RESUMO
Combretum leprosum Mart. is a plant of the Combretaceae family, widely distributed in the Northeast region of Brazil, popularly used as an anti-inflammatory agent, and rich in triterpenes. This study evaluated in vitro and in silico potential osteogenic of two semisynthetic triterpenes (CL-P2 and CL-P2A) obtained from the pentacyclic triterpene 3ß,6ß,16ß-trihydroxylup-20(29)-ene (CL-1) isolated from C. leprosum. Assays were carried out in cultured murine osteoblasts (OFCOL II), first investigating the possible toxicity of the compounds on these cells through viability assays (MTT). Cell proliferation and activation were investigated by immunohistochemical evaluation of Ki-67, bone alkaline phosphatase (ALP) activity, and mineralization test by Von Kossa. Molecular docking analysis was performed to predict the binding affinity of CL-P2 and CL-P2A to target proteins involved in the regulation of osteogenesis, including: bone morphogenetic protein 2 (BMP-2), proteins related to Wingless-related integration (WNT) pathway (Low-density lipoprotein receptor-related protein 6-LRP6 and sclerostin-SOST), and receptor activator of nuclear factor (NF)-kB-ligand (RANK-L). Next, Western Blot and immunofluorescence investigated BMP-2, WNT, RANK-L, and OPG protein expressions in cultured murine osteoblasts (OFCOL II). None of the CL-P2 and CL-P2A concentrations were toxic to osteoblasts. Increased cell proliferation, ALP activity, and bone mineralization were observed. Molecular docking assays demonstrated interactions with BMP-2, LRP6, SOST, and RANK-L/OPG. There was observed increased expression of BMP-2, WNT, and RANK-L/OPG proteins. These results suggest, for the first time, the osteogenic potential of CL-P2 and CL-P2A.
RESUMO
To investigate de effect of PAb gel on the bone tissue of rats submitted to Bisphosphonate-related osteonecrosis of the jaws (BRONJ). Initially, 54 animals were submitted to BRONJ model by Zoledronic Acid (ZA) (0.1 mg/kg 3x/wk for 9 wk, ip), followed by the 1st upper left molar extraction at the 8th wk. After tooth removal, the animals were divided into 3 groups, ZA that received placebo gel or PAb gel that received 1% PAb gel, inside the dental alveolus. The control Group (CONTROL) received 0.1 mg/kg of 0.9% saline and then placebo gel. Three weeks after tooth extraction, the animals were euthanized, and maxillae were colleted for macroscopic, radiographic, histological and Raman spectomery assays. Additionally, GSK3b, beta-catenin, and Runx2 mRNA expressions were determined. Blood samples were collected for the analysis of Bone-specific alkaline phosphatase (BALP) levels. PAb gel improved mucosal healing, increased the number of viable osteocytes, while it reduced the number of empty lacunae, as well as the amount of bone sequestration. Furthermore, PAb gel positively influenced the number and functionality of osteoblasts by stimulating Wnt signaling, thereby inducing bone remodeling. Additionally, PAb gel contributed to improved bone quality, as evidenced by an increase in bone mineral content, a decrease in bone solubility, and an enhancement in the quality of collagen, particularly type I collagen. PAb gel mitigated bone necrosis by stimulating of bone remodeling through Wnt signaling and concurrently improved bone quality. PAb gel emerges as a promising pharmacological tool for aiding in BRONJ therapy or potentially preventing the development of BRONJ.
Assuntos
Agaricus , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos , Conservadores da Densidade Óssea , Animais , Ratos , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/tratamento farmacológico , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/etiologia , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/patologia , Difosfonatos , Maxila/patologia , Extração Dentária , Via de Sinalização Wnt , Ácido ZoledrônicoRESUMO
Statement of the Problem: Periodontitis is an inflammatory disease that causes bone loss. Some patients do not respond well to the classic treatment and need therapies that minimize bone loss, the main sequel of the disease. Chenopodium ambrosioides L. has stood out due to its anti-inflammatory and anti-oxidative activities. However, no study has yet investigated its effect on periodontitis. Purpose: This study aimed to evaluate the bone protective effect of Chenopodium ambrosioides L. (CAL) extract on ligature-induced periodontitis model in rats. Materials and Method: For this, a pre-clinical assay was performed, using male Wistar rats divided into 3 groups: Naive (N) (n=6), not submitted to any procedure; Saline (SAL) (n=6), submitted to ligature-induced periodontitis and receiving 2 ml/kg of 0.9% saline solution; and CAL extract, which was subdivided into 3 subgroups (n=6/subgroup) receiving the CAL at 3 (CAL3), 10 (CAL10) or 30 mg/kg (CAL30). All agents were given, by oral gavage, 30 min before periodontitis induction and daily until euthanasia (11th day). By then, maxillae were removed for macroscopic, histological, and histometric analyses. Kidneys, liver, and stomach were collected to evaluate the safety of CAL extract. High-performance liquid chromatography (HPLC) assay was used to investigate the flavonoid content in the extract. Results: Chenopodium ambrosioides L. extract at 30mg/kg showed a reduction by 58% in bone loss marked by an increase (+35%) in the number of osteoblasts and a reduction (-51%) on the number of osteoclasts (p< 0.05). No significant alteration in the liver, kidney, or stomach was seen. Rutin was the main flavonoid found. Conclusion: In summary, it was observed that Chenopodium ambrosioides L. extract has shown important anti-inflammatory and bone anabolic and anti-resorptive properties without causing toxicity in the main organs. Rutin, as the main flavonoid of the extract, seems to be responsible for the beneficial effect of this agent.
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The canonical Wnt pathway participates in inflammatory diseases and it is involved in neuropathic pain. This study evaluated the immunoexpression of the canonical Wnt signaling pathway in the articular cartilage of the temporomandibular joint (TMJ) and along the nociceptive trigeminal pathway in arthritic rats. For this, male Wistar rats were divided into Control (C) and Arthritic (RA) groups. Arthritis induction was performed through subcutaneous injection of methylated bovine serum albumin (mBSA) and complete Freund Adjuvant (CFA)/ Incomplete Freund Adjuvant (IFA) on the first 14 days (once a week), followed by 3 weekly intra-articular injections of mBSA (10 µl/joint; left TMJ). The following parameters were evaluated: nociceptive threshold, inflammatory infiltrate, type I and III collagen birefringence, immunohistochemistry for IL-1ß, TNF-α, IL-6, Wnt10b, ß-catenin, cyclin-D1 in articular cartilage, c-Myc in synovial membrane, and immunofluorescence analysis for c-Fos, Wnt-10b and ß-catenin in the trigeminal ganglion and the trigeminal subnucleus caudalis. The RA group showed intense articular cartilage damage with proliferation of type III collagen, increased immunoexpression of proinflammatory cytokines and Wnt-10b, ß-catenin and cyclin-D1 in the articular cartilage and c-Myc in the synovial membrane. In the RA group, a reduction in the nociceptive threshold was observed, followed by a significant increase in the expression of Wnt-10b in neurons and ß-catenin in satellite cells of the trigeminal ganglion. c-Fos immunoexpression was observed in neurons, peripherally and centrally, in arthritic rats. Our data demonstrated that TMJ arthritis in rats causes articular cartilage damage and nociceptive behavior, with increased immunoexpression of canonical Wnt pathway in the articular cartilage and trigeminal ganglion.
Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos , Conservadores da Densidade Óssea , Osteonecrose , Animais , Atorvastatina/efeitos adversos , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/tratamento farmacológico , Conservadores da Densidade Óssea/efeitos adversos , Difosfonatos/farmacologia , Arcada Osseodentária , Osteonecrose/induzido quimicamente , Osteonecrose/tratamento farmacológico , Ratos , Ácido Zoledrônico/efeitos adversosRESUMO
OBJETIVE: This study aimed to evaluate the anti-resorptive activity of a semi-synthetic coumarin derivative from Platymiscium floribundum, named 6,7-dimethoxy-3-nitrocoumarin. MATERIAL AND METHODS: Molecular docking studies were performed to test the binding performance of the derivative against targets associated with alveolar bone loss (TNF-α, IL-1ß, and catalase) and a target considered an antioxidant defense (HO-1) during periodontitis. Periodontitis was induced by placing a nylon ligature around the second molars. The rats received for 11 days 6,7-dimethoxy-3-nitrocoumarin (0.01, 0.1, or 1 mg/kg) or vehicle. We investigated by RT-qPCR analysis (TNF-α, IL-1ß, and HO-1 mRNA expression levels) and by colorimetric assay (catalase activity) the mechanism of action mediated by 6,7-dimethoxy-3-nitrocoumarin. The in vivo toxicity of 6,7-dimethoxy-3-nitrocoumarin was evaluated. RESULTS: 6,7-Dimethoxy-3-nitrocoumarin (0.1 or 1 mg/kg) reduced alveolar bone loss (1.05 ± 0.24), when compared to vehicle-treated group (3.05 ± 0.30). The interactions of 6,7-dimethoxy-3-nitrocoumarin and the four targets (TNF-α, IL-1ß, catalase, and HO-1) showed firm bonds above 6.0 kcal/mol. 6,7-dimethoxy-3-nitrocoumarin (1 mg/kg) lowered mRNA expression levels of TNF-α (2.33 ± 0.56) and IL-1ß (19.87 ± 2.9), while it increased both the mRNA expression levels of HO-1 (43.40 ± 1.05) and the catalase activity (46.42 ± 4.59), when compared to vehicle-treated group (46.29 ± 8.43; 37.83 ± 4.38; 1.58 ± 0.11; 8.93 ± 1.86, respectively). The animals did not show any signs of toxicity. CONCLUSION: 6,7-Dimethoxy-3-nitrocoumarin decreased inflammatory bone loss in the ligature-induced periodontitis in rats, and the activation of the HO-1 pathway may contribute, at least partially, to its protective effects by reducing TNF-α and IL-1ß mRNA levels and increasing catalase activity. CLINICAL RELEVANCE: 6,7-Dimethoxy-3-nitrocumarin could be used as an adjunct to subgingival instrumentation during active and supportive periodontal treatment.
Assuntos
Perda do Osso Alveolar , Cumarínicos , Fabaceae/química , Periodontite , Animais , Cumarínicos/farmacologia , Heme Oxigenase (Desciclizante) , Heme Oxigenase-1 , Interleucina-1beta , Simulação de Acoplamento Molecular , Periodontite/tratamento farmacológico , Compostos Fitoquímicos/farmacologia , Ratos , Fator de Necrose Tumoral alfaRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation in the joints. Although methotrexate (MX) is the first-line treatment, side effects are common. This study aimed to investigate the effects of quercetin (QT) and/or MX on inflammation and systemic toxicity in a rat model of RA. Male Wistar rats were divided into control (C), RA, QT, MX, and QT + MX groups (n=6). The RA induction consisted of three intra-articular injections of methylated bovine serum albumin (1×/week) in the temporomandibular joint (TMJ). QT (25 mg/kg) and/or MX (0.75 mg) administration occurred by oral gavage daily. We performed mechanical hyperalgesia in TMJ, leukocyte recruitment in synovial fluid, histopathology, and immunohistochemistry (TNF-α, IL-17, and IL-10) in synovial membrane and toxicity parameters. The RA showed a reduction in the nociceptive threshold (p<0.001), increase in leukocyte recruitment in synovial fluid (p<0.001), intense inflammatory infiltrate (p<0.001), and intense immunoexpression of TNF-α, IL-17, and IL-10 in the synovial membrane (p<0.001) compared to C (p<0.001). QT and/or MX therapy reduced inflammatory parameters (p<0.001). However, downregulation of IL-10 was observed only in the groups that received MX (p<0.001). Leukocytosis was seen in RA (p<0.05), but QT and/or MX reversed it (p<0.05). MX was associated with pathological changes in the liver and higher levels of transaminases when compared to the other groups (p<0.05). QT co-administered with MX reversed this hepatotoxicity (p<0.05). There were no alterations in the kidney between the groups (p>0.05). QT has potential to support MX therapy, showing anti-inflammatory and hepatoprotective effects in this model.
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Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Artrite Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Quercetina/uso terapêutico , Soroalbumina Bovina/toxicidade , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Hiperalgesia/prevenção & controle , Fígado/metabolismo , Masculino , Quercetina/farmacologia , Ratos , Ratos Wistar , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Resultado do TratamentoRESUMO
Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.
Assuntos
Processo Alveolar/efeitos dos fármacos , Conservadores da Densidade Óssea/efeitos adversos , Ligamento Periodontal/efeitos dos fármacos , Técnicas de Movimentação Dentária , Raiz Dentária/efeitos dos fármacos , Ácido Zoledrônico/efeitos adversos , Animais , Conservadores da Densidade Óssea/administração & dosagem , Reabsorção Óssea/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Masculino , Osteoporose/tratamento farmacológico , Ratos Wistar , Ácido Zoledrônico/administração & dosagemRESUMO
OBJECTIVE: This work aimed to study the role of inflammation in medication-related osteonecrosis of the jaw (MRONJ) in rats with focus on Wnt signaling. METHODS: A total of 36 female Wistar rats (12 weeks ± 200 g) were divided into 2 groups (n = 6) in 3 experiments: saline (SAL) and zoledronic acid (ZOL). For MRONJ induction, rats received 0.1 mg/kg of ZOL (ip) 3×/week for 9 weeks. Animals from the SAL group received 0.1 mg/kg of 0.9% SAL, ip 3×/week for 9 weeks. On the 8th week, 3 left upper molars were extracted, and on the 11th week, they were euthanized. Maxillae were evaluated by macroscopic and histopathological analyses; scanning electron microscopy (SEM); immunohistochemistry for DKK-1, Wnt 10b, and caspase-3; and Raman spectrometry. Gingiva was also collected for TNF-α e IL-1ß quantification. RESULTS: Bone necrosis was confirmed by healing impairment, reduced number of viable osteocytes, increased caspase-3 immunoexpression, and increased number of empty lacunae (p < 0.05). ZOL enhanced inflammation and increased gingival levels of IL-1ß and TNF-α (p < 0.05). Irregular indentations were seen on bone after ZOL administration. Bone necrosis was marked by reduced amount of total and type I collagen. ZOL reduced the mineral/matrix ratio and increased carbonate/phosphate ratio. It was observed a significant reduction on Wnt10b and beta-catenin immunolabeling in the bone tissue of ZOL group. CONCLUSION: In summary, MRONJ model caused bone necrosis due to intense inflammation. Wnt signaling seems to play an important role in this process. CLINICAL RELEVANCE: New therapeutic strategies focusing on Wnt pathway can provide an interesting approach for future treatments.
Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos , Conservadores da Densidade Óssea , Animais , Conservadores da Densidade Óssea/toxicidade , Difosfonatos/toxicidade , Feminino , Maxila , Ratos , Ratos Wistar , Via de Sinalização Wnt , Ácido Zoledrônico/toxicidadeRESUMO
OBJECTIVE: To investigate the participation of canonical Wnt and NF-κB signaling pathways in an experimental model of chronic arthritis induced by methylated bovine serum albumin (mBSA) in rat temporomandibular joint (TMJ). MATERIALS AND METHODS: Wistar rats were sensitized by mBSA+Complete Freund Adjuvant (CFA)/Incomplete Freund Adjuvant (IFA) on the first 14 days (1 ×/week). Subsequently, they received 1, 2 or 3 mBSA or saline solution injections into the TMJ (1 ×/week). Hypernociceptive threshold was assessed during the whole experimental period. 24 h after the mBSA injections, the TMJs were removed for histopathological and immunohistochemical analyses for TNF-α, IL-1ß, NF-κB, RANKL, Wnt-10b, ß-catenin and DKK1. RESULTS: The nociceptive threshold was significantly reduced after mBSA injections. An inflammatory infiltrate and thickening of the synovial membrane were observed only after mBSA booster injections. Immunolabeling of TNF-α, IL-1ß and Wnt-10b was increased in the synovial membrane in arthritic groups. The immunoexpression of nuclear ß-catenin was significantly higher only in the group that received 2 booster TMJ injections. However, NF-κB, RANKL and DKK1 immunoexpression were increased only in animals with 3 mBSA intra-articular injections. CONCLUSION: Our results suggest that canonical Wnt and NF-κB signaling pathways participate in the hypernociception and inflammatory response in TMJ synovial membrane during the development of rheumatoid arthritis in rats.