Lycopene effect in the induced periodontal disease in rats with type 2 diabetes mellitus

• Grant number: 22/11244-7
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: November 01, 2023
• End date: March 31, 2026
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Paulo Sergio Cerri
• Grantee: Arles Naisa Amaral Silva
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Periodontitis (P) and type 2 diabetes mellitus (T2DM) are complex and multifactorial chronic diseases, characterized by a hyper-inflammatory state. The immunoinflammatory state promotes the recruitment of specific cells that release mediators, such as interleukin-1² (IL-1²), IL-6, IL-7, tumor necrosis factor-alpha (TNF-±), and matrix metalloproteinases (MMPs). Therefore, the modulation of the immunoinflammatory response may be a complementary approach in the treatment of P and control of T2DM. Lycopene, a carotenoid hydrocarbon, has anti-inflammatory and antioxidant effects. Thus, it is possible that lycopene plays a beneficial role in P and T2DM control. The aim of this study will be to evaluate whether the systemic administration of lycopene reduces the deleterious effects promoted by induced periodontitis in rats with T2DM. 252 Holtzman rats will be randomly distributed into 7 groups: 1) Control Group (CG; healthy rats), 2) T2DM+P (rats with type 2 diabetes mellitus and periodontitis induced), 3) T2DM+P+L (rats with type 2 diabetes mellitus, periodontitis induced and treated with lycopene), 4) T2DM (rats with T2DM), 5) T2DM+L (rats with T2DM and treated with lycopene), 6) P (rats with periodontitis), 7) P+L (rats with periodontitis and treated with lycopene). T2DM will be induced with a high fat diet and streptozotocin (25 mg/kg). After 7 days of administration of streptozotocin, the P will be induced with the insertion of a cotton thread in the cervical colon of the upper 1st molars. On the P-induction day, the rats will receive 20 mg/kg body weight of lycopene or physiological solution by gavage for 7, 35, and 70 days. In each period, the blood will be collected and lipids parameters, the concentration of insulin, HOMA-IR, glycated hemoglobin, C-reactive protein, and oxidative stress (MDA - malondialdehyde) levels will be measured in the serum. The liver and pancreas will be removed for histopathological evaluation. For paraffin-embedded, the maxilla fragments will be fixed in formaldehyde (pH 7.2), decalcified, and then processed for paraffin embedding. Fragments of maxilla will also be fixed glutaraldehyde/formaldehyde and embedded in Araldite medium for ultrastructural analysis. From each maxilla, sections will be stained with HE for morphological and quantitative analyses. Moreover, the possible interference on pro-inflammatory cytokines will be evaluated by immunohistochemistry detection of IL-1², TNF-±, IL-6, IL-7 e IL7-R. To evaluate whether lycopene reduces bone loss, the maxillae will be analyzed by micro-CT and the osteoclasts number in the alveolar surface will be computed in the sections subjected to the TRAP, used as osteoclast marker. Detection of caspase-3, the TUNEL method, and ultrastructural features will be performed to evaluate whether lycopene induces osteoclast apoptosis. The immunohistochemical reactions anti-RANKL, -OPG, and -NF-kB p65 will be performed to evaluate whether the lycopene interferes in osteoclast formation. To evaluate whether lycopene reduces the extracellular components breakdown, the birefringent collagen will be measured and detection of MMP-1, MMP-8 and MMP-9 by immunohistochemical reactions will be performed in the gingival mucosa. The lycopene effect during the periodontitis progression will be also investigated by the detection of proteins associated with bone formation (osterix and alkaline phosphatase) and IL-10, an anti-inflammatory cytokine associated with tissue repair. Gingival mucosa samples will be removed and stored at -80º C to evaluate the expression of Nf-kb, STAT3, and DC-STAMP, required factors for osteoclast differentiation by real-time RT-PCR. The data will be statically analyzed in the GraphPad Prism 8.4.3 program in addition to multiple and linear logistic regressions and correlations of the proposed analyzes for all groups and experimental periods.