Resumen de Cimetidine Reduces Interleukin-6, Matrix Metalloproteinases-1 and -9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease
Priscila Aparecida de Oliveira, José Paulo de Pizzol Júnior, Renata Longhini, Estela Sasso Cerri, Pauolo Sérgio Cerri
Background: Histamine seems to act, via H2 receptor, on inflammatory processes by stimulating interleukin (IL)-6 and matrix metalloproteinase (MMP) release. As cimetidine is an H2 receptor antagonist, the authors hypothesize that this antiulcer drug reduces IL-6, MMP-1, and MMP-9 immunoexpression in gingiva with induced periodontal disease (PD). To confirm a possible modulatory role of IL-6 on MMPs, the relationship between IL-6/MMP-1 and IL-6/MMP-9 immunoexpression was evaluated.
Methods: Forty-six male rats were distributed into the cimetidine group (CimG: received daily intraperitoneal injections of 100 mg/kg of body weight of cimetidine) or the saline group (SG). PD was induced by cotton ligature around the maxillary left first molars (PDSG and PDCimG). The right molars were used as controls (SG and CimG). After 7, 15, 30, and 50 days, maxillary fragments were processed for paraffin embedding or for transmission electron microscopy. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the alveolar process surface and number of IL-6, MMP-1, and MMP-9-immunolabeled cells in the gingival mucosa were quantified. Statistical analyses were performed (P ≤0.05).
Results: In PDSG and PDCimG, gingival mucosa exhibited few collagen fibers among numerous inflammatory cells. In PDCimG, the number of TRAP-positive osteoclasts and IL-6, MMP-1, and MMP-9-immunolabeled cells was significantly lower than in PDSG at all periods. A positive correlation between IL-6/MMP-1 and IL-6/MMP-9 was detected in PDSG and PDCimG.
Conclusion: Cimetidine decreases bone loss through reduction of osteoclast number and induces reduction of IL-6, MMP-1, and MMP-9 immunoexpression, reinforcing the idea that the beneficial effect of cimetidine in PD may be due to reduction of IL-6 immunolabeling in the inflamed gingival mucosa.
Periodontitis is an inflammatory disease that affects periodontal tissues by action of pathogenic microorganisms in dental biofilm.1 Although periodontal bacteria are the causative agents in periodontitis, progression and severity of periodontal disease (PD) relies on host immune response.2,3 Toxic and pathogenic products released in gingival mucosa induce an inflammatory response. Inflammatory cells and resident cells produce several mediators, such as interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandins, and histamine.4,5 These released mediators trigger a cascade of cellular and molecular events that culminate with destruction of soft and mineralized tissues.6,7 In addition to the well-known role of histamine in the allergic process, several studies have suggested an important participation of this mediator in the inflammatory process.1,8,9 Histamine is secreted and released mainly by mast cells, although neutrophils and eosinophils also release histamine in inflammation.8 Histamine can exert its biologic action by binding to a specific superfamily of G-protein-coupled transmembrane receptors: histamine H1 receptor (H1R), H2R, H3R, and H4R.9 H2R may play a pivotal role in regulating histamine-mediated inflammatory reactions.2,10 These receptors are expressed on several cell types that can be present in the inflammation process, including lymphocytes,11 monocyte-derived dendritic cells,12 macrophages, and neutrophils.10 In addition, histamine via H2R inhibits production of immunoglobulins (Igs) by B-cell, mainly IgG and IgM, interfering in immune response.13 Histamine also stimulates production of several cytokines such as IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor.14,15 IL-6 is a multifunctional cytokine produced by different cell types, including gingival fibroblasts,16 endothelial cells,15 macrophages, plasma cells, mast cells,17 monocytes, epithelial cells, and T- and B-cells.4 Among other biologic activities, IL-6 has been associated with regulation of immune response, inflammation, and hematopoiesis.18 Enhanced IL-6 levels have been observed in gingival crevicular fluid in patients with periodontitis19 and in inflamed gingival mucosa.20 High messenger RNA expression and protein expression of IL-6 has also been detected in patients with PD.3 Furthermore, IL-6 stimulates secretion of receptor activator of nuclear factor-kappa B ligand (RANKL),21 a cytokine produced by osteoblasts, fibroblasts,22,23 and inflammatory cells23 that promotes osteoclast formation and, consequently, bone resorption.21,23,24 It is known that matrix metalloproteinases (MMPs) are proteolytic enzymes involved in extracellular matrix (ECM) degradation and remodeling in both physiologic and pathologic conditions.25-27 Enhanced production of collagenases such as MMP-1, MMP-8, and MMP-13 causes degradation to collagen fibril, which is the main component of periodontal ligament, cementum, and alveolar bone. MMP-2 and MMP-9, known as gelatinases, also have marked participation in the breakdown of ECM during periodontitis.28,29 Cimetidine, a specific histamine H2R antagonist, binds to H2-receptors of gastric parietal cells and is clinically used as antacid and antiulcer drug.30 As histamine also binds to H2R in endothelial cells and stimulates vascular endothelial growth factor (VEGF) production,31 the histamine-induced VEGF production is inhibited by cimetidine.32 Thus, this drug has also played an antiangiogenic role and has been used clinically to suppress tumor growth by inhibiting tumor-associated angiogenesis.33 Besides the specific antagonist H2 action of cimetidine, this drug has induced changes in male reproductive organs of rats, which has been related to an antiandrogenic effect.34,35 In rats with arthritis, this drug has demonstrated reduction in inflammatory response and bone resorption.36 Topical administration of cimetidine in patients with gingivitis enhanced the antibacterial function of neutrophils of crevicular fluid. Moreover, significant clinical improvement was verified in patients treated with cimetidine.37 In rabbits with induced periodontitis, inflammatory reaction and bone resorption were reduced in response to topically applied cimetidine.2 In the gingival mucosa of rat molars with induced PD, cimetidine caused significant reduction in inflammatory reaction and RANKL/osteoprotegerin ratio, leading to a decrease of alveolar process loss.23 Thus, evidence indicates that accentuated histamine levels in inflamed periodontium stimulates IL-638 and MMP39 secretion. Moreover, IL-6 seems to stimulate MMP release in inflamed periodontal tissues.4,38 Based on the hypothesis that cimetidine may alleviate the inflammatory process by inhibiting histamine-stimulated IL-6 and MMPs, the current study investigates the effect of cimetidine, an H2R antagonist, on IL-6, MMP-1, and MMP-9 immunoexpression in ligature-induced gingival inflammation in rat molars. Possible correlation between IL-6 and MMP-1 and/or MMP-9 immunoexpression is also evaluated.
