Effect of omega-3 associated with vitamin D and calcium supplementation in the inflammatory response and bone resorption of rat molars with periodontitis

• Grant number: 24/15030-7
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: July 01, 2025
• End date: March 31, 2028
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Paulo Sergio Cerri
• Grantee: Gabriella de Oliveira
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Periodontitis is a public health issue that can lead to tooth loss due to alveolar bone resorption, potentially causing nutritional and psychosocial problems. Since the resorption of periodontal tissues is mediated by inflammatory cytokines such as interleukins (IL), prostaglandins, and matrix metalloproteinases (MMPs), studies have been conducted to develop therapies that modulate the immunoinflammatory response in order to reduce the expression of inflammatory mediators, as well as other proteins that regulate the formation and activity of osteoclasts and osteoblasts. The potential benefits of omega-3 (O3) and vitamin D (VitD) with calcium (Ca) have been evaluated. While vitamin D contributes to increased Ca absorption in the intestine, omega-3 is a natural anti-inflammatory agent. The objective of this research will be to evaluate whether omega-3, combined with vitamin D and calcium supplementation, reduces the expression of inflammatory mediators and bone loss, affecting the formation, activity, and survival of osteoclasts and osteoblasts. For this study, 90 Holtzman rats will be divided into 5 groups: control group (CG), periodontitis sham group (PSG), group with periodontitis treated with vitamin D and Ca (GPDC; VitD: 6.7 IU/kg body weight; Ca: 10 mg/kg body weight), group with periodontitis treated with omega-3 (GPO3; 40 mg/kg body weight), and group with periodontitis treated with vitamin D, Ca, and omega-3 (GPDCO3; VitD: 6.7 IU/kg body weight + Ca: 10 mg/kg body weight + O3: 40 mg/kg body weight). Corn oil will be use as dilution vehicle, and the rats will receive a daily dose administered via gavage. The animals in the PSG will receive daily gavage of corn oil in the same volume administered to the animals in the GPDCO3, GPO3, and GPDC. The treatment will begin on the day of periodontitis induction. Periodontitis will be induced by placing a ligature around the second upper molars (left and right), which will be maintained throughout the experiment. After 7 and 35 days, the animals will be anaesthetized, and blood will be collected via cardiac puncture for analysis of serum levels of Ca, alkaline phosphatase (ALP), total cholesterol, HDL and LDL. After blood collection, the animals will be euthanized with an anaesthetic overdose and the left hemimaxilla of six animals from each group/period will be processed for paraffin embedding, while the right hemimaxilla will be used for the collection of gingival mucosa for RT-qPCR analysis. Subsequently, these right hemimaxillae will be stored in formaldehyde for MicroCT analysis. Additionally, 3 animals/group/period will be allocated for ultrastructural analysis. Paraffin sections will be stained with haematoxylin and eosin and Masson's trichrome for morphological and morphometric analyses. Three sections from each hemimaxilla will be subjected to the histochemical reaction for detection of tartrate-resistant acid phosphatase (TRAP) to estimate the number of osteoclasts in the bone surface. Other sections will be used for immunohistochemistry and immunofluorescence reactions to detect TNF-¿, IL-1¿, IL-6, MMP-9, IkBa, CD86 and CD206 macrophages, RANKL, OPG, M-CSF, c-FMS, V-ATPase, cathepsin K, ALP, and IL-10. The TUNEL method and caspase-3 detection, combined with ultrastructural analysis, will be used to characterize the osteoclast cell death process. The gene expression of DC-STAMP, BMP2, and NFkB will be evaluated using RT-qPCR. Quantitative data will be subjected to two-way ANOVA and Tukey's post-test (p < 0.05). RT-qPCR will be conducted in duplicates, and relative quantification will be obtained using the ¿¿CT method and one-way ANOVA with Tukey's post-test for each period. (AU)