Investigation of Anti-inflammatory Activities of Methanol Extract of Nigella Sativa Seed and Thymoquinone in Human Gingival Fibroblasts

  • Ayokulehin Muse Kosoko

Student thesis: Doctoral Thesis


Studies have revealed the role of inflammation in the onset, progression and pathogenesis of gingivitis and periodontitis. Nigella sativa (NS) is known for its anti-inflammatory activities. This study therefore investigated the role of NS methanol extract and its bioactive component, thymoquinone (TQ) on gingival inflammation in Porphyromonas gingivalis lipopolysaccharide (P.g.LPS)-stimulated human gingival fibroblasts (HGFs). HGFs in culture were stimulated with P.g.LPS (100 ng/mL) following pre-treatment with NS (12.5, 25 and 50 μg/mL) or TQ (2.5, 5 and 10 μM). Levels of NO., PGE2, IL-1β, IL-6, IL-8 and TNF-α production were measured from culture supernatants by ELISA. Protein levels of COX-2, iNOS, phosphorylated-p38, Nrf2, HO-1 and NQO-1 were determined by western blot. Phosphorylation of NF-κB-p65 sub-unit protein expression was determined by In-cell western and ELISA. Phosphorylated-IκB-α, total-IκB, phosphorylated-ERK1/2, phosphorylated-JNK1/2, pro-and active-MMP-9 from extracts were also determined by ELISA. HGFs were transfected with Promega pGL4.32[luc2P/NF-κB-RE/Hygro] vector and reporter gene assays were employed to explore the effect of NS on NF-κB luciferase activity in the nucleus. NS methanol extract blocked the production of NO., PGE2, IL-6 and TNF-α in P.g.LPS-stimulated HGFs. Protein levels of phosphorylated-NF-κB-p65 sub-unit as well as phosphorylated-IκB-α were significantly suppressed by NS while an elevation in total-IκB protein level was observed. NF-κB binding as well as its transactivational activities were markedly inhibited by NS in P.g.LPS-stimulated HGFs. TQ significantly inhibited the production of IL-1β, IL-6, IL-8 and TNF-α. COX-2 and iNOS mediated production of PGE2and NO. respectively were significantly blocked by TQ in P.g.LPS-stimulated HGFs. Increases in P.g.LPS-mediated phosphorylation of ERK1/2, p38 and JNK1/2 MAPKs was significantly blocked by TQ. Pro-MMP-9 protein as well as the secretion of active enzyme was also significantly suppressed by TQ in P.g.LPS-stimulated HGFs. Protein levels of Nrf2, HO-1 and NQO-1 were significantly elevated by TQ in HGFs. These results suggest that N Sand TQ suppressed gingival inflammation through mechanisms involving NF-κB and MAPK and activation of antioxidant defense mechanisms. This study also demonstrates that bioactive TQ and NS are potential adjunctive therapeutic candidates in the prevention and treatment of gingivitis.
Date of Award28 Jul 2022
Original languageEnglish
SupervisorOlumayokun Olajide (Main Supervisor) & Karl Hemming (Co-Supervisor)

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