Unveiling the therapeutic potential of Tanshinone-IIa sulfonate in the prevention and treatment of experimental periodontitis in mice

Abstract

Periodontitis (PD), a highly prevalent multicausal chronic inflammatory and destructive disease, develops as a result from complex host-parasite interactions. The dysbiotic bacterial biofilm in contact with the gingival tissues initiates a cascade of inflammatory events, mediated and modulated by the host immune response, characterized by increased expression of innumerous inflammatory mediators such as cytokines and chemokines in the connective tissue. PD results in the destruction of supporting tissues around the teeth, including periodontal ligament, cementum, and alveolar bone. This process depends on the differentiation and activity of osteoclasts, the cells responsible for resorbing the bone tissue. Thus, inhibiting the differentiation or activity of these cells is a viable strategy for controlling bone resorption during the development of PD. Tanshinones are a new class of cathepsin K inhibitors that have recently emerged as promising drugs in reducing the osteoporotic phenotype in mice. Tanshinona-IIa sulfonate is a derivative of Tanshinona IIA, a natural substance from Salvia miltiorrhiza (Danshen) with diverse biological properties. Such compounds can specifically inhibit the collagenolytic activity of Cathepsin K, an enzyme secreted by osteoclasts that participates in the degradation of the organic phase of the bone matrix, without interfering with osteoclast differentiation. Considering the biological potential of tanshinona IIa sulfonate and the site-specific nature of PD, this project aims to unveiling the therapeutic potential of this compound on bone resorption, inflammation progression, in the immunoinflammatory profile and in its osteogenic potential in preventive and therapeutic approach in a mouse model of ligature-induced bone loss. The outcomes to be analyzed include: evaluation of the inflammatory process in the connective tissue through histological analysis, assessment of alveolar bone loss inhibition by micro-computed tomography, quantification of osteoclasts number (TRAP) by histochemistry, immunolocalization and quantification of bone markers (OPG, RANKL and Cathepsin K) and transcription factors (RUNX-2, NFATc1, and NF-ºB), by immunohistochemistry and the immunoinflammatory profile (anti-CD80, anti-CD206, antiF4/80, anti-CD4, anti-CD8, anti-CD45, e anti-CD3) by immunofluorescence; quantification of bone marker expression (RANKL, OPG, c-fos, NFATc1, OSCAR, cathepsin K, and IL-6), and the expression of immunoinflammatory markers (TGF-²1, TNF-±, IL-1², IL-10, IL-4, INF-g, e Arg2) by RT-qPCR; protein levels of NFATc1, c-fos, and IkB± by western blot; the functional activity of Cathepsin K by fluorimetric assay; and finally, the bone mineral apposition rate (MAR) by dynamic histomorphometry through fluorescence microscopy and serum levels of CTx1, P1NP, OCN, and OSX by ELISA, and the structure and organization of collagen fibers in the periodontal ligament under bright-field and polarized microscopy. (AU)