Thymol Inhibits LPS-induced Inflammation in Rat Gingival Fibroblasts by Regulating Inflammatory Pathways

doi:10.13701/j.cnki.kqyxyj.2025.09.008

Abstract

Abstract: Objective: To investigate the effect of thymol on Porphyromonas gingivalis (P. g) and lipopolysaccharide (LPS)-induced inflammatory response in rat gingival fibroblasts. Methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of thymol against P. g were determined by microdilution method. Biofilm adhesion reduction-crystal violet assay was used to determine the anti-biofilm activity of thymol against P. g. Bacteria were detected by scanning electron microscopy and confocal scanning laser microscopy (CSLM). Rat gingival fibroblasts were used to establish periodontitis models in vitro: control group, LPS + different concentrations of thymol (0, 10, 20, and 40 μg/mL) groups. ELISA was used to measure the expression of related proteins in the supernatant, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), osteoclastogenesis inhibitory factor (OPG) and soluble receptor activator of nuclear factor-κB ligand (sRANKL), and OPG/sRANKL ratio was calculated. Western blot was used to detect the phosphorylation of NF-κB p65 and IκBα in the nuclear protein of gingival fibroblasts. Results: Thymol attenuated P. g toxicity and biofilm formation. Thymol had no adverse effect on LPS-induced growth of rat gingival fibroblasts. Compared with LPS group, the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the cell supernatant were significantly decreased after Thymol stimulation, while the expression level of anti-inflammatory factor IL-10 was significantly increased. The ratio of OPG/sRANKL was increased. LPS-induced phosphorylation of NF-κB p65 and IκBα was significantly decreased(P＜0.001). Conclusion: Thymol inhibits P. g toxicity and biofilm formation, and inhibits LPS-induced inflammation in GFS by regulating the expression of related inflammatory factors (TNF-α, IL-1β, IL-6, IL-10, OPG/sRANKL) and inflammatory pathways (NF-κB p65, IκBα).

Key words: thymol, Porphyromonas gingivalis, lipopolysaccharide, gingival fibroblasts, NF-κB signaling pathway

GUO Jia, GAO Mengjie, NIU Hui, LIU Danfeng, CHEN Xi. Thymol Inhibits LPS-induced Inflammation in Rat Gingival Fibroblasts by Regulating Inflammatory Pathways[J]. Journal of Oral Science Research, 2025, 41(9): 781-787.

References

[1] Bui FQ, Almeida-da-Silva CLC, Huynh B, et al. Association between periodontal pathogens and systemic disease[J]. Biomed J, 2019, 42(1):27-35.
[2] Dye BA. Global periodontal disease epidemiology[J]. Periodontol 2000, 2012, 58(1):10-25.
[3] Li YY, Cai Q, Li BS, et al. The effect of Porphyromonas gingivalis lipopolysaccharide on the pyroptosis of gingival fibroblasts[J]. Inflammation, 2021, 44(3):846-858.
[4] Sahoo CR, Paidesetty SK, Padhy RN. The recent development of thymol derivative as a promising pharmacological scaffold[J]. Drug Dev Res, 2021, 82(8):1079-1095.
[5] Marchese A, Orhan IE, Daglia M, et al. Antibacterial and antifungal activities of thymol: A brief review of the literature[J]. Food Chem, 2016, 210:402-414.
[6] Sapkota M, Li L, Kim SW, et al. Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMM cells and LPS-induced bone loss in mice[J]. Food Chem Toxicol, 2018, 120:418-429.
[7] Alfonso García SL, Parada-Sanchez MT, Arboleda Toro D. The phenotype of gingival fibroblasts and their potential use in advanced therapies[J]. Eur J Cell Biol, 2020, 99(7):151123.
[8] Jentsch HF, Eckert FR, Eschrich K, et al. Antibacterial action of chlorhexidine/thymol containing varnishes in vitro and in vivo[J]. Int J Dent Hyg, 2014, 12(3):168-173.
[9] Chatrath A, Gangwar R, Kumari P, et al. In vitro anti-biofilm activities of citral and thymol against Candida tropicalis[J]. J Fungi (Basel), 2019, 5(1):13.
[10] Kirkwood KL, Li F, Rogers JE, et al. A p38alpha selective mitogen-activated protein kinase inhibitor prevents periodontal bone loss[J]. J Pharmacol Exp Ther, 2007, 320(1):56-63.
[11] Naruishi K, Nagata T. Biological effects of interleukin-6 on gingival fibroblasts: Cytokine regulation in periodontitis[J]. J Cell Physiol, 2018, 233(9):6393-6400.
[12] Lopez-Castejon G, Brough D. Understanding the mechanism of IL-1β secretion[J]. Cytokine Growth Factor Rev, 2011, 22(4):189-195.
[13] Pan W, Wang Q, Chen Q. The cytokine network involved in the host immune response to periodontitis[J]. Int J Oral Sci, 2019, 11(3):30.
[14] Saraiva M, O'Garra A. The regulation of IL-10 production by immune cells[J]. Nat Rev Immunol, 2010, 10(3):170-181.
[15] Sojod B, Chateau D, Mueller CG, et al. RANK/RANKL/OPG signalization implication in periodontitis: New evidence from a RANK transgenic mouse model[J]. Front Physiol, 2017, 8:338.
[16] Liu B, Wu Y, Wang Y, et al. NF-κB p65 knock-down inhibits TF, PAI-1 and promotes activated protein C production in lipopolysaccharide-stimulated alveolar epithelial cells type Ⅱ[J]. Exp Lung Res, 2018, 44(4-5):241-251.