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Effects of Gossypetin from Hibiscus sabdariffa on Interleukin-6 Production in Porphyromonas gingivalis Lipopolysaccharide-Stimulated Human Gingival Fibroblasts
J Dent Hyg Sci 2023;23:296-301
Published online December 31, 2023;  https://doi.org/10.17135/jdhs.2023.23.4.296
© 2023 Korean Society of Dental Hygiene Science.

Ke Huang1 and Ki-Rim Kim2,†

1Department of Animal Science and Biotechnology, Research Institute for Innovative Animal Science, Kyungpook National University, Sangju 37224, 2Department of Dental Hygiene, Kyungpook National University, Sangju 37224, Korea
Correspondence to: Ki-Rim Kim, https://orcid.org/0000-0002-5967-6537
Department of Dental Hygiene, Kyungpook National University, 2559 Gyeongsang-daero, Sangju 37224, Korea
Tel: +82-54-530-1422, Fax: +82-54-530-1429, E-mail: rim0804@knu.ac.kr
Received November 6, 2023; Revised November 17, 2023; Accepted November 21, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Periodontal disease is a major cause of tooth loss in adults and is a representative oral disease commonly suffered by most people around the world. Mainly the proliferation of Gram-negative bacteria and secreted virulence factors cause an inflammatory response and destroy periodontal tissue. Gossypetin, isolated from Hibiscus sabdariffa L, is known to have various pharmacological effects, including antibacterial and anticancer activities. We aimed to confirm the anti-inflammatory effect of gossypetin through interleukin-6 (IL-6) regulation in human gingival fibroblasts (HGFs) stimulated with lipopolysaccharide (LPS) of Porphyromonas gingivalis, a major cause of adult periodontitis.
Methods: CCK-8 assay was performed to confirm the concentration-dependent cytotoxicity of gossypetin against HGFs. The secretion level and mRNA expression of IL-6, an inflammation-related cytokine, and the effect of gossypetin on these in HGFs stimulated with P. gingivalis LPS were confirmed by ELISA and qRT-PCR analysis, respectively.
Results: Up to a concentration of 100 μM gossypetin with or without P. gingivalis LPS, the survival rate for HGFs was maintained at over 95% and showed no toxicity. ELISA and qRT-PCR analysis results showed that P. gingivalis LPS increased IL-6 secretion and mRNA levels in HGFs compared to the control group. However, this increase in IL-6 was significantly down-regulated by gossypetin treatment in a dose-dependent manner. In particular, 80 μM gossypetin inhibited IL-6 production to the level of the control group.
Conclusion: These results indicated that gossypetin attenuated IL-6 production in HGFs stimulated by P. gingivalis LPS, which may ultimately suppress the inflammatory response in periodontal tissue. Therefore, gossypetin may have potential as a natural ingredient for the prevention and treatment of periodontal disease.
Keywords : Gingival fibroblasts, Gossypetin, Interleukin-6, Lipopolysaccharides, Porphyromonas gingivalis
Introduction

1.Background

Periodontal disease, including gingivitis and periodontitis, is a representative oral disease that affects many people worldwide1). If the inflammatory condition in which the gingiva swells and bleeds persists, it can progress to peri-odontitis, which destroys the alveolar bone that supports the teeth, ultimately leading to tooth loss and systemic inflammation1,2). Periodontal disease is associated with not only local risk factors such as dental plaque, a biofilm caused by oral microorganisms, but also various systemic factors such as smoking, diabetes, stress, and genetic fac-tors3,4). Therefore, compared to other oral diseases, perio-dontal disease is not easy to cure and is one of the diseases with a very high tendency to relapse.

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is one of the main causative bacteria of chronic periodontitis, and produces many toxic factors, worsening inflammation and directly or indirectly destroying perio-dontal tissue5). Lipopolysaccharide (LPS) from P. gingivalis plays an important virulence factor that causes periodontal disease progression by causing an inflammatory response in gingival tissue6). Human gingival fibroblasts (HGFs) are the most abundant cells in gingival connective tissue and contribute to the development of periodontitis caused by periodontal pathogens such as LPS7). HGFs induce an inflammatory response by producing inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1b, and IL-6 through stimulation of P. gingivalis LPS, and it leads to the activation of osteoclasts and destroys perio-dontal tissue7,8). Previous studies have reported that HGFs increased IL-6 production in periodontitis lesions and that large amounts of IL-6 were produced locally in periodon-titis patients9,10). Therefore, the amounts of cytokines are important in controlling inflammation in periodontal tissue.

Gossypetin, a flavonoid from Hibiscus sabdariffa that has been used as traditional herbal medicine due to its various pharmacological properties, is known to exhibit antio-xidant, anticancer, and antibacterial effects11-14). However, the effects of gossypetin on periodontitis have not yet been reported.

2.Objectives

Periodontal disease, which causes an extensive inflam-matory response in periodontal tissue, is the main cause of tooth loss in adults2). It is not easy to treat because it pro-gresses chronically due to an imbalance between the multi- species microflora and the host immune response15). There-fore, it is very important to control inflammation and maintain oral care consistently during the gingivitis stage, before periodontitis progresses. To this end, it is necessary to develop products for the prevention and treatment of periodontitis using natural substances with low toxicity and high biosafety. There is a lot of evaluation of the effe-ctiveness of various natural products related to various systemic diseases such as diabetes and cancer, but because research related to oral diseases is insufficient, the develop-ment of medicines for these diseases is not active. Acco-rdingly, we investigated the IL-6 regulatory effect of goss-ypetin, a flavonoid component of Hibiscus sabdariffa, in HGFs stimulated with P. gingivalis LPS, and aimed to provide basic data for the development of future perio-dontitis-related products.

Materials and Methods

1.Reagents

Gossypetin (purity: ≥93%) was purchased from INDO-FINE chemical Company (Hillsborough, NJ, US) and dissol-ved in dimethyl sulfoxide (DMSO). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phos-phate-buffered saline, antibiotic–antimycotic mixture contai-ning 100 U/ml of penicillin and 100 mg/ml of streptomycin, and 0.25% trypsin-ethylenediaminetetraacetic acid solution were obtained from Gibco BRL (Grand Island, NY, USA). LPS from P. gingivalis was purchased from InvivoGen (San Diego, CA, USA).

2.Cell culture

HGFs were obtained from the Department of Oral Bio-logy, College of Dentistry, Yonsei University (Seoul, Korea). HGFs were cultured in DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic mixture at 37°C in a humidified atmosphere with 5% CO2. In this experiment, HGFs from passages 3 to 6 was used.

3.Cell viability assay

HGFs (1×104 cells/well) were seeded into the 96-well plate and cultured in a complete medium overnight. Cells were incubated in serum-free media treated various con-centrations of gossypetin with or without 1 mg/ml P. gingivalis LPS for 24 hours. Viability of the cells was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instruc-tions. Absorbance was measured at 450 nm using a micro-plate reader (Thermo Fisher Scientific, Waltham, MA, USA).

4.Enzyme-linked immunosorbent assay (ELISA) assay

HGFs (2×105 cells/well) were plated into a 24-well plate and incubated with 1 mg/ml P. gingivalis LPS and various concentrations of gossypetin for 24 hours. IL-6 in the cell culture supernatants (100 ml/well) was quantified using an ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s protocols.

5.Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

HGFs were plated in 60 mm culture dishes and incu-bated with 1 mg/ml P. gingivalis LPS and various concen-trations of gossypetin for 24 hours. Cells were lysed and total RNA contents were isolated using a TRIzol reagent (Thermo Fisher Scientific), and total RNA was reverse transcribed into cDNA using the PrimeScrip 1st strand cDNA Synthesis Kit (Takara Bio Inc., Tokyo, Japan) acco-rding to the manufacturer’s protocol. qPCR was performed with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) using a StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific). The sequences of pri-mer used are as follows: IL-6 (5’-CGC CTT CGG TCC AGT TGC C-3’ forward, 5’-GCC AGT GCC TCT TTG CTG CTT T-3’ reverse); b-actin (5’-GAC TTA GTT GCG TTA CAC CCT TTC TTG-3’ forward, 5’-CTG TCA CCT TCA CCG TTC CAG TTT T-3’ reverse).

6.Statistical analysis

Data are expressed as the means±standard error from three independent experiments and analyzed by the Student’s t-test and one-way analysis of variance using SPSS statistical software version 25.0 (SPSS Inc., Chicago, IL, USA). Stati-stical significance was determined at p-value <0.05.

Results

1.Effect of gossypetin on the cell viability of HGFs

A CCK-8 assay was used to determine the cytotoxicity of gossypetin on HGFs. As shown in Fig. 1, cell viability was maintained above approximately 95% by treatment with 20 to 100 mM gossypetin for 24 hours compared to the control group treated with 0 mM gossypetin group. 1 mg/ml P. gingivalis LPS also showed no change in the viability of HGFs, and simultaneous treatment with LPS and gossy-petin did not affect the cell viability (Fig. 1).

Fig. 1. Effect of gossypetin from Hibiscus sabdariffa on the viability of human gingival fibroblasts (HGFs). HGFs were treated with gossypetin (0, 20, 40, 80 and 100 mM) with or without 1 mg/ml Porphyromonas gingivalis LPS for 24 hours, and cell viability was determined by Cell Counting Kit-8 assay. Data are expressed as the mean±standard error from three independent experiments. LPS: lipopolysaccharide. ap<0.05 versus 0 mM gossypetin group (control), bp<0.05 vs 0 mM gossypetin+P. gingivalis LPS group.

2.Effects of gossypetin on IL-6 production in P. gingivalis LPS-stimulated HGFs

To investigate the effects of gossypetin on the produc-tion of proinflammatory cytokine induced by P. gingivalis LPS in HGFs, the secretion level and mRNA expression of IL-6 were determined using ELISA and quantitative real- time PCR, respectively. As shown in Fig. 2A, the level of IL-6 in the culture medium of HGFs stimulated with P. gingivalis LPS increased compared to the control, and goss-ypetin dose-dependently inhibited the production of IL-6 induced by P. gingivalis LPS. In particular, 80 mM goss-ypetin suppressed IL-6 secretion to a level similar to that of the control group. In HGFs with P. gingivalis LPS treat-ment, the expression of IL-6 mRNA was up-regulated 4.0-fold compared to the control group, but only 1.5-, 1.3-, and 1.2-fold in the 20, 40, and 80 mM gossypetin-treated groups (Fig. 2B). Thus, gossypetin down-regulated the P. gingivalis LPS-increased IL-6 mRNA level in a concen-tration-dependent manner.

Fig. 2. Effect of gossypetin from Hibiscus sabdariffa on IL-6 production in Porphyromonas gingivalis lipopolysaccharide (LPS)-treated human gingival fibroblasts (HGFs). Cells were treated with 1 mg/ml P. gingivalis LPS and gossypetin (0, 20, 40, 80 mM) for 24 hours. (A) The level of IL-6 in the culture supernatant was detected using ELISA. (B) IL-6 mRNA expression was determined by qRT-PCR analysis, and bars was indicated as a fold change compared with the control. Data are expressed as the mean±standard error from three independent experiments. IL-6: interleukin-6. **p<0.005 vs P. gingivalis LPS alone group.
Discussion

1.Interpretation and comparison to previous studies

Periodontal disease is a chronic inflammatory and infec-tious disease caused by the complex toxicity of various oral microorganisms1). P. gingivalis, Aggregatibacter acti-nomycetemcomitans, and Tanerella forsythia, which are known to cause periodontitis, are mostly anaerobic Gram- negative bacteria, and periodontal disease-related various microorganisms, including these bacteria, interact and affect each other’s growth and activity3,16). In particular, LPS in the outer membrane of Gram-negative bacteria acts as an important endotoxin factor that stimulates the host’s immune system and causes inflammation17). It was reported that in P. gingivalis, the main bacterium in adult periodontal disease, the lipid A structure of LPS activates the Toll-like receptor 4 (TLR4)-mediated nuclear factor- kappa B (NF-kB) pathway in HGFs and regulates the expression of inflammatory cytokines IL-618). In addition, a previous study reported that P. gingivalis LPS induces an immune-inflammatory response by activating TLR2/4- mediated NF-kB/STAT3 pathways in microglial cells, and this result suggested the possibility that these periodontitis pathogens invade the central nervous system and induce neuroinflammation, causing cognitive disorders such as Alzheimer’s disease19). In this way, bacteria and their viru-lence factors increased in inflammatory lesions of perio-dontal tissue can affect not only diseases such as cardio-vascular disease and cancer, but also the central nervous system, causing neurological disorders20). Therefore, con-trolling the inflammatory response of periodontal disease can be a factor in lowering the risk of other systemic diseases.

Calprotectin, a molecule associated with inflammatory diseases, is also produced by neutrophils in inflamed peri-odontal tissues and induces the production of IL-6 from HGFs and the soluble form of the IL-6 receptor (sIL-6R) from macrophages10). The IL-6/sIL-6R complex induces the production of various periodontal tissue destructive substances from HGFs, thereby promoting periodontitis10,21). Therefore, because IL-6 is an important inflammatory cytokine as a biomarker in the pathophysiology of perio-dontitis, it is important to suppress the production of IL-6 in controlling inflammation.

2.Suggestion and limitations

In our study, IL-6 protein secretion and mRNA expre-ssion were obviously increased in HGFs by stimulation of P. gingivalis LPS, and were suppressed by gossypetin treatment in a concentration-dependent manner. Based on the report that P. gingivalis LPS activates the TLR4- mediated NF-kB pathway in HGFs and our previous study that gossypetin shows anticancer effects by downregu-lating the expression of phospho-NF-kB in oral cancer, it can be expected that the efficacy of gossypetin in modula-ting IL-6 would be the result of a mechanism that inhibits activation of the NF-kB pathway12,18). Additionally, we confirmed the anti-inflammatory effect of gossypetin in an animal study using a mouse ear edema model, which is commonly used to test anti-inflammatory activity (data not shown). As a result, topical application of gossypetin significantly attenuated ear edema induced by 12-O-tetra-decanoylphorbol-13-acetate, a known tumor promoter that induces the production of various inflammatory mediators through activation of mitogen-activated protein kinases and NF-kB22). Although the NF-kB pathway is important in the inflammatory response, our study is limited in that it did not confirm the molecular biological mechanism of the NF-kB signaling pathway of gossypetin in HGFs, so further research on this is required. Furthermore, it is necessary to confirm the action of gossypetin on the clinical symptoms and its mechanism studies through in vivo research using a periodontitis animal model.

3.Conclusion

We confirmed the effects of gossypetin derived from Hibiscus sabdariffa on the inflammatory response in peri-odontal disease. In HGFs stimulated with P. gingivalis LPS, the secretion amount and mRNA expression of IL-6, an inflammation-related cytokine, increased. However, gossypetin statistically significantly inhibited the production of IL-6 increased by P. gingivalis LPS in a concentration- dependent manner. Consequently, the IL-6 regulating effect of gossypetin can be expected to have the potential to be developed as a natural ingredient in periodontitis-related prevention and treatment products.

Acknowledgments

None.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Ethical Approval

Not applicable.

Author contributions

Conceptualization, funding, supervision, and writing- review & editing: Ki-Rim Kim. Experiments, Data acqui-sition, formal analysis, and writing–original draft: Ke Huang.

Funding

This research was supported by Kyungpook National University Research Fund, 2021.

Data availability

Data supporting the results of this study are available from the corresponding author upon reasonable request.

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