The Concomitant Relationship And Protective Effects Of Paeoniflorin On Alveolar Bone Resorption And Soft-tissue Breakdown In Experimental Periodontitis

Periodontitis is caused by bacterial plaque and is manifested by inflammatory responses. This disease is characterized by the breakdown of connective tissues and the resorption of alveolar bone and the loss of periodontal supporting tissues, finally resulting in the loss of teeth. The precise pathogenesis of periodontitis has not been fully studied till now. Previous studies have reported that periodontitis is significantly associated with inflammatory destruction and immune reactions. Hard-tissue breakdown and soft-tissue destruction may be mediated by the synthesis and release of physiological or pathological factors like reactive oxygen, inflammatory cytokines, proteolytic enzymes, compounds and prostaglandins (PGs) as an inflammatory disease. However, the concomitant relationship of timing phase between inflammation of soft tissue and the alveolar alterations from demineralization (reduction of bone mineral density(BMD)) to breakdown of bone is not well understood.Bone resorption and soft-tissue breakdown are major pathological factors in periodontitis. MMPs are a family of proteolytic enzymes that break down almost all the components of extracellular matrix and basement membranes. MMP-2 and MMP-9 have been found to play important roles in periodontal tissue breakdown. Elevated activities of MMP-2 and MMP-9 have been observed in gingival crevicular fluid, salivary samples and gingival tissue in periodontal disease. Inducible nitric oxide synthase(iNOS) and cyclooxygenase-2(COX-2) are pleiotropic molecules with diverse effects on vasodilatation, the immune response and bone resorption. At the site of inflammation, over-expression of iNOS and COX-2, followed by the production of NO and PGE2, respectively, participate in the immunoinflammatory response by mediating connective tissue alteration and bone resorption.The OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappa-B/receptor activator of nuclear factor kappa-B ligand) system is the most influential bone metabolism regulation system currently known. Most tissues and cells generate OPG/RANK/RANKL, all other cytokines also directly or indirectly affect bone metabolism through this system.Electron probe microanalysis (EPMA) provides highly sensitive, non-invasive measurements and can detect multiple elements in a compound in a single test. The output data are quantitative with a detection limit of approximately 100μg/g. EPMA is not frequently applied in biochemical assessments and is used even more rarely in elemental bone analysis.Paeoniflorin, a monoterpene glycoside(C23H28O11),is the main active ingredient in Radix Paeoniae. Paeoniflorin possesses various biological effects, such as anti-inflammatory, antioxidative and immune-regulatory activities. The biological effects of paeoniflorin suggest that it may be a potential anti-inflammatory agent for periodontitis. However, the overall effects of paeoniflorin on alveolar bone and soft tissue in periodontitis are still unknown. Moreover, the molecular mechanisms by which paeoniflorin exerts its anti-inflammatory and immune-regulatory properties still remain to be investigated.We designed this study based on the concomitant relationship between soft tissue inflammation and alveolar degradation and the potential mechanism of paeoniflorin on alveolar bone resorption and soft-tissue breakdown in experimental periodontitis. Experiment 1:The expression of MMP-2, MMP-9, iNOS and COX-2 in ligature-induced periodontitis in rats.EXPERIMENT 2:The concomitant relationship between soft tissue inflammation and alveolar degradation in ligature-induced periodontitis in rats. EXPERIMENT 3:Protective effects of paeoniflorin on alveolar bone resorption and soft-tissue breakdown in experimental periodontitis.EXPERIMENT 1 The expression of MMP-2, MMP-9, iNOS and COX-2 in ligature-induced periodontitis in ratsBackground and Objective:This study is to set up the ligature induced experimental periodontitis in rats and to detective the expression level of mmp-2, MMP-9, iNOS and COX-2.Material and Methods:Fourteen rats were randomly placed into the following two groups:healthy group; periodontitis group. Periodontitis was induced in rats by placing ligatures around the lower first molars. The body weight of each rat was measured and recorded before and after the experiment. The animal were executed on the seventh day. Bone resorption was evaluated using micro-computed tomography. Soft-tissue destruction and collagen fiber degradation were assessed by histologic analysis and picrosirius red staining. The expression levels of MMP-2, MMP-9, iNOS and COX-2 in gingiva were detected by western blotting.Results:Periodontal tissues were intact in the healthy group. Alveolar bone level was significantly reduced, and evident inflammation of soft tissues was observed in the periodontitis group. The results of the western blot analyses revealed that the levels ofMMP-2,MMP-9, iNOS and COX-2 were significantly higher in the periodontitis group than in the healthy group.Conclusion:This study provides that the ligature-induced periodontitis could result in the acute soft tissue inflammation and alveolar bone resorption.EXPERIMENT 2 The concomitant relationship between soft tissue inflammation and alveolar degradation in ligature-induced periodontitis in ratsBackground and Objective:In this study, we interfered with ligature-induced periodontitis in a rat model to observe the sequence of soft tissue inflammation and alveolar bone degradation and their concomitant relationship during the progression and recovery of periodontitis.Material and Methods:The rats were euthanized on day 0 and on day 1,3,5,7,9 and 11after the procedure of experimental periodontitis. Hematoxylin and eosin (HE) staining was used to observe soft tissue inflammation and alveolar bone destruction. Micro-computed tomography (Micro-CT) and Electron probe microanalysis (EMPA) was used to assess the degree and depth of local demineralization of alveolar bones. Reverse transcription-PCR (RT-PCR) was used to assess the expression of genes such as receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG).Results:HE staining revealed that soft tissue inflammation occurred immediately after the ligature, while alveolar bone destruction developed slightly later. After the ligature suture was removed, the soft tissue inflammation subsided and the alveolar bone destruction stopped simultaneously. Micro-CT revealed the same result. EMPA analysis revealed that the bone rapidly demineralized and brokedown from the surface, but the demineralization became less obvious beyond 30μm in depth. The difference was not statistically significant (P>0.05). After the soft tissue inflammation stopped, the bone stopped demineralizing, and bone density rapidly recovered. RT-PCR results revealed that the levels of mRNA transcription of RANKL and OPG genes and the ratio RANKL to OPG in soft tissue did not significantly change (P>0.05).However, the level of RANKL gene in alveolar bone rapidly increased after the soft tissue inflammation occurred and immediately decreased after it stopped. The change in OPG gene expression was synchronized with RANKL gene expression and relatively smaller. The ratio of RANKL to OPG gradually increased after the inflammation started and immediately reduced with the inflammation stopped.Conclusion:In the periodontitis progression, the demineralization of alveolar bone occurs later than soft tissue inflammation and stopped accompany with it ceased. This result suggests that the time interval for periodontal follow-up examination should be less than the required period for alveolar bone breakdown. It is necessary to maintain the health of periodontal soft tissue to avoid possible further alveolar bone loss.EXPERIMENT 3 Protective effects of paeoniflorin on alveolar bone resorption and soft-tissue breakdown in experimental periodontitisBackground and Objective:Paeoniflorin is the main active ingredient in Radix Paeoniae and has been reported to exhibit obvious anti-inflammatory and immune-regulatory activities. This study aimed to investigate the protective effects of paeoniflorin on alveolar bone destruction and soft-tissue breakdown in experimental periodontitis. As supplementary evidence, we evaluated the related expression of MMP-2, MMP-9, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Material and Methods:Twenty-eight rats were randomly placed into the following four groups:healthy group; periodontitis group; periodontitis plus 30 mg/kg of paeoniflorin (P30) group; and periodontitis plus 60 mg/kg of paeoniflorin (P60) group. Periodontitis was induced in rats by placing ligatures around the lower first molars. The body weight of each rat was measured and recorded before and after the experiment. Bone resorption was evaluated using micro-computed tomography. Soft-tissue destruction and collagen fiber degradation were assessed by histologic analysis and picrosirius red staining. The expression levels of MMP-2, MMP-9, iNOS and COX-2 in gingiva were detected by western blotting.Results:Periodontal tissues were intact in the healthy group. Alveolar bone level was significantly reduced, and evident inflammation of soft tissues was observed in the periodontitis group. Paeoniflorin significantly prevented alveolar bone loss and inflammatory infiltration in a dose-dependent manner. The collagen fiber fractions were significantly higher in the P30 and P60 groups than in the periodontitis group. The results of the western blot analyses revealed that both 30 and 60 mg/kg of paeoniflorin significantly down-regulated the levels ofMMP-2,MMP-9, iNOS and COX-2.Conclusion:This study provides the first evidence that paeoniflorin significantly down-regulates inflammatory infiltration and prevents alveolar bone loss and soft-tissue destruction in experimental periodontitis. The anti-inflammatory mechanism of paeoniflorin was further supported, in part, by its inhibitory effect on MMP-2, MMP-9,iNOS and COX-2.