Curcumin inhibits inflammatory response and bone loss during experimental periodontitis in rats

Abstract

Objective. Curcumin, an active ingredient of turmeric, is proved to be a potential candidate of controlling inflammation and bone resorption, but few reports are on the periodontitis. The purpose of this study was to evaluate whether the intra-gastric administration of curcumin could inhibit the in?ammation and alveolar bone resorption in rats following ligature-induced experimental periodontitis. Materials and method. Male Wistar rats were randomly divided into three groups: no ligature placement and administration of vehicle, ligature placement and administration of vehicle, ligature placement and administration of curcumin. After the animals were sacrificed, their mandibles were collected for morphological, histological and immunohistochemical analysis; their gingival tissues were collected for cytokine measurements. Results. Bone resorption was significantly higher in the experimental periodontitis animals treated with vehicle compared with the curcumin-treated group or the control group. Furthermore, receptor activator of nuclear factor-κB ligand (RANKL), receptor activator of nuclear factor-κB (RANK), osteoprotegerin (OPG), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression levels were higher in the experimental periodontitis animals treated with vehicle compared with the curcumin treated group or the control group. Conclusions. Curcumin may decrease alveolar bone loss in the experimental periodontitis rats via suppressing the expression of RANKL/RANK/OPG and its anti-inflammatory properties.     

Genistein suppresses Prevotella intermedia lipopolysaccharide-induced inflammatory response in macrophages and attenuates alveolar bone loss in ligature-induced periodontitis

ObjectiveGenistein is a major isoflavone subclass of flavonoids found in soybean and a potent tyrosine kinase inhibitor. The present study aimed to assess the effect of genistein on the production of proinflammatory mediators in murine macrophages stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen associated with different forms of periodontal disease, and to evaluate its possible influence on alveolar bone loss in ligature-induced periodontitis using micro-computed tomography (micro-CT) analysis as well.DesignLPS was isolated from P. intermedia ATCC 25611 by using the standard hot phenol–water method. Culture supernatants were analyzed for nitric oxide (NO) and interleukin-6 (IL-6). Inducible NO synthase (iNOS) protein expression was evaluated by immunoblot analysis. Real-time PCR was carried out to measure iNOS and IL-6 mRNA expression. In addition, effect of genistein on alveolar bone loss was evaluated in a rat model of experimental periodontitis using micro-CT analysis.ResultsGenistein significantly attenuated P. intermedia LPS-induced production of iNOS-derived NO and IL-6 with attendant decrease in their mRNA expression in RAW264.7 cells. In addition, when genistein was administered to rats, decreases in alveolar bone height and bone volume fraction induced by ligature placement were significantly inhibited. Genistein administration also prevented ligature-induced alterations in the microstructural parameters of trabecular bone, including trabecular thickness, trabecular separation, bone mineral density and structure model index.ConclusionsWhile additional studies are required, we suggest that genistein could be utilized for the therapy of human periodontitis in the future.     

iNOS-derived nitric oxide stimulates osteoclast activity and alveolar bone loss in ligature-induced periodontitis in rats

Background: Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long‐term iNOS inhibition, and the differentiation and activity of OC from iNOS‐knockout (KO) mice in vitro. Methods: Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild‐type C57BL/6 mice. OC were counted 6 days later after tartrate‐resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. Results: Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature‐induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild‐type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. Conclusion: Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature‐induced periodontitis, thus confirming that iNOS‐derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.     

Neurogenic component in ligature-induced periodontitis in the rat

Abstract. Effect of ligation on the vascular permeability in the gingiva and alveolar mucosa encircling the mandibular left 1st molar was studied in rats with and without capsaicin pretreatment. Vascular permeability was assessed by the Evans blue extravasation. Ligation caused a significant augmentation in vascular permeability of the gingivomucosal tissue at day 8 (right: 18.14±1.68 μg g^?1; left (ligature): 38.21 ±2.43 μg g^?1, n=8, p(0.001) and at day 14 (right: 20.31 ±1.71 μg g^?1: left (ligature): 36.98±2.73 μg^?1, n= 8, p(0.001). 4 days after ligation, no difference could be observed in vascular permeability in the oral mucosa of the ligated side (left: 23.14±1.21 μg^?1) as compared to the side without ligature (right: 23.5±1.45μg g^?1, n=8, NS). There was no elevation of vascular permeability of gingivomuscosal tissue around the ligation in rats pretreated with capsaicin either in newborn age (right: 23.92±1.76 μg g^?1; left (ligature): 23.51±2.16 μg g^?1, n=8, NS) or in adult age (right: 20.61±1.62 μg g^?1; left (ligature): 20.85±1.07 μg g^?1, n=8, NS). Light microscopical studies of oral mucosa revealed, that 8 and 14 days after the ligature placed around the mandibular left 1st molar of the rat, there resulted an accumulation of inflammatory cells in the connective tissue. There was no inflammatory reaction in the gingivomucosal tissue of the side of the ligature 4 days after ligature, neither could inflammatory reaction be detected 14 days following ligation in the oral mucosa of rats systemically pretreated with capsaicin either neonatally or in the adult. Based on these observations, it can be assumed that neurogenic mechanism plays a crucial role in the development of inflammation in the oral cavity induced by mechanical and/or bacterial stimulation frequently found in clinical practice.     

A model of periodontitis in the rat: effect of lipopolysaccharide on bone resorption, osteoclast activity, and local peptidergic innervation

OBJECTIVE: To establish and characterise a rat model of periodontitis that reiterates the features of human disease. METHODS: Periodontal inflammation was induced by a single injection of 10 microg liposaccharide (LPS) (Salmonella typhimurium) in 1 microl saline into rat mandibular gingiva at the buccomesial aspect of the second molar. Animals were killed after 3, 7 and 10 days, mandibles dissected and sectioned for histological and immunocytochemical analysis. RESULTS: LPS injection resulted in a significant gingival and periodontal inflammation with inflammatory infiltrate, apical migration of the junctional epithelium, interdental bone loss, and activation of osteoclasts at the site of injection 7 and 10 days after injection. At 10 days post injection, there was a significant trend for bone loss on both sides of the mandible. Periodontal inflammation was associated with alteration in the levels of calcitonin gene-related peptide-like immunoreactivity in nerve terminals innervating the inflamed gingival papilla. CONCLUSION: Intragingival injection of LPS in the rat provides an easily induced reproducible experimental model of periodontal inflammation that reiterates features of human disease.     

iTRAQ-based quantitative analysis of alveolar bone resorption in rats with experimental periodontitis

ObjectivePeriapical periodontitis results in alveolar bone resorption around the root apex. During the progression of inflammation, host cells release various inflammatory mediators and pro-inflammatory cytokines through immune responses. However, the pathological mechanisms associated with periapical bone destruction remain unclear. This study was objected to identify differentially regulated proteins in periapical periodontitis via a quantitative proteomics approach using isobaric tags for relative and absolute quantification (iTRAQ) labelling of peptides.MethodsA model of periapical periodontitis by sealing LPS into the pulp chambers of rats was established. iTRAQ was employed to screen differentially expressed proteins in alveolar bone between periapical lesions and healthy controls. These proteins were further analysed by bioinformatics. And four proteins were validated by western bolt.ResultsWe identified 4398 proteins, of which 7 were up-regulated and 151 were down-regulated in periapical periodontitis compared to normal tissue. Using bioinformatics tools such as GO and KEGG pathway analysis, we found that our proteomics strategy could identify and quantify differentially expressed proteins that were not described in previous studies examining periapical periodontitis; these proteins included hexokinase, legumain and members of the keratin family.ConclusionIn summary, our results represent potential biomarkers for the detection of periapical periodontitis and demonstrate that quantitative proteomics is a robust discovery tool for the identification of differentially regulated proteins in periapical periodontitis.     

Clinical and histological findings in ligature-induced experimental periodontitis in dogs A pilot study

Prior to experimental periodontal regenerative procedures in animals, the process of destruction due to periodontitis is often simulated by placing ligatures around the necks of teeth. 2 models, one using cotton floss ligatures and the other elastic rubber ligatures, have been widely used. The aim of this study was to compare these 2 methods using clinical and histometric criteria. Premolars on both sides of the mandibular jaw were used in 2 beagle dogs. Periodontal tissue breakdown was induced during a 48-day period using cotton floss ligatures on one side of the mandible and contra-laterally by elastic rubber ligatures. At weekly intervals, the ligatures were changed and pocket depth and loss of probing attachment were recorded. All the ligatures were removed after 7 weeks. The animals were fed a soft, plaque-promoting diet during the experimental period. 20 days after ligature removal, the teeth were hemisected and the 2 roots extracted. From each tooth, 1 root was prepared for scanning electron microscopy and one for light microscopy. At the time of extraction, no differences regarding pocket depth or probing attachment level were found between the 2 groups. Root resorption was observed in all elastic ligature teeth, whereas only minor alterations at the root surface were observed on cotton ligature teeth. This finding may be of particular interest to investigators designing studies evaluating the healing sequalae of regenerative surgery in animals. As was observed in this study, root resorption can already be present in the pre-experimental phase of periodontal tissue breakdown, particularly if elastic ligatures are used.     

Low-dose doxycycline inhibits bone resorption associated with apical periodontitis

Aim To test the effect of low‐dose doxycycline on bone resorption associated with apical periodontitis. Methodology Apical periodontitis was induced by occlusal pulp exposure in the mandibular first molars of 36 rats. Animals were divided into three groups of 12: group A received doxycycline in drinking water at a dose of 5.85 mg day^?1; group B received a dose of 1.48 mg day^?1 (one‐quarter of the original dose); and group C received no medicament and served as the control. A bioassay determined the doxycycline serum levels. After 21 days, the mandibles were removed, radiographed and the radiographs scanned to generate digital images. These images were analysed morphometrically and the total area of the periapical bone resorption of the mesial and distal roots of each tooth was determined and used to compare the groups. Statistical analysis was completed using anova with repeated measures. Results The mean doxycycline serum level in group A was 0.22(±0.03) μg mL^?1 and in group B below the detection level of the assay (<0.062 μg mL^?1). The mean area of the periapical bone resorption in the control group C was 2.91(±0.61) mm². In animals treated with a low‐dose doxycycline, the mean size of the bone resorption was significantly smaller at 1.59(±0.59) mm² (group A) and 1.72(±0.85) mm² (group B) (P = 0.001). No significant difference was found in the area of the bone resorption between these two groups A and B. Conclusions Low‐dose doxycycline reduced the area of bone resorption associated with apical periodontitis in the mandibular first molar teeth of rats.     

A novel murine model for chronic inflammatory alveolar bone loss

Oz HS, Ebersole JL. A novel murine model for chronic inflammatory alveolar bone loss. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01207.x. © 2009 John Wiley & Sons A/S
Background and Objective:  Chronic inflammatory bowel disease (IBD) demonstrates some similarities to the dysregulated chronic immunoinflammatory lesion of periodontitis. Trinitrobenzene sulphonic acid (TNBS) and dextran sodium sulphate (DSS) administered to rodents have been shown to elicit inflammatory responses that undermine the integrity of the gut epithelium in a similar manner to IBD in humans. The objective of this study was to evaluate the ability of these chemicals to elicit periodontal inflammation as a novel model for alveolar bone loss.
Material and Methods:  Mice were treated by oral application of TNBS twice a week, or with DSS in the diet over a period of 18 weeks. Alveolar bone loss was assessed on the defleshed skull using morphometric measures for area of bone resorption.
Results:  The TNBS-treated animals tolerated oral administration with no clinical symptoms and gained weight at a similar rate to normal control animals. In contrast, DSS exerted a systemic response, including shortening of colonic tissue and liver enzyme changes. Both TNBS and DSS caused a localized action on periodontal tissues, with alveolar bone loss observed in both maxilla and mandibles, with progression in a time-dependent manner. Bone loss was detected as early as week 7, with more severe periodontitis increasing over the 18 weeks ( p  < 0.001). Young (7-month-old) and old (12-month-old) mice with severe combined immunodefiency were treated with TNBS for a period of 7 weeks and did not develop significant bone loss.
Conclusion:  These data show that oral administration of TNBS or DSS provokes alveolar bone loss in concert with the autochthonous oral microbiota.     

Salivary biomarkers: Relationship between oxidative stress and alveolar bone loss in chronic periodontitis

Objectives. Oxidative stress is implicated in the pathogenesis of many systemic and oral diseases such as periodontal disease. The main aim of this study is to explore a possible association between salivary markers of OS and alveolar bone loss. Materials and methods. The study included 20 patients with chronic periodontitis and 20 controls. Salivary OS biomarkers 8-hidroxy-desoxguanosine (8-HOdG), malondialdehyde (MDA), uric acid, total antioxidant capacity (TAC) and glutathione peroxidase (GPx) were evaluated. Bone loss markers such as C-terminal telopeptide of type I collagen (CTX I), matrix metalloproteinases-8 (MMP-8), osteocalcin and 25-hydroxy vitamin D₃ (25- OH D) were detected in this study. The methods included general biochemical tests and ELISA. Results. Salivary 8-OHdG, MDA levels were significantly higher in the chronic periodontitis group compared with controls (p < 0.05). Salivary activities for uric acid, TAC and GPx were significantly decreased in patients with chronic periodontitis vs controls (p < 0.05). Salivary levels for CTX I, MMP-8, 25-OH D and Osteocalcin were significantly higher in the chronic periodontitis group compared to the controls (p < 0.05). A significant positive correlation was observed between salivary levels of MDA and CTX I. Significant negative correlations between uric acid and CTX I and between MMP-8 and uric acid have been found. Significant positive correlations were observed between CTX I, MMP-8, 25-OH D, osteocalcin and clinical parameters of periodontal disease. Conclusions. Important oxidative stress associated with alveolar bone loss biomarkers can be detected in saliva of patients with periodontal disease.     

Juvenile periodontitis Localization of bone loss in relation to age, sex, and teeth

The distribution of bone loss in 156 patients, 12-32 years old, with juvenile periodontitis was analyzed according to age, sex, and teeth affected. The criteria for bone loss were: vertical or horizontal bone loss involving more than one-third of the root as judged by radiographs. Three age groups were established: 12-18, 19-25, and 26-32 years old. Three types of bone loss localization were defined: I. First molars and/or incisors. II. First molars, incisors and some additional teeth (total less than 14 teeth). III. General involvement . There was a dominance of female patients. The ratio females: males decreased from 5.3:1 in the youngest age group to 1.5:1 in the oldest. The mean number of involved teeth increased with age from 5.3 teeth in the youngest group to 11.6 in the oldest. The frequency of type I bone loss decreased from 55% in the youngest group to 7% in the oldest. Type II occurred with the same frequency (55-58%) in all three age groups. Type III was not seen in the youngest group whereas it increased from 17% in the middle to 35% in the oldest group. Of the total number of involved teeth, the first molars were most frequently affected, followed by the incisors. Maxillary teeth were involved to a slightly higher degree than mandibular teeth, and there was a strong "mirror effect" between involved teeth of right and left jaw halves.     

睾酮水平对牙周炎小鼠模型炎症性骨吸收的影响及机制探讨

目的：探讨睾酮水平对牙周炎小鼠模型炎症性骨吸收的影响及机制。方法：48只SD小鼠随机分为未结扎组、假手术组、去势组、去势+睾酮组4组,每组各12只,分别进行空白处理、去势模型和牙周炎模型构建。于结扎术后6周采集小鼠内眦静脉血,测定血清睾酮水平。处死小鼠后,取左侧上颌骨组织,进行苏木精-伊红染色和亚甲蓝染色,比较各组小鼠牙槽骨丧失量（alveolar bone loss,ABL）和牙槽骨吸收面积。利用实时荧光定量PCR测定牙龈组织中炎性细胞因子mRNA的表达,同时对血清睾酮水平与ABL、牙槽骨吸收面积及细胞因子进行相关性分析。采用SPSS 20.0软件包对数据进行统计学分析。结果：假手术组、去势组和去势+睾酮组小鼠血清睾酮水平显著低于未结扎组（P<0.05）;去势+睾酮组小鼠血清睾酮水平显著高于假手术组和去势组（P<0.05）;去势组小鼠血清睾酮水平显著低于假手术组（P<0.05）;去势+睾酮组小鼠ABL显著大于未结扎组、假手术组和去势组（P<0.05）,去势组小鼠ABL显著小于假手术组（P<0.05）;去势+睾酮组小鼠牙槽骨吸收面积显著大于未结扎组、假手术组和去势组（P<0.05）,去势组小鼠牙槽骨吸收面积显著小于假手术组（P<0.05）;假手术组、去势组和去势+睾酮组小鼠牙龈组织中白介素1β（IL-1β）mRNA、白介素6（IL-6）mRNA及肿瘤坏死因子α（TNF-α）mRNA水平显著高于未结扎组,白介素10（IL-10）mRNA水平显著低于未结扎组（P<0.05）;假手术组和去势组小鼠牙龈组织中IL-1β mRNA水平显著低于去势+睾酮组,去势组显著低于假手术组（P<0.05）;血清睾酮水平与ABL、牙槽骨吸收面积、IL-1β呈正相关（P<0.05）。结论：牙周炎小鼠睾酮水平降低,睾酮长期耗竭状态可减少牙槽骨炎症性骨吸收,可能通过降低IL-1β水平而实现。合理降低睾酮水平,有可能成为减少牙周炎患者牙槽骨吸收的治疗新思路。     

Enhanced alveolar bone loss in a model of non-invasive periodontitis in rice rats

Oral Diseases (2012) 18 , 459–468 Objective: The rice rat (Oryzomys palustris) develops periodontitis‐like lesions when fed a diet rich in sucrose and casein (H‐SC). We aimed to establish whether this model can accurately mimic the development of human periodontitis. Materials and Methods: For this purpose, 28‐day‐old rice rats (15/group) were assigned to standard (STD) or H‐SC diets and sacrificed after 6, 12, and 18 weeks. Jaws were processed for morphometric, histometric, histologic, histomorphometric, and micro‐CT analyses. Results: We found a progressive increase in horizontal alveolar bone loss (ABL) with age in maxillae of rats fed the STD diet as determined by morphometry. The H‐SC diet exacerbated horizontal ABL at the palatal surface at 12 and 18 weeks. Furthermore, increased vertical ABL was detected in mandibles and maxillae of rats fed the H‐SC diet for 12 and/or 18 weeks by histometry and micro‐CT. Remarkably, the H‐SC diet significantly increased bone remodeling at the interproximal alveolar bone of mandibles from rats fed for 6 weeks, but not in those fed for longer periods. Conclusions: These findings indicate that the H‐SC diet induced a transient increase in alveolar bone remodeling, which is followed by ABL characteristic of moderate periodontitis.     

Inhibition of 5‐lipoxygenase attenuates inflammation and bone resorption in lipopolysaccharide‐induced periodontal disease

1 Background

Arachidonate‐5‐lipoxygenase (5‐LO) activity and increased leukotriene B4 (LTB4) production have been implicated in various inflammatory conditions. Increased production of leukotrienes has been associated with periodontal diseases; however, their relative contribution to tissue destruction is unknown. In this study, an orally active specific 5‐LO inhibitor is used to assess its role in inflammation and bone resorption in a murine model of lipopolysaccharide (LPS)‐induced periodontal disease.

2 Methods

Periodontal disease was induced in Balb/c mice by direct injections of LPS into the palatal gingival tissues adjacent to the maxillary first molars three times per week for 4 weeks. Animals were treated with biochemical inhibitor (2 mg/kg/daily) or the same volume of the vehicle by oral gavage. Microcomputed tomography analysis was used to assess bone resorption. Enzyme immunoassay determined LTB4, and enzyme‐linked immunosorbent assays quantified tumor necrosis factor (TNF), interleukin (IL)‐12, and IL‐10 in gingival tissues. Histologic sections were used for the morphometric analysis (number of neutrophils and mononuclear cells). Osteoclasts were counted in tartrate‐resistant acid phosphatase–stained sections.

3 Results

Administration of 5‐LO inhibitor effectively reduced production of LTB4 (23.7% decrease) and significantly reduced TNF and IL‐12 levels in gingival tissues. Moreover, reduction of LTB4 levels in gingival tissues was associated with a significant decrease in bone resorption and a marked reduction in number of osteoclasts and inflammatory cells.

4 Conclusion

5‐LO activity plays a relevant role in inflammation and bone resorption associated with the LPS model of experimental periodontal disease.