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Impact of lycopene in the immunoexpression of cathepsin K and MMP-9 in osteoclasts of rats with periodontitis and type 2 diabetes mellitus

Grant number: 25/08688-9
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Start date: July 01, 2025
End date: February 28, 2026
Field of knowledge:Biological Sciences - Morphology - Histology
Principal Investigator:Paulo Sergio Cerri
Grantee:Lays Cristina Gouvea
Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Periodontitis (P) is a chronic inflammatory disease characterized by the destruction of tooth-supporting tissues, including bone resorption mediated by osteoclasts. Systemic diseases such as type 2 diabetes mellitus (T2DM), cardiovascular, and immunological disorders can aggravate its progression by dysregulating the immune response and increasing susceptibility to infection, in a bidirectional relationship. Among systemic diseases, T2DM promotes persistent hyperglycemia and systemic inflammation, enhancing the progression of P through inflammatory cytokines such as interleukin 1 beta (IL-1¿), tumor necrosis factor-alpha (TNF-¿), and interleukin 6 (IL-6). These cytokines activate macrophages, neutrophils, lymphocytes, and resident connective tissue cells, stimulate the activation of matrix metalloproteinases (MMPs), and promote the recruitment of inflammatory cells and the release of RANKL, inducing osteoclast formation and activation, consequently increasing bone resorption. In this context, adjunctive therapies with antioxidant and anti-inflammatory properties, such as lycopene (LYC), a carotenoid found in tomatoes, have been investigated. LYC neutralizes reactive oxygen species (ROS), modulates inflammatory cytokines (IL-1¿, TNF-¿, IL-6), inhibits MMPs, and reduces oxidative stress, aiding in tissue preservation. This carotenoid may also inhibit the activity of enzymes involved in bone resorption, such as cathepsin K - expressed by osteoclasts and responsible for the degradation of type I collagen, and MMP-9, which contributes to the removal of the organic matrix during bone resorption. This study aims to evaluate the effects of LYC in the immunoexpression of cathepsin K and MMP-9 in an experimental model of P associated with T2DM in rats. A total of 126 adult male Holtzman rats were distributed into 7 groups: (1) Control group (CG; n=18), (2) T2DM + P group (T2DM+PG; n=18), (3) T2DM + P group treated with LYC (T2DM+P+LG; n=18), (4) T2DM group (T2DMG; n=18), (5) T2DM group treated with LYC (T2DM+LG; n=18), (6) Periodontitis group (PG; n=18), and (7) Periodontitis group treated with LYC (P+LG; n=18). T2DM was induced by a high-fat diet for 28 days. On the 28th day, rats received an intraperitoneal dose of 35 mg/kg streptozotocin (Sigma). After 7 days, animals with blood glucose levels above 250 mg/dL were considered diabetic. On this same day, P was induced in groups PG, P+LG, T2DM+PG, and T2DM+P+LG by placing a ligature around the upper first molar. The rats in groups T2DM+P+LG, T2DM+LG, and P+LG received daily oral gavage of 20 mg/kg LYC (Galena®, Campinas, Brazil), diluted in corn oil, for 7, 35, and 70 days. After the experimental periods, the maxillae will be removed and the specimens will be embedded in paraffin for histological analysis. Sections of 6 µm thickness will be adhered on glass slides for hematoxylin and eosin (HE) staining for morphological analysis and detection of cathepsin K (Abcam) and MMP-9 (Abcam MMP-9). Sections for cathepsin K detection will be incubated with Alexa Fluor® 488-conjugated secondary antibody (goat anti-rabbit IgG), and sections for MMP-9 will be incubated with Alexa Fluor® 647 (Abcam, donkey antibody to rabbit IgG). Reactions will be evaluated, and immunoexpression of cathepsin K and MMP-9 in osteoclasts will be quantified using a Leica fluorescence microscope (model DM4000 B LED) and Leica Application Suite software (LAS 4.3). The total area of 20 osteoclasts per specimen (n=6/group/period) will be measured, followed by quantification of cathepsin K and MMP-9 immunofluorescent areas using the image analysis software. The immunofluorescent area for cathepsin K and MMP-9 in osteoclasts will be estimated. Data will be analyzed using ANOVA followed by Tukey's post hoc test, with significance set at p ¿ 0.05.

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