Diacerein impact on osteoclast formation, activity and survival of alveolar process of rat molars with induced periodontal disease

• Grant number: 21/05968-0
• Support Opportunities: Regular Research Grants
• Start date: March 01, 2022
• End date: May 31, 2024
• Field of knowledge: Biological Sciences - Morphology - Histology
• Principal Investigator: Paulo Sergio Cerri
• Grantee: Paulo Sergio Cerri
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Associated researchers: Estela Sasso Cerri

Abstract

Periodontitis (P), triggered by subgingival bacterial accumulation, causes the periodontal tissues degradation (gingiva, cementum, periodontal ligament and bone alveolar). Bone loss is considered as hallmark of progression of P. Therefore, the bone loss resulting of P is closely associated with osteoclast formation, activity and survival. Cytokines such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-±), receptor activator of nuclear factor-kB ligand (RANKL), largely secreted by resident and inflammatory cells under stimuli of lipopolysaccharide (LPS), induce the osteoclastogenesis and resorptive activity. Considering that diacerein inhibits the IL-1 and TNF-±, it is conceivable to suggest that this anti-inflammatory interferes in the inflammatory cells types in the gingival mucosa (leukocytes, lymphocytes T and macrophages) and inhibits the cytokines involved in the osteoclast formation, activity and survival. Moreover, will be evaluated whether diacerein interferes in the factors immunoexpression associated with osteoblast differentiation, alkaline phosphatase and osterix, as well as IL-10, a cytokine associated with tissue repair. One hundred and thirty-two Holtzman rats will be distributed into PDG (periodontitis diacerein-treated group), PDS (periodontitis sham group) and CG (Control group; healthy periodontium). P will be induced with the insertion of a cotton thread in the cervical colon of the upper right 1st molars. After 7 days, the ligature will be removed and the rats with induced periodontal disease will receive 100 mg/kg body weight of diacerein (PDG) by gavage or physiological solution (PSG) for 7, 15 and 30 days. For paraffin-embedded, 6 animals per group (PDG, PSG and CG) will be sacrificed in each period. The maxilla fragments will be fixed in formaldehyde (pH 7.2), decalcified in 7% EDTA and then processed for paraffin embedding. From each maxilla, non-serial sagittal sections will be stained with hematoxylin and eosin (HE) and Masson's trichrome for morphological analyses. Three sections of each specimen will be subjected to the tartrate-resistant acid phosphatase (TRAP) and the number of osteoclasts will be computed. Bone area occupied by interdental alveolar process, osteoclast area, number of nuclei/osteoclast and bone resorption surface (in close contact with osteoclast) will be also measured. Cathepsin K, V-ATPase and matrix metalloproteinase-9 (MMP-9), enzymes associated with osteoclast activity, will be also detected. To evaluate whether diacerein interferes in the osteoclast survival, sections will be submitted to immunohistochemistry for detection of caspase-3 and to the TUNEL method, and the frequency of caspase-3- and TUNEL-positive osteoclasts will be estimated. Sections will be submitted to immunohistochemistry or immunofluorescence reactions for detection of CD45, CD4, CD8, mac387, IL-1², TNF-±, IL-6, RANKL, OPG, NF-kB (nuclear factor kappa B), fosfatase alcalina, osterix e IL-10, and the number of immunopositive cells will be obtained. In order to verify whether diacerein promotes ultrastructural changes in the osteoclasts, samples (n=3 rats/group/period) will be fixed in glutaraldehyde/formaldehyde and embedded in Araldite. Ultrathin sections will be examined using a TECNAI transmission electron microscope. Gingival mucosa samples will be removed (from 4 animals/group) and stored at -80º C to evaluate the expression of inflammatory cytokines Tnf-± and Il-1² as well as Nf-kb, required factors for osteoclast differentiation by real time RT-PCR. TNF-±, IL-1², NF-kB, IL-6, IL-10, RANKL, OPG e MMP-9 will also be evaluated by Western blot (4 samples/group). The quantitative data will be submitted to two-way ANOVA and Tukey post-test (p < 0.05). (AU)