted by anti-inflammatory screening agents due to its multiple roles in inflammation [41]. Interleukin-β1 induces secretion of interleukin-6 and interleukin-8 which also play a role as pro-inflammatory cytokines and are important for the initiation and increase of the inflammatory response to microbial infection [42]. In addition, GTE anti-inflammatory effect was confirmed in animal studies on LPS-induced retinal inflammation [43,44].
Regarding the possible wound healing effect of green tea via our in vitro scratch wound healing assay, increase in difference with regard to the control was found with the GTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml at 24 h, while 1 mg/ml stimulation was non-significant. Despite GTE Fig. 4 The effect of GTE on the activation of Nrf2 and the expressions of HO-1 in LPSstimulated oral keratinocytes. The total proteins of the cells were prepared and the expressions of Nrf2 (a) and HO-1 (b) were analyzed using western blot. Results are expressed as mean ± standard deviation of three independent experiments (n = 3). *p < 0.05 component's EGCG association with wound healing in pharmacological animal studies [45,46], in vitro studies with cancer cells showed that EGCG significantly decreased or inhibited cell migration in a dose-dependent manner [47,48]. Based on our experimental observations, normal gingival keratinocytes showed an ≈ 50% increase in cell migration or change in migrating behavior after exposure to GTE (Fig. 3). In contrast to its effect on normal keratinocytes, GTE component's EGCG was found to be specially antiproliferative and apoptotic on cancer cells [48][49][50][51][52]. However, it has been suggested that these effects of EGCG are cancer specific and EGCG shows pronounced growth inhibitory effect on cancerous cells but not on normal cells [53]. Another study demonstrated that, in cultured skin keratinocytes, EGCG acts to enhance differentiation without triggering apoptosis [54]. Topical application of EGCG on human skin which has been identified and reported earlier results in increased cell proliferation and reduced keratinocyte apoptosis [55]; however, to the best of our knowledge, there is no report of green tea on oral cells. Our results suggest that oral gingival epithelial keratinocytes presented significant decrease in cell mobility/ migration activity challenged with P. gingivalis LPS (1 μg/ml) at 24 h (Fig. 3). Moreover, GTE at concentrations of 2.5 mg/ml, 5 gm/ml, and 10 mg/ml could successfully stimulate cells in an inflammatory environment when exposed to LPS. The unchanged migration found after 1 mg/ml GTE exposure could be explained by its lower concentration and EGCG content, which reduces green tea stimulation potency. And it indicates that there is possibly a threshold for gingival keratinocytes' responsiveness to green tea. We also have to consider that any difference in green tea effects on migration likely reflects relative sensitivity of specific cell types to LPS. But further examinations of molecular basis are still needed to support this hypothesis. Regarding the signaling pathway analysis, our results showed that GTE may activate the Nrf2 pathway, bringing into play its anti-inflammatory effect on cells exposed to LPS (Fig. 4a). Also, the Nrf2-dependent anti-oxidant gene HO-1 was also increased (Fig. 4b). Some reports previously confirmed that the activation of the Nrf2 pathway prevents LPS-induced upregulation of proinflammatory cytokines, such as IL−β1, IL-6, and TNFα analyzed here [20,56]. Drugs used to activate Nrf2 pathway could be considered for use as potential oral therapies for oral inflammatory diseases, such as gingivitis or periodontitis. Until today, there are few Nrf2 activating medicaments used in the dental clinic. Therefore, GTE could be used to develop new and safer Nrf2 activators for clinical use in dentistry.
This study has taken a step in the direction of defining the effect of green tea extract on inflammation suppression associated with wounding. However, the results are neither viewed nor are they presented as conclusive. In addition, it is important to emphasize that problems in methodological research design of in vitro assays place limitations on interpretations.
Further, the potential for green tea catechins to aid in epithelial formation bears further study. Although this study did not support the finding of earlier animal studies [57], other research designs may yet show a benefit in its use. In addition, in vivo angiogenesis and granulation tissue augmentation by GTE have been demonstrated [30]. To our knowledge, there is a lack on studies evaluating GTE's effect on oral epithelial healing. Therefore, further animal model research is needed.

Conclusions
In summary, our results sustained the hypothesis that GTE attenuates the inflammation in gingival epithelial keratinocytes treated with LPS by downregulating inflammatory cytokines in a dose-dependent manner. The results from our experiment support that GTE can be considered a potent anti-inflammatory agent with a potential as an oral therapeutic against inflammation.