RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Peel of Citrus reticulata, a Chinese herbal drug with functions of regulating Qi and expelling phlegm, has been used for the treatment of lung related diseases in Chinese medicine for a long time. Its detailed effects on collagen in anti-idiopathic pulmonary fibrosis (IPF) is still unclear. AIM OF THE STUDY: To explore the effects of citrus alkaline extract (CAE) on collagen synthesis, crosslinking and deposition in pulmonary fibrosis and understand the possible signal pathways involved in the activity. MATERIALS AND METHODS: CAE was prepared from C. reticulata. Bleomycin-induced pulmonary fibrosis mouse model was applied. Pulmonary fibrosis of lung was estimated with histopathology analysis, and collagen deposition was evaluated with immunohistochemistry. Collagen crosslinking related biomarkers and enzymes were analyzed with chemical methods, immunohistochemical and western blot analyses. RESULTS: CAE oral administration lowered hydroxyproline content, inhibited the collagen deposition including expressions of collagen I and III, and relieved bleomycin-induced pulmonary fibrosis in mice model. The productions of a collagen crosslink pyridinoline and crosslinking related enzymes including lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1) in lung were suppressed by CAE treatment. Furthermore, the protein expressions of TGF-ß1 and Smad3 levels in lungs were also downregulated by CAE. CONCLUSIONS: This study demonstrated that CAE inhibited collagen synthesis, crosslinking and deposition, and ameliorated bleomycin-induced pulmonary fibrosis. Preliminary mechanism study revealed that CAE exerted its bioactivity at least via downregulation of TGF-ß1/Smad3 pathway. Our findings provided a great potential in fighting IPF based on CAE.
Assuntos
Citrus/química , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Extratos Vegetais/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Administração Oral , Álcalis/química , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Animais , Bleomicina/toxicidade , Colágeno Tipo III/genética , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/metabolismo , Hidroxiprolina/metabolismo , Camundongos Endogâmicos C57BL , Extratos Vegetais/administração & dosagem , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteína-Lisina 6-Oxidase/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
Cyclovirobuxine D (CVBD) is an alkaloid, which is mainly derived from Buxus microphylla. It has been reported that CVBD has positive effects on breast cancer, gastric cancer and other malignant tumors. However, to the best of our knowledge, there are no reports regarding the effects of CVBD on colorectal cancer (CRC). The purpose of the present study was to determine the anticancer effects of CVBD and further elucidate its molecular mechanism(s). DLD1 and LoVo cell lines were selected to evaluate the antitumor effect of CVBD. Cytotoxicity, viability and proliferation were evaluated by the MTT and colony formation assays. Flow cytometry was used to detect the effects on apoptosis and the cell cycle in CVBDtreated CRC cells. The migration and invasion abilities of CRC cells were examined by wound healing and Transwell assays. In addition, RNA sequencing, bioinformatics analysis and western blotting were performed to investigate the target of drug action and clarify the molecular mechanisms. A xenograft model was established using nude mice, and ultrasound was employed to assess the preclinical therapeutic effects of CVBD in vivo. It was identified that CVBD inhibited the proliferation, migration, stemness, angiogenesis and epithelialmesenchymal transition of CRC cells, and induced apoptosis and Sphase arrest. In addition, CVBD significantly inhibited the growth of xenografts. It is notable that CVBD exerted anticancer effects in CRC cells partly by targeting collagen triple helix repeat containing 1 (CTHRC1), which may be upstream of the AKT and ERK pathways. CVBD exerted anticancer effects through the CTHRC1AKT/ERKSnail signaling pathway. Targeted therapy combining CTHRC1 with CVBD may offer a promising novel therapeutic approach for CRC treatment.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Medicamentos de Ervas Chinesas/uso terapêutico , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , RNA-Seq , Fatores de Transcrição da Família Snail/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To address whether matrix Gla protein (MGP) can inhibit mineralization in normal rat kidney tubular cells (NRK-52E) under high concentration of calcium. MATERIALS AND METHODS: NRK-52E cells were treated with high concentration of calcium. The viability and apoptosis of cells were detected by cell counting kit-8 and flow cytology, respectively. Real-time-polymerase chain, Western blotting, and immunofluorescence analysis were conducted to detect the expression of MGP. Cells were transfected with plasmid-MGP or siRNA-MGP for up- or down-regulation of the expression of MGP, respectively. Rat recombinant MGP was also used as supplementation of exogenous MGP. Alizarin red staining was conducted to detect the adherent and deposition of calcium salt. RESULTS: High concentration of calcium suppressed MGP expression in NRK-52E cells. There was significant mineralization when NRK-52E cells were treated with high concentration of calcium. Supplementation with exogenous rat recombinant MGP and overexpression of endogenous MGP both decreased the adherent and deposition of calcium salt to NRK-52E cells, while silence of MGP showed reverse results. CONCLUSION: MGP plays an inhibitory role in the stone formation. However, high concentration of calcium significantly inhibits the expression of MGP and then promotes mineralization in NRK-52E cells.
Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/biossíntese , Cálcio/metabolismo , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/biossíntese , Nefropatias/etiologia , Animais , Calcinose/etiologia , Células Cultivadas , Ratos , Proteína de Matriz GlaRESUMO
Alterations in the extracellular matrix (ECM) production and remodeling of smooth muscle cells (SMCs) have been implicated in processes related to the differentiation in atherosclerosis. Due to the anti-atherosclerotic properties of the tetracyclines, we aimed to investigate whether cholesterol supplementation changes the effect of doxycycline over the ECM proteins synthesis and whether isoprenylated proteins and Rho A protein activation are affected. SMC primary culture isolated from chicks exposed to atherogenic factors in vivo (a cholesterol-rich diet, SMC-Ch), comparing it with control cultures isolated after a standard diet (SMC-C). After treatment with 20 nM doxycycline, [H3]-proline and [H3]-mevalonate incorporation were used to measure the synthesis of collagen and isoprenylated proteins, respectively. Real-time PCR was assessed to determine col1a2, col2a1, col3a1, fibronectin, and mmp2 gene expression and the pull-down technique was applied to determine the Rho A activation state. A higher synthesis of collagens and isoprenylated proteins in SMC-Ch than in SMC-C was determined showing that doxycycline inhibits ECM production and remodeling in both SMC types of cultures. Moreover, preliminary results about the effect of doxycycline on protein isoprenylation and Rho A protein activation led us to discuss the possibility that membrane G-protein activation pathways could mediate the molecular mechanism.
Assuntos
Doxiciclina/farmacologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Animais , Células Cultivadas , Galinhas , Colesterol/farmacologia , Colágeno/biossíntese , Colágeno/genética , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Fibronectinas/biossíntese , Fibronectinas/genética , Expressão Gênica/efeitos dos fármacos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Músculo Liso Vascular/citologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.
Assuntos
Cemento Dentário/efeitos dos fármacos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Hormônio Paratireóideo/farmacologia , Vitamina D/farmacologia , Animais , Western Blotting , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Cemento Dentário/fisiologia , Regulação para Baixo/efeitos dos fármacos , Proteínas da Matriz Extracelular/fisiologia , Fator de Crescimento de Fibroblastos 23 , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Camundongos , Hormônio Paratireóideo/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Vitamina D/fisiologiaRESUMO
Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response.
Assuntos
Anti-Inflamatórios/farmacologia , Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tiamina/análogos & derivados , Animais , Ácido Araquidônico/metabolismo , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Epoprostenol/antagonistas & inibidores , Epoprostenol/biossíntese , Epoprostenol/metabolismo , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inflamação/genética , Inflamação/metabolismo , Leucotrienos/biossíntese , Leucotrienos/metabolismo , Lipopolissacarídeos/efeitos adversos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Tiamina/farmacologia , Tromboxano B2/antagonistas & inibidores , Tromboxano B2/biossíntese , Tromboxano B2/metabolismoRESUMO
UNLABELLED: Calciphylaxis is a rare, usually fatal vasculopathic disorder characterized by cutaneous ischemia and necrosis due to calcification of arterioles. Although calciphylaxis is most frequently associated with end-stage renal disease (ESRD) and secondary hyperparathyroidism, it has been reported infrequently among patients on warfarin. No standard treatment has been established for atypical calciphylaxis; however, a potentially beneficial treatment is hyperbaric oxygen therapy (HBOT). A high degree of clinical suspicion, early diagnosis, and understanding the pathophysiology of this disease promotes the optimal management of this extremely morbid and often fatal condition. CASE REPORT: We present a 63-year-old Polynesian woman with biopsy-proven calciphylaxis in the absence of ESRD or elevated serum calcium levels while taking warfarin. Therapeutic dose enoxaparin was substituted for warfarin and she received 40 sessions of HBOT during which lower extremity ulcers resolved. DISCUSSION: Warfarin has been implicated when calciphylaxis presents in an atypical fashion. No guidelines exist for treatment of atypical calciphylaxis in the setting of concomitant warfarin therapy. Up to 80% of calciphylaxis patients die within 1 year of diagnosis. Our patient was changed to low-molecular-weight heparin and received HBOT. CONCLUSION: We present what we believe is the first case of atypical calciphylaxis thought to be attributable to warfarin treated with a therapeutic substitution of anticoagulant and HBOT leading to resolution of cutaneous lesions.
Assuntos
Anticoagulantes/efeitos adversos , Calciofilaxia/induzido quimicamente , Oxigenoterapia Hiperbárica , Úlcera da Perna/etiologia , Varfarina/efeitos adversos , Antibacterianos/uso terapêutico , Anticoagulantes/uso terapêutico , Calciofilaxia/diagnóstico , Calciofilaxia/cirurgia , Calciofilaxia/terapia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Celulite (Flegmão)/diagnóstico , Celulite (Flegmão)/tratamento farmacológico , Terapia Combinada , Desbridamento , Diabetes Mellitus Tipo 2/complicações , Proteínas da Matriz Extracelular/antagonistas & inibidores , Feminino , Humanos , Úlcera da Perna/cirurgia , Úlcera da Perna/terapia , Pessoa de Meia-Idade , Obesidade Mórbida/complicações , Fatores de Risco , Trombofilia/tratamento farmacológico , Varfarina/uso terapêutico , Proteína de Matriz GlaRESUMO
Betel quid chewing is a popular oral habit in India, South Africa, and many Southeast Asian countries. The effects of areca nut (AN) extract on the growth, attachment, and protein synthesis of healthy human gingival fibroblasts (GF) were investigated to determine why betel quid (BQ) chewers have higher prevalence of periodontal disease than non-chewers. Twenty-four hour exposure of human GF to AN extract (> 200 microg/ml) in culture led to the formation of numerous intracellular vacuoles. As analyzed by modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay, AN extract significantly suppressed the growth of GF over 5 days of incubation in a dose-dependent manner. At concentrations of 50 and 300 microg/ml, AN extract suppressed the growth of GF with 30% and 57% (P < 0.05), respectively. AN extract also significantly suppressed the synthesis of [3H]proline incorporation into trichloroacetic acid (TCA) precipitated proteins. At concentrations of 200, 400, and 600 microg/ml, AN extract suppressed the protein synthesis with 33%, 58%, and 63% of inhibition (P < 0.05), respectively. Preincubation of cells in a medium containing AN extract for 2 hours inhibits the subsequent attachment of cultured GF to type I collagen at the 50% inhibitory concentration (IC50) which is about 720 to 798 microg/ml. Considering the frequent consumption of BQ throughout the day, impairment of sequential fibroblast functions by BQ ingredients is a potential mechanism through which BQ chewing exert a deleterious effect to the gingival tissues.