RESUMO
The genetically transformed hairy root line LRT 7.31 obtained by infecting leaf explants of Lopezia racemosa Cav with the Agrobacterium rhizogenes strain ATCC15834/pTDT, was evaluated to identify the anti-inflammatory and cytotoxic compounds reported previously for the wild plant. After several subcultures of the LRT 7.31 line, the bio-guided fractionation of the dichloromethane-methanol (1:1) extract obtained from dry biomass afforded a fraction that showed important in vivo anti-inflammatory, and in vitro cytotoxic activities. Chemical separation of the active fraction allowed us to identify the triterpenes ursolic (1) and oleanolic (2) acids, and (23R)-2α,3ß,23,28-tetrahydroxy-14,15-dehydrocampesterol (3) as the anti-inflammatory principles of the active fraction. A new molecule 3 was characterized by spectroscopic analysis of its tetraacetate derivative 3a. This compound was not described in previous reports of callus cultures, in vitro germinated seedlings and wild plant extracts of whole L. racemosa plants. The anti-inflammatory and cytotoxic activities displayed by the fraction are associated to the presence of compounds 1-3. The present study reports the obtaining of the transformed hairy roots, the bioguided isolation of the new molecule 3, and its structure characterization.
Assuntos
Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Colesterol/análogos & derivados , Germinação/efeitos dos fármacos , Onagraceae/química , Fitosteróis/farmacologia , Agrobacterium/química , Agrobacterium/genética , Anti-Inflamatórios/química , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/crescimento & desenvolvimento , Colesterol/química , Colesterol/farmacologia , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Fitosteróis/química , Raízes de Plantas/química , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plântula/efeitos dos fármacosRESUMO
The expression of transforming growth factor-beta 1 (TGF-ß1) inside the callus cells of diabetic rats and the impact of insulin therapy on its expression and biomechanics was investigated. The rats were randomly divided as follows: an insulin therapy group (IT), a diabetic model group (DM), and a non-diabetic control group (NC). Bone specimens from each group were extracted at different times for immunohistochemical observation of the expression of TGF-ß1. Concurrently, the destruction torque and torsional stiffness were detected at different times. One to four weeks after fracture, TGF-ß1 was widely expressed in fractured callus cells and periosteal proliferating cells, while the expression inside diabetic cells was significantly reduced. The expression of TGF-ß1 decreased over the first 68 weeks, and the mature bone cells never expressed TGF-ß1. The destruction torque (Nm) detected in the 6th week revealed that there was a statistically significant difference between the DM, NC, and IT groups (P < 0.01). In conclusion, TGF-ß1 expression was significantly reduced inside the callus cells of diabetic rats. Insulin therapy increased TGF-ß1 expression inside the callus cells of diabetic rats and improved the biomechanical characteristics of the callus.
Assuntos
Calo Ósseo/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fraturas da Tíbia/tratamento farmacológico , Fator de Crescimento Transformador beta1/genética , Animais , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Proliferação de Células , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Consolidação da Fratura/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Dureza , Masculino , Periósteo/efeitos dos fármacos , Periósteo/metabolismo , Periósteo/patologia , Ratos , Ratos Wistar , Fraturas da Tíbia/complicações , Fraturas da Tíbia/genética , Fraturas da Tíbia/patologia , Torque , Fator de Crescimento Transformador beta1/metabolismoRESUMO
We studied the effects of different media for callus induction and differentiation, and pre-culture period of immature wheat embryo culture on biolistic transformation efficiency for including antifreeze gene KN2 and bar conferring resistance to the herbicide bialaphos. The percentage of plantlets generated from induction and differentiation media without Cu2+ was lower than those cultured on differentiation media with Cu2+ (71.15%) or induction media with Cu2+ (68.45%) and both induction and differentiation media with Cu2+ (52.17%). The combinations of Nor medium for callus induction and Cu2+ medium for regeneration, and Cu2+ medium for induction and R medium for regeneration were superior for biolistic transformation. The calli induced on Cu2+ medium and pre-cultured for 4 d before biolistic transformation, and cultured on R medium after biolistic transformation produced the highest percentage (65%) of transgenic plantlets with the KN2 gene. Overall, about 50% plantlets regenerated from calli pre-cultured 4d before bombardment carried the KN2 gene; 44.7% of the plantlets carried the bar gene, which was higher than for any other treatment, followed by pre-culture 1d with 31.43% transformation rate for the KN2 gene and 20% transformation rate for the bar gene.
Assuntos
Proteínas Anticongelantes/genética , Resistência a Herbicidas/genética , Transformação Genética , Triticum/genética , Biolística , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/crescimento & desenvolvimento , Diferenciação Celular/genética , Cobre/química , Compostos Organofosforados/toxicidade , Plantas Geneticamente Modificadas , Triticum/efeitos dos fármacos , Triticum/crescimento & desenvolvimentoRESUMO
BACKGROUND: Methotrexate (MTX) is an anti-metabolite used in rheumatology and oncology. High doses are indicated for oncological treatment, whereas low doses are indicated for chronic inflammatory diseases. This study evaluated the effect of two MTX treatment schedules on the bone healing of the temporomandibular joint fracture in rats. METHODS: Seventy-five adult male Wistar rats were used to generate an experimental unilateral medially rotated condylar fracture model that allows an evaluation of bone healing and the articular structures. The animals were subdivided into three groups that each received one of the following treatments intraperitoneally: saline (1 mL/week), low-dose MTX (3 mg/kg/week) and high-dose MTX (30 mg/kg). The histological study comprised fracture site and temporomandibular joint evaluations and bone neoformation was evaluated by histomorphometric analysis. A biochemical parameter of bone formation was also assessed. RESULTS: When compared with saline, high-dose MTX delayed bone fracture repairs. In this latter group, after 90 days, the histological analysis revealed atrophy of the fibrocartilage and the presence of fibrous tissue in the joint space. The histomorphometric analysis revealed diminished bone neoformation. The alkaline phosphatase levels also decreased after MTX treatment. CONCLUSION: It was concluded that high-dose MTX impaired mandibular condyle repair and induced degenerative articular changes.
Assuntos
Consolidação da Fratura/efeitos dos fármacos , Imunossupressores/uso terapêutico , Côndilo Mandibular/efeitos dos fármacos , Fraturas Mandibulares/tratamento farmacológico , Metotrexato/uso terapêutico , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos dos fármacos , Animais , Atrofia , Remodelação Óssea/efeitos dos fármacos , Reabsorção Óssea/patologia , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/patologia , Modelos Animais de Doenças , Fibrocartilagem/efeitos dos fármacos , Fibrocartilagem/patologia , Imunossupressores/administração & dosagem , Injeções Intraperitoneais , Masculino , Côndilo Mandibular/lesões , Côndilo Mandibular/patologia , Fraturas Mandibulares/patologia , Metotrexato/administração & dosagem , Osteogênese/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Wistar , Articulação Temporomandibular/efeitos dos fármacos , Articulação Temporomandibular/lesões , Sobrevivência de Tecidos/efeitos dos fármacosRESUMO
BACKGROUND: Intermittent administration of parathyroid hormone (PTH) promotes new bone formation in patients with osteoporosis and bone fractures. It was shown previously that PTH also reduces periodontitis-related bone loss. The aim of this study is to evaluate the effect of treatment with PTH on periodontal healing in rats. METHODS: Fenestration defects were created at the buccal surface of the distal root of the mandibular first molars, and both periodontal ligament (PDL) and cementum were removed. Animals were then assigned to two groups (eight animals per group): group 1: control, placebo administration; and group 2: test, human PTH (hPTH) 1-34 administration at a concentration of 40 µg/kg. For both groups, the animals were injected every 2 days, and the animals were sacrificed at 14 and 21 days after surgery. Specimens were harvested and processed for routine decalcified histologic sections. The following parameters were assessed: 1) remaining bone defect extension (RBDE); 2) newly formed bone density (NFBD); 3) total callus area (TCA); 4) osteoclast number (ON) in the callus region; and 5) newly formed dental cementum-like tissue (NFC). Birefringence of root PDL reattachment was also evaluated. RESULTS: Birefringence analysis showed root PDL reattachment for both groups 21 days after treatment. Intermittent hPTH 1-34 administration decreased RBDE (P <0.01) and increased NFBD (P <0.01), TCA (P <0.01), area of NFC (P <0.01), and ON in the callus region (P <0.01). CONCLUSION: Within the limits of the present study, intermittent administration of hPTH 1-34 led to an enhanced periodontal healing process compared with non-treated animals.
Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Hormônio Paratireóideo/uso terapêutico , Fosfatase Ácida/análise , Administração Metronômica , Animais , Densidade Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Contagem de Células , Cementogênese/efeitos dos fármacos , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/patologia , Injeções Subcutâneas , Isoenzimas/análise , Masculino , Doenças Mandibulares/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/administração & dosagem , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/patologia , Placebos , Ratos , Ratos Wistar , Fosfatase Ácida Resistente a Tartarato , Fatores de Tempo , Raiz Dentária/efeitos dos fármacos , Raiz Dentária/patologia , Cicatrização/efeitos dos fármacosRESUMO
INTRODUCTION: Osteoporotic fractures are common during osteoporotic states. Piper sarmentosum extract is known to possess antioxidant and anti-inflammatory properties. OBJECTIVES: To observe the radiological changes in fracture calluses following administration of a Piper sarmentosum extract during an estrogen-deficient state. METHODS: A total of 24 female Sprague-Dawley rats (200-250 g) were randomly divided into 4 groups: (i) the sham-operated group; (ii) the ovariectomized-control group; (iii) the ovariectomized + estrogen-replacement therapy (ovariectomized-control + estrogen replacement therapy) group, which was supplemented with estrogen (100 µg/kg/day); and (iv) the ovariectomized + Piper sarmentosum (ovariectomized + Piper sarmentosum) group, which was supplemented with a water-based Piper sarmentosum extract (125 mg/kg). Six weeks after an ovariectomy, the right femora were fractured at the mid-diaphysis, and a K-wire was inserted. Each group of rats received their respective treatment for 6 weeks. Following sacrifice, the right femora were subjected to radiological assessment. RESULTS: The mean axial callus volume was significantly higher in the ovariectomized-control group (68.2 ± 11.74 mm³) than in the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups (20.4 ± 4.05, 22.4 ± 4.14 and 17.5 ± 3.68 mm³, respectively). The median callus scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups had median (range, minimum - maximum value) as 1.0 (0 - 2), 1.0 (1 - 2) and 1.0 (1 - 2), respectively, which were significantly lower than the ovariectomized-control group score of 2.0 (2 - 3). The median fracture scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups were 3.0 (3 - 4), 3.0 (2 - 3) and 3.0 (2 - 3), respectively, which were significantly higher than the ovariectomized-control group score of 2.0 (1 - 2) (p<0.05). CONCLUSION: The Piper sarmentosum extract improved fracture healing, as assessed by the reduced callus volumes and reduced callus scores. This extract is beneficial for fractures in osteoporotic states.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Consolidação da Fratura/efeitos dos fármacos , Fraturas por Osteoporose/tratamento farmacológico , Piper/química , Extratos Vegetais/uso terapêutico , Animais , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Estrogênios/deficiência , Feminino , Consolidação da Fratura/fisiologia , Fraturas por Osteoporose/diagnóstico por imagem , Ovariectomia , Radiografia , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Osteoporotic fractures are common during osteoporotic states. Piper sarmentosum extract is known to possess antioxidant and anti-inflammatory properties. OBJECTIVES: To observe the radiological changes in fracture calluses following administration of a Piper sarmentosum extract during an estrogen-deficient state. METHODS: A total of 24 female Sprague-Dawley rats (200-250 g) were randomly divided into 4 groups: (i) the sham-operated group; (ii) the ovariectomized-control group; (iii) the ovariectomized + estrogen-replacement therapy (ovariectomized-control + estrogen replacement therapy) group, which was supplemented with estrogen (100 μg/kg/day); and (iv) the ovariectomized + Piper sarmentosum (ovariectomized + Piper sarmentosum) group, which was supplemented with a water-based Piper sarmentosum extract (125 mg/kg). Six weeks after an ovariectomy, the right femora were fractured at the mid-diaphysis, and a K-wire was inserted. Each group of rats received their respective treatment for 6 weeks. Following sacrifice, the right femora were subjected to radiological assessment. RESULTS: The mean axial callus volume was significantly higher in the ovariectomized-control group (68.2 + 11.74 mm³) than in the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups (20.4 + 4.05, 22.4 + 4.14 and 17.5 + 3.68 mm³, respectively). The median callus scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups had median (range, minimum - maximum value) as 1.0 (0 - 2), 1.0 (1 - 2) and 1.0 (1 - 2), respectively, which were significantly lower than the ovariectomized-control group score of 2.0 (2 - 3). The median fracture scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups were 3.0 (3 - 4), 3.0 (2 - 3) and 3.0 (2 - 3), respectively, which were significantly higher than the ovariectomized-control group score of 2.0 (1 - 2) (p<0.05). CONCLUSION: The Piper sarmentosum extract improved fracture healing, as assessed by the reduced callus volumes and reduced callus scores. This extract is beneficial for fractures in osteoporotic states.
Assuntos
Animais , Feminino , Ratos , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Consolidação da Fratura/efeitos dos fármacos , Fraturas por Osteoporose/tratamento farmacológico , Piper/química , Extratos Vegetais/uso terapêutico , Calo Ósseo/efeitos dos fármacos , Calo Ósseo , Estrogênios/deficiência , Consolidação da Fratura/fisiologia , Ovariectomia , Fraturas por Osteoporose , Distribuição Aleatória , Ratos Sprague-DawleyRESUMO
BACKGROUND: Bisphosphonates have become the treatment of choice for a variety of bone diseases in which excessive osteoclastic activity is an important pathologic feature. However, inhibition of osteoclastic activity could lead to inhibition of remodeling during bone healing or repair. The objective of this study is to investigate the effect of zoledronate (the most potent bisphosphonate) in the biological process of bone healing. METHODS: Thirty immature male rabbits were divided into two groups (control and experimental) of 15 animals each. Both groups were submitted to fibular osteotomy. Only in the experimental group a single dose of zoledronate was administered. After 1, 2, and 4 weeks, animals of both groups were euthanized and the osteotomy site was histomorphometrically evaluated. The associated parameters analyzed were tissue volume (TV), fractional trabecular bone volume (BV/TV), fractional woven bone volume (WoV/TV), fractional periosteal fibrous volume (FbV/TV), and medullary fibrous volume (MaV/TV). RESULTS: The first week of healing was characterized by small callus area (experimental group) and less periosteal fibrosis. The second week was characterized by a large quantity of woven bone and marked decrease in periosteal fibrosis in the two groups. In the control group there was also a significant increase in trabecular bone. The fourth week was characterized by increased amount of woven bone and trabecular bone in the experimental group; there was increased medullary fibrosis in the two groups, while there continued to be significantly less periosteal fibrosis in the experimental group. CONCLUSIONS: Zoledronate does not prevent bone healing. However, the effect of zoledronate was characterized by accentuated stimulation of primary bone production and probably inhibition of remodeling, leading to retention of trabecular bone.