Date on Master's Thesis/Doctoral Dissertation

12-2018

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Degree Program

Oral Biology, MS

Committee Chair

Liang, Shuang

Committee Co-Chair (if applicable)

Scott, David

Committee Member

Scott, David

Committee Member

Sandell, Lisa

Author's Keywords

grape components; NF-κB pathway; periodontal diseases; inflammation; alveolar bone loss; P. gingivalis

Abstract

Background: Half of the adult American population (47 %) are victims of periodontitis, a chronic inflammatory disease which leads to destruction of the tissues supporting the tooth. Periodontitis is initiated by periodontal pathogens and caused by excessive or prolonged host mediated inflammation. Porphyromonas gingivalisis one important agent implicated in the etiopathogenesis of chronic periodontitis. Pro-inflammatory cytokines induced by periodontal pathogens can cause alveolar bone loss and periodontitis. Bone loss is caused by the imbalance of bone formation and bone resorption. Osteoclasts are major cells that cause bone resorption. Moreover, periodontal inflammation also leads to bone resorption. Grapes are rich in several components like catechin and pro-anthocyanidins, which possess extensive biologic properties including inhibitory effects on oral bacteria and the ability to suppress inflammation. Grape components may intensely inhibit osteoclastogenesis by suppressing osteoclast differentiation and thus may be beneficial in anti-inflammatory treatment accompanying bone destruction. Grapes may thus prove as a promising natural therapeutic agent for periodontitis therapy. They may exhibit their anti-inflammatory and immunomodulatory properties by suppressing the activation of NF-κB signal transduction pathways (Lee, Kim et al. 2017)(Chu, Tang et al. 2016). The focus of this study was to determine the effects of grape components on bacterial growth, inflammation, and osteoclastogenesis in vitroand gingival inflammation and bone loss in vivo. Hypothesis: We hypothesize that grape powder extract will inhibit P. gingivalisgrowth, suppress pro-inflammatory cytokines and inhibit osteoclastogenesis in vitro. We also hypothesize that grape powder inhibits gingival inflammation and prevents bone loss in a murine model. Methods:In vitro– the effect of grape powder extract on bacterial growth was tested by inoculatingP. gingivalisinto 1.5 ml microcentrifuge tubes containing different concentrations (0.0625, 0.125, 0.5 µg/ml) of grape powder extract and incubating the tubes at 37°C for 24 h. Controls included no grape powder treatment. The bacterial number was determined by plating on blood agar for CFU. 105 Murine RAW 264.7 cells were treated with grape powder extract (50, 100, 200 µg/ml) or no grape powder extract and stimulated with 1 µg/ml P. gingivalisLPS overnight and tested for the expression of IL-6 by ELISA. Similarly, 105THP1-Blue cells™ were treated with grape powder extract (50, 100, 200 µg/ml)or no grape powder extract, stimulated with 10 µg/ml P. gingivalisLPS overnight. Activity of NF-κB-inducible alkaline phosphatase in THP1-Blue cells™ was determined by colorimetric activation. 103RAW 264.7 cells were stimulated with 100 ng/ml RANKL and treated with 200µg/ml grape powder extract. The cells with no grape powder extract treatment were used as controls. Seven days later, the cultures were fixed and stained with TRAP for 15 mins, an osteoclast marker and the number of TRAP positive cells were counted microscopically. In vivo- Grape powder was tested in a 7-day murine ligature-induced periodontitis model where mice received a ligation around the second molar and also a baker model, where P. gingivaliswas orally infected and mice were kept for 2 months. The mice consumed a grape powder or no grape powder diet. Maxillary gingival tissue was tested for mRNA expression of pro-inflammatory and anti-inflammatory cytokines by Quantitative real-time PCR in the ligature model. Alveolar bone loss was determined by measuring the CEJ-ABC distance in the baker model. The statistical analysis used was a two-tailed t test. (GraphPad Prism Software, San Diego, CA, USA). Differences were considered statistically significant at a P value of Results: There was a decrease in the number of oral bacteria (CFU) with increasing concentration of grape powder extract. Thus, grape powder extract inhibited P. gingivalisgrowth. RAW 264.7 cells stimulated with P. gingivalisonlyshowed maximum levels of IL-6 production. However, in the cells that received grape powder extract, a decrease in the amount of IL-6 was noted. THP1-Blue cells™ stimulated with P. gingivalisLPS and treated with grape powder extract showed reduction in the level of NF-κB activity. Cells without RANKL stimulation and grape powder extract treatment showed no formation of TRAP-positive cells. Cells treated with grape powder extract showed a significant reduction in the number of TRAP positive cells. Thus, grape powder extract inhibited P. gingivalisgrowth, osteoclastogenesis and P. gingivalis-induced pro-inflammatory cytokine production in vitro. There was a reduction in the levels of pro-inflammatory cytokines IL-6 and TNF-αafter treatment with grape powder in ligature model mice. However, there were no significant changes in the levels of IL-17, IL-1b, IL-10 and TGF-b. CEJ-ABC distance was also reduced which indicated bone loss in the baker model. Overall, grape powder reduced gingival inflammation and bone loss in murine models. Conclusion and practical implications: Grape components or grape consumption can be beneficial in the prevention or treatment of periodontal disease. Grape components can be useful for further development as a potential and safe therapeutic agent in periodontal disease treatment.

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