Assessment of the Vanillin Anti-Inflammatory and Regenerative Potentials in Inflamed Primary Human Gingival Fibroblast

Abstract

Background: Inflammatory responses have been associated with delayed oral mucosal wound healing and the pathogenesis of the periodontal disease. The invasion of microbes into the tissues and the establishment of a chronic infection may be due to impaired healing. The protracted inflammatory phase may delay wound healing and probably support tissue fibrosis and reduce tissue regeneration. Vanillin is a well-known natural compound with potential anti-inflammatory capacity. Hence, we hypothesized that Vanillin could accelerate wound healing reducing inflammation and especially cytokine production making the oral tissue repair process easier.

Methods: Our hypothesis was tested using primary human gingival fibroblast (HGF) cell pretreated with Vanillin and primed with IL-1β, as inductor of proinflammatory environment. After 24 hours of treatments, the gene expression and production of IL-6, TNF-α, IL-8, COX-2, iNOS, and nitric oxide (NO) generation and the wound healing rate were determined.

Results: In IL-1β-primed cells, preincubation with Vanillin reduced IL-6, IL-8, COX-2, and iNOS expression and NO release, compared to IL-1β-primed cells. Moreover, Vanillin determines the increased gene expression of nAChRα7, leading us to hypothesize a role of Vanillin in the activation of the cholinergic anti-inflammatory pathway. Furthermore, in presence of mechanical injury, the Vanillin preincubation, wound closure may be reducing the expression and release of IL-6 and TNF-α and upregulation of COX-2 and IL-8.

Conclusion: Together, the results of this study highlight the anti-inflammatory and tissue repair ability of Vanillin in IL-1β-primed HGF. Therefore, Vanillin shows a potential therapeutic interest as an inflammatory modulator molecule with novel application in periodontal regeneration and oral health.

Figure 1

MTT viability assay in HGF cell treated for 24 h with Vanillin (100, 200, and 300 μM) or IL-1β (0.1, 1, and 10 ng/ml). Absorbance values are given as media ±SD of three independent experiments.

Figure 2

TNF-α, IL-6, and IL-8 levels in HGF cell culture supernatant. The mean values ± SD were reported, ^∗∗∗p < 0.001. All experiments were repeated in triplicate.

Figure 3

Gene expression of TNF-α, IL-6, IL-8, and nAChRα7 in HGF cells. Data are reported as mean and 95% CI, of three independent experiments. ^∗p < 0.05. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.

Figure 4

Gene expression of COX-2 and iNOS in HGF cells. Data are reported as mean and 95% CI, of three independent experiments, ^∗p < 0.05. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pre-treatment) plus IL-1β.

Figure 5

Levels of NO in HGF cells were determined based on Griess reagent reaction. Experiments were conducted in duplicate, and changes were reported as mean ± SD; ^∗∗∗p < 0.001. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.

Figure 6

Wound healing of scratched oral fibroblast monolayer photographs and measurement of wound area were made immediately after the scratch (T0) and after 24 h (T24). (a) Representative images of wound-healing assay. (b) Wound repair was evaluated measuring the remaining cell-free area after 24 h and expressed as a percentage of the initial wound size (T0) assumed as 100%. ^∗∗p < 0.01; ^∗∗∗p < 0.001. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.

Figure 7

Levels of TNF-α, IL-6, and IL-8 in supernatant of HGF scratched cells. Data are reported as mean and 95% CI, of three independent experiments. ^∗∗p < 0.01; ^∗∗∗p < 0.001. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.

Figure 8

Gene expression of TNF-α, IL-6, and IL-8 in HGF scratched cells. Data are reported as mean and 95% CI, of three independent experiments. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.

Figure 9

Gene expression of COX-2 and iNOS in HGF cells. Data are reported as mean and 95% CI, of three independent experiments. ^∗∗p < 0.01; ^∗∗∗p < 0.001. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.

Figure 10

Levels of NO in HGF cells were determined based on Griess reagent reaction. Experiments were conducted in duplicate, and changes were reported as mean ± SD. ^∗∗p < 0.01; ^∗∗∗p < 0.001. IL-1β: IL-1β-primed cells; Van: Vanillin; Van (2 h)+IL-1β: Vanillin (2 h pretreatment) plus IL-1β.