Periodontitis (P) is a multifactorial disease triggered by the host response to the accumulation of bacteria in the gingival sulcus, which can be influenced by several factors, among them systemic, environmental, trauma and drugs. The periodontitis progression is mediated by some bioactive agents such as interleukins (IL), matrix metalloproteinases (MMP) and transcription factors that interact through signaling pathways to stimulate osteoclastogenesis and inhibit the osteoblast activity, favoring resorptive activity. Diacerein is an anti-inflammatory drug capable of inhibiting mediators such as TNF-± and IL-1 widely used for osteoarthritis treatment, which has a pathophysiology similar to periodontitis. The aim of the study will be to evaluate the effects of diacerein on induced periodontitis in rat molars, particularly on the types of inflammatory cells in the gingival mucosa (leukocytes, T lymphocytes and/or macrophages) and cytokines involved in the formation and activity of osteoblasts and osteoclasts. 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 embedding, 6 animals per group (PDG, PSG and CG) will be sacrificed in each period. The maxilla fragments will be fixed in formaldehyde, decalcified in EDTA and then processed for paraffin embedding. From each maxilla, non-serial sagittal sections will be stained with hematoxylin and eosin (HE) for morphological analyses. Sections will be submitted to immunohistochemistry or immunofluorescence reactions for detection of inflammatory cell types (CD45, CD4, CD8 and mac387), cytokines involved with osteoclastogenesis (IL-1², TNF-± and NF-kB - nuclear factor kappa B), enzymes associated with osteoclast activity (V-ATPase and MMP-9), as well as factors and enzymes involved with osteoblast formation, differentiation and activity (osterix, phosphatase alkaline and IL-10); the number of immunolabelled cells (for each immunolabelling) will be obtained. In order to verify whether diacerein promotes ultrastructural changes in the alveolar process surface, particularly in osteoclasts and osteoblasts, samples (n=3 rats/group/period) will be fixed in glutaraldehyde/formaldehyde and embedded in Araldite. 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, Rankl and OPG required factors for osteoclast differentiation by real time RT-qPCR. 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).
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