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1.
Eur J Histochem ; 64(1)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31988531

RESUMO

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent sinonasal mucosa inflammatory disease with still unclear pathophysiologic mechanisms that imply events of tissue repair and structural remodelling. Several cascades seem to have a considerable role in the onset and progression of mucosa hyperproliferation in nasal polyps including transforming growth factor ß/Small mother against decapentaplegic (TGFß/Smads), mitogenactivated protein kinases (MAPKs), advanced glycosylation end-products (AGEs) together with epithelial-tomesenchymal transition (EMT). Since many inflammatory mediators are reported to play important roles in the development of nasal polyps (NP) disease, this study aimed to analyse the correlation between the AGEs/receptor of advanced glycosylation end-products (RAGE)/extracellular signal-regulated kinase (ERK) signalling pathway and the main markers of EMT to better understand the influence that they exert on the remodelling of nasal mucous membranes in patients affected by CRSwNP vs normal controls. A total of 30 patients were enrolled in this study. Immunohistochemical analysis, using AGE, RAGE, p-ERK, MMP-3, TGF-ß1, Smad2/3, Collagen I-III, α-SMA, E-cadherin, IL-6 and Vimentin antibodies, was performed. AGE, RAGE, ERK, p-ERK and MMP3 were also evaluated using western blot analysis. We observed an overexpression of the AGE/RAGE/p-ERK and the main mesenchymal markers of EMT (Vimentin and IL-6) in CRSwNP vs controls whereas the TGF-ß/Smad3 pathway did not show any significant differences between the two groups of patients. These observations suggest a complex network of processes in the pathogenesis of NP, and the AGE/RAGE/ERK pathway and EMT might work together in promoting tissue remodelling in the formation of CRSwNP.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Pólipos Nasais/etiologia , Sinusite/etiologia , Adulto , Doença Crônica , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Masculino , Mucosa Nasal/patologia , Pólipos Nasais/patologia , Pólipos Nasais/fisiopatologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Sinusite/patologia , Sinusite/fisiopatologia , Vimentina/metabolismo
2.
J Agric Food Chem ; 68(1): 193-205, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31826610

RESUMO

Gynostemma pentaphyllum possesses neuroprotective bioactivity. However, the effect of gypenosides on hypoxia-induced neural damage remains obscure. In this study, Gyp, the active fraction extracted from G. pentaphyllum and its bioactive compounds as well as the underlying molecular mechanisms were investigated. Eighteen dammarane-type saponins were isolated from Gyp. The absolute configurations of six unreported compounds (13-18) were assessed via electron capture detection (ECD) analyses. The results of cell viability assay showed that Gyp and its bioactive compounds (13-16 and 18) effectively protected PC12 cells from hypoxia injury. Gyp pretreatment also improved mice spatial memory impairment caused by hypoxia exposure. At the molecular level, Gyp and its bioactive compounds could activate the signaling pathways of ERK, Akt, and CREB in vitro and in vivo. In summary, Gyp and its bioactive compounds could prevent hypoxia-induced injury via ERK, Akt, and CREB signaling pathways.


Assuntos
Proteína de Ligação a CREB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gynostemma/química , Hipóxia/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteína de Ligação a CREB/genética , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Células PC12 , Extratos Vegetais/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Saponinas/administração & dosagem
3.
Fitoterapia ; 140: 104416, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704261

RESUMO

Vitiligo is a common depigmentary disease characterized as diagnosis simplicity and cure difficulty in view of the ambiguity of etiology, thus novel and effective treatments are urgently needed. Paeoniflorin, the major active compound extracted from the root of Paeonia lactiflora Pall, a traditional Chinese medicine, has been validated pharmacological properties such as antioxidant stress, a theory participating in the occurrence of vitiligo, but the effect on melanogenesis is still unclear. In this study, melanosythesis effect of paeoniflorin and the potential mechanism were evaluated. We found that treatment with paeoniflorin at the concentration of 10 µg/ml significantly increased melanin content and intracellular tyrosinase activity of human melanocytes, in accordance with the elevation of protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TRP-1). In addition, we also investigated that paeoniflorin promoted phosphorylation of cAMP-response element binding (CREB) and extracellular signal-regulated kinase (ERK) without affecting p38 and c-Jun N-terminal kinase (JNK). These results demonstrated that paeoniflorin had a synergistic effect on normal human melanocytes via ERK/CREB pathway with up-regulation of MITF and TRP-1, enhancing melanin synthesis. Meanwhile, the milder pathological changes in vitiligo mice treat with paeoniflorin also confirmed its potential in treating vitiligo. To sum up, we suggest that paeoniflorin may be a potential medicine of vitiligo treatment in clinical.


Assuntos
Glucosídeos/farmacologia , Melanócitos/efeitos dos fármacos , Monoterpenos/farmacologia , Vitiligo/tratamento farmacológico , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição Associado à Microftalmia/metabolismo , Oxirredutases/metabolismo , Fosforilação , Distribuição Aleatória
4.
Toxicol Lett ; 321: 103-113, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31706003

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no effective medication. Andrographolide (Andro), extracted from Chinese herbal Andrographis paniculata, could attenuate bleomycin (BLM)-induced pulmonary fibrosis via inhibition of inflammation and oxidative stress, however, the anti-fibrotic mechanisms have not been clarified. Myofibroblasts are the primary cell types responsible for the accumulation of extracellular matrix (ECM) in fibrotic diseases, and targeting fibroblast proliferation and differentiation is an important therapeutic strategy for the treatment of IPF. Hence, this study aimed to investigate the effects of Andro on the fibroblast proliferation and differentiation in the in vivo and in vitro models. The results showed that Andro improved pulmonary function and inhibited BLM-induced fibroblast proliferation and differentiation and ECM deposition in the lungs. In vitro, Andro inhibited proliferation and induced apoptosis of TGF-ß1-stimulated NIH 3T3 fibroblasts and primary lung fibroblasts (PLFs). Andro also inhibited TGF-ß1-induced myofibroblast differentiation and ECM deposition in both cells. We also found that Andro suppressed TGF-ß1-induced Smad2/3 and Erk1/2 activation, suggesting that Smad2/3 and Erk1/2 inactivation mediates Andro-induced effects on TGF-ß1-induced fibroblast proliferation and differentiation. These results indicated that Andro has novel and potent anti-fibrotic effects in lung fibroblasts via inhibition of the proliferation and myofibroblast differentiation of fibroblasts and subsequent ECM deposition, which are modulated by TGF-ß1-mediated Smad-dependent and -independent pathways.


Assuntos
Bleomicina , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diterpenos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose Pulmonar Idiopática/prevenção & controle , Pulmão/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células NIH 3T3 , Ratos Sprague-Dawley , Transdução de Sinais
5.
J Biochem Mol Toxicol ; 33(12): e22409, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617652

RESUMO

Melanoma is the most aggressive type of cutaneous tumor and the occurrence of metastasis makes it resistant to almost all available treatment and becomes incorrigible. Hence, identifying metastasis-related biomarkers and effective therapeutic targets will assist in preventing metastasis and ameliorating cutaneous melanoma. In our present study, we reported kinesin family member 18B (KIF18B) as a novel contributor in cutaneous melanoma proliferation and metastasis, and it was found to be of great significance in predicting the prognosis of cutaneous melanoma patients. Bioinformatics analysis based on ONCOMINE, The Cancer Genome Atlas, and Genotype-Tissue Expression database revealed that KIF18B was highly expressed in cutaneous melanoma and remarkably correlated with unfavorable clinical outcomes. Consistently, the results of the quantitative real-time polymerase chain reaction exhibited that the expression of KIF18B was significantly higher in cutaneous melanoma cell lines than that in normal cells. In vitro, biological assays found that knockdown of KIF18B in cutaneous melanoma cells noticeably repressed cell proliferation, migration, and invasion, while inducing cell apoptosis. Moreover, the protein expression of E-cadherin was enhanced while the expression of N-cadherin, vimentin, and Snail was decreased in M14 cells after knocking down KIF18B. In addition, the phosphorylation of phosphoinositide 3-kinase (PI3K) and extracellular-signal-regulated kinase (ERK) was significantly suppressed in M14 cells with silenced KIF18B. Above all, our results indicated that the repression of cutaneous melanoma cell migration and proliferation caused by KIF18B depletion suggested an oncogenic role of KIF18B in cutaneous melanoma, which acts through modulating epithelial-mesenchymal transition and ERK/PI3K pathway.


Assuntos
Proliferação de Células , Cinesina/metabolismo , Melanoma/enzimologia , Melanoma/secundário , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/secundário , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Cinesina/genética , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Prognóstico , Fatores de Transcrição da Família Snail/metabolismo , Vimentina/metabolismo
6.
Nat Commun ; 10(1): 4804, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641113

RESUMO

Immunotherapy involving checkpoint blockades of inhibitory co-receptors is effective in combating cancer. Despite this, the full range of mediators that inhibit T-cell activation and influence anti-tumor immunity is unclear. Here, we identify the GTPase-activating protein (GAP) Rasal1 as a novel TCR-ZAP-70 binding protein that negatively regulates T-cell activation and tumor immunity. Rasal1 inhibits via two pathways, the binding and inhibition of the kinase domain of ZAP-70, and GAP inhibition of the p21ras-ERK pathway. It is expressed in activated CD4 + and CD8 + T-cells, and inhibits CD4 + T-cell responses to antigenic peptides presented by dendritic cells as well as CD4 + T-cell responses to peptide antigens in vivo. Furthermore, siRNA reduction of Rasal1 expression in T-cells shrinks B16 melanoma and EL-4 lymphoma tumors, concurrent with an increase in CD8 + tumor-infiltrating T-cells expressing granzyme B and interferon γ-1. Our findings identify ZAP-70-associated Rasal1 as a new negative regulator of T-cell activation and tumor immunity.


Assuntos
Proteínas Ativadoras de GTPase/imunologia , Proteínas Ativadoras de GTPase/metabolismo , Melanoma Experimental/imunologia , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Ativação Linfocitária , Masculino , Melanoma Experimental/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Proteína-Tirosina Quinase ZAP-70/genética
7.
Acta Odontol Latinoam ; 32(2): 103-110, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31664301

RESUMO

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inflamação , Plasticidade Neuronal , Núcleo Inferior Caudal do Nervo Trigêmeo/fisiopatologia , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Núcleos do Trigêmeo
8.
Neurochem Res ; 44(11): 2643-2657, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31606837

RESUMO

Schwann cells (SCs) play an important role in producing myelin for rapid neurotransmission in the peripheral nervous system. Activation of the differentiation and myelination processes in SCs requires the expression of a series of transcriptional factors including Sox10, Oct6/Pou3f1, and Egr2/Krox20. However, functional interactions among several transcription factors are poorly defined and the important components of the regulatory network are still unknown. Until now, available evidence suggests that SCs require cAMP signaling to initiate the myelination program. Heat shock protein 90 (Hsp90) is known as a chaperone required to stabilize ErbB2 receptor. In recent years, it was reported that cAMP transactivated the ErbB2/ErbB3 signaling in SCs. However, the relationship between Hsp90 and cAMP-induced differentiation in SCs is undefined. Here we investigated the role of Hsp90 during cAMP-induced differentiation of SCs using Hsp90 inhibitor, geldanamycin and Hsp90 siRNA transfection. Our results showed that dibutyryl-cAMP (db-cAMP) treatment upregulated Hsp90 expression and led to nuclear translocation of Gab1/ERK, the downstream signaling pathway of the ErbB2 signaling mechanism in myelination. The expression of myelin-related genes and nuclear translocation of Gab1/ERK following db-cAMP treatment was inhibited by geldanamycin pretreatment and Hsp90 knockdown. These findings suggest that Hsp90 might play a role in cAMP-induced differentiation via stabilization of ErbB2 and nuclear translocation of Gab1/ERK in SCs.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Células de Schwann/fisiologia , Animais , Benzoquinonas/farmacologia , Bucladesina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Lactamas Macrocíclicas/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos Sprague-Dawley , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Células de Schwann/citologia , Regulação para Cima
9.
Med Sci Monit ; 25: 6836-6845, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31509521

RESUMO

BACKGROUND Ginkgo biloba extract (EGb761), a standard extract of the Chinese traditional medicine Ginkgo biloba, plays an anti-tumor role in various cancers. However, whether EGb761 is involved in the invasion and metastasis of gastric cancer remains unclear. MATERIAL AND METHODS In the current study, cell viability assay, Western blotting, wound-healing assay, Transwell invasion assay, and orthotopic transplantation model were performed to explore the effects of EGb761 on gastric cancer. RESULTS In vitro, the results showed that EGb761 suppressed the proliferation of gastric cancer cells in a dose-dependent manner. Furthermore, the migration and invasiveness were weakened and the protein levels of p-ERK1/2, NF-kappaB P65, NF-kappaB p-P65, and MMP2 were decreased by EGb761 or U0126 (an inhibitor of ERK signaling pathway) exposure in gastric cancer cells. Moreover, the combined treatment with EGb761 and U0126 significantly inhibited ERK, NF-kappaB signaling pathway, and the expression of MMP2 than those of single drug. In vivo, EGb761 inhibited the tumor growth and hepatic metastasis of gastric cancer in the mouse model. Results of immunohistochemistry indicated that the expression of ERK1/2, NF-kappaB P65 and MMP2 were decreased by EGb761 in the tumor tissues. CONCLUSIONS EGb761 plays a vital role in the suppression of metastasis and ERK/NF-kappaB signaling pathway in gastric cancer.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , NF-kappa B/metabolismo , Extratos Vegetais/uso terapêutico , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Animais , Butadienos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Nitrilos/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
10.
Chem Biol Interact ; 313: 108826, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31545954

RESUMO

BACKGROUND: Despite of the most effective surgical removal of malignant tumors, metastasis makes cancer treatment difficult. The studies on natural compounds to inhibit this metastasis have been actively performed until now. However, the effect of tomatidine on metastasis remains unclear. METHOD: The effect of tomatidine on antioxidative activity was measured with DPPH radical assay and reducing power assay. After treatment with tomatidine, the viability of human fibrosarcoma cells (HT1080 cells) was evaluated with MTT assay. The effect of tomatidine on the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-9, gelatinases related to metastasis, was analyzed using gelatin zymography, western blot and immunofluorescence staining. Cell invasion assay was used to investigate anti-metastasis activity of tomatidine. RESULT: Tomatidine showed no DPPH radical scavenging effect and showed 8% of reduction power at 8 µM. Furthermore, tomatidine below 8 µM showed more than 80% of cell viability in MTT assay. The inhibition of tomatidine on MMP-2 activity and its protein expression levels were observed by gelatin zymography, western blot and immunofluorescence. It was observed that tomatidine inhibited not only p38 and ERK but also cell invasion. CONCLUSION: Above results suggest that tomatidine could use as a potential candidate for cancer prevention and metastasis through the inhibitory effect on gelatinase.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Tomatina/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Depuradores de Radicais Livres/farmacologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Tomatina/farmacologia , Fator de Transcrição AP-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
11.
Invest Ophthalmol Vis Sci ; 60(12): 3854-3862, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31529118

RESUMO

Purpose: Subconjunctival injection of antagomir-21 attenuates the progression of corneal neovascularization. We examined the underlying mechanism by investigating the regulation of microRNA (miR)-21 expression and the involvement of miR-21 in the homeostasis of corneal epithelial cells. Methods: Corneal epithelial cells were cultured with TGF-ß1 and/or under hypoxia conditions. miR-21 expression was measured by quantitative PCR. The direct targets of miR-21 were validated by the 3'-UTR luciferase reporter assay. Alterations of proangiogenic signaling and the epithelial-mesenchymal transition (EMT) phenotype after miR-21/Sprouty2 (SPRY2) knockdown were examined by Western blotting. The effect of conditioned medium on angiogenesis was assessed using the tube formation assay. Wound healing was evaluated by the migration and scratch assays. Results: TGF-ß1 or hypoxia upregulated miR-21, and miR-21 silencing abolished TGF-ß1/hypoxia-induced hypoxia inducible factor (HIF)-1α and VEGF expression. miR-21 inhibited SPRY2 by directly targeting its 3'-UTR. Simultaneous silencing of miR-21 and SPRY2 significantly upregulated p-ERK, HIF-1α, and VEGF and promoted angiogenesis. Induction of miR-21 or inhibition of SPRY2 reduced the levels of cytokeratin (CK)-3 and CK-12 and promoted EMT. Transwell and wound healing assays indicated that miR-21 promoted cell migration. Conclusions: TGF-ß1 or hypoxia induced miR-21 and inhibited SPRY2, thereby enhancing proangiogenic signaling, suppressing the epithelial phenotype, and promoting wound healing in corneal epithelial cells.


Assuntos
Epitélio Anterior/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/fisiologia , MicroRNAs/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Animais , Western Blotting , Movimento Celular/fisiologia , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Epitélio Anterior/efeitos dos fármacos , Hipóxia/metabolismo , Queratina-12/metabolismo , Queratina-3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Fator de Crescimento Transformador beta1/farmacologia
12.
Phytother Res ; 33(11): 2948-2959, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31478281

RESUMO

The balance between the osteoblasts and the osteoclasts is important for the maintenance of the skeleton of the human body. The osteoclasts absorb bone after differentiated into polymorphonuclear cells by the fusion of monocytes/macrophages. We have found that 6,7,4'-Trihydroxyflavone (THF), a compound from the heartwood of Dalbergia Odorifera inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, actin ring formation, and bone resorption in RAW 264.7 cells and bone marrow macrophage. THF significantly inhibited the c-Jun-N-terminal kinase signaling pathway without affecting extracellular signal-regulated kinase, p38, and AKT signaling. Moreover, THF inhibited the expression of c-Fos, nuclear factor-activated T cells cytoplasm 1, cathepsin K, and c-src by RANKL. We used a lipopolysaccharide (LPS)-induced bone loss model in mice. Consequently, bone volume per tissue volume, trabecular number's reduction was recovered in THF-treated mice, and trabecular separation's augmentation was also attenuated by THF administration. In summary, THF inhibits RANKL-induced osteoclast differentiation by MAPK signaling pathway and inhibits bone resorption by destroying the actin ring in mature osteoclasts. THF also prevented LPS-induced bone loss in a mice model. Thus, THF may be useful in the treatment of bone diseases associated with excessive osteoclast differentiation and bone resorption.


Assuntos
Reabsorção Óssea/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Isoflavonas/farmacologia , Osteoclastos/efeitos dos fármacos , Animais , Células Cultivadas , Dalbergia/química , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
13.
Biol Pharm Bull ; 42(9): 1510-1516, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474711

RESUMO

The ability of dermal fibroblasts to synthesize collagen decreases with ages. The integrity of collagen fibers severely decreases in aged skin, causing its characteristic morphological changes such as wrinkles and sagging. To prevent and improve skin aging, the stimulation of collagen synthesis in dermal fibroblasts is important. Potato peels contain many biofunctional compounds, but not much is known about their effects on human skin physiology. To characterize the potential effects of a potato peel extract (PPE) against skin aging, we examined its effects on the synthesis of type I collagen by normal human dermal fibroblasts (NHDFs). Treatment with the PPE significantly increased the expression of type I collagen mRNA in NHDFs and their secretion of type I collagen. To elucidate the mechanism involved, we examined the signaling pathway controlled by transforming growth factor-ß (TGF-ß), which regulates the synthesis of type I collagen. Treatment of NHDFs with the PPE significantly increased the expression of TGF-ß receptor mRNA. TGF-ß signaling involves Smad-dependent and Smad-independent pathways, like phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). The PPE did not activate Smad, but significantly activated Akt and ERK. These results demonstrate that the PPE activates PI3K/Akt and MAPK/ERK signals via TGF-ß receptors, which stimulate the synthesis of type I collagen in NHDFs. These results suggest that the PPE could be a novel and effective antiaging material.


Assuntos
Colágeno Tipo I/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pele/citologia , Solanum tuberosum/química , Técnicas de Cultura de Células , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Extratos Vegetais/isolamento & purificação , Raízes de Plantas/química , Transdução de Sinais , Pele/metabolismo , Envelhecimento da Pele/efeitos dos fármacos
14.
Tissue Cell ; 59: 51-61, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383289

RESUMO

We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.


Assuntos
Neoplasias Ósseas/metabolismo , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Osteossarcoma/metabolismo , Receptores Notch/metabolismo , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Camundongos , Osteossarcoma/patologia , Fosforilação
15.
J Exp Clin Cancer Res ; 38(1): 335, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370872

RESUMO

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilcolantreno/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/química , Receptores Estrogênicos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
16.
Int J Nanomedicine ; 14: 5215-5228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371957

RESUMO

Background: Resveratrol (RSV) has attracted interest as an alternative drug for the treatment of acute lung injury (ALI) and other pulmonary diseases, but its poor oral bioavailability is a limitation. In this study, we employed drug delivery nanotechnology to improve the stability, lung localization and efficacy of orally administered resveratrol to control lung damage leading to ALI. Methods and materials: RSV-loaded lipid-core nanocapsules (RSV-LNCs), prepared by interfacial deposition of biodegradable polymers, were given orally to A/J mice prior to lipopolysaccharide (LPS) intranasal instillation. Inflammatory changes, oxidative stress and lung tissue elastance were assessed 24 h after LPS challenge. Results: RSV-LNCs (5 mg/kg), given 1, 4, 6 or 12 h but not 24 h before provocation, inhibited LPS-induced leukocyte accumulation in the bronchoalveolar fluid (BALF), whereas unloaded nanocapsules (ULNCs) or free RSV (5 mg/kg) were ineffective. RSV-LNCs (2.5-10 mg/kg) but not ULNCs or RSV improved lung function and prevented total leukocyte and neutrophil accumulation equally in both BALF and lung tissue when given 4 h before LPS challenge. Similar findings were seen concerning the generation of a range of pro-inflammatory cytokines such as IL-6, KC, MIP-1α, MIP-2, MCP-1 and RANTES in lung tissue. In addition, only RSV-LNCs inhibited MDA levels and SOD activity in parallel with blockade of the ERK and PI3K/Akt pathways following LPS provocation. Conclusion: Nanoformulation of RSV in biodegradable oil-core polymers is an effective strategy to improve the anti-ALI activity of RSV, suggesting that the modified-release formulation of this plant polyphenol may be of great value in clinical conditions associated with ALI and respiratory failure.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Nanocápsulas/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol/administração & dosagem , Resveratrol/uso terapêutico , Transdução de Sinais , Lesão Pulmonar Aguda/complicações , Administração Oral , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Masculino , Camundongos Endogâmicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Hipersensibilidade Respiratória/complicações , Hipersensibilidade Respiratória/patologia , Resveratrol/farmacologia
17.
Nucleic Acids Res ; 47(16): 8606-8619, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372646

RESUMO

Epithelial-mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate 'indirect' miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon-Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-ß or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.


Assuntos
Transição Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Transcrição Genética , Linhagem Celular , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Éxons , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Íntrons , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Transfecção , Fator de Crescimento Transformador beta/farmacologia
18.
Artif Cells Nanomed Biotechnol ; 47(1): 3500-3510, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31432697

RESUMO

Salidroside is an active ingredient extracted from Rhodiola rosea that has anti-tumor activities. The current paper attempted to assess the impact of Salidroside on gastric cancer (GC) and explore the potential mechanism. GC cell lines (SNU-216 and MGC803) and gastric epithelial cell line GES-1 were treated with Salidroside. CCK-8 assay, colony formation assay, flow cytometry and Transwell assay were respectively performed to evaluate GC cells phenotype. qRT-PCR and western blot were conducted to reveal the downstream genes and signaling of Salidroside. We found that 800 µM Salidroside was capable of reducing GC cells viability, while has no such impacts on GES-1 cells. Salidroside inhibited GC cells proliferation, migration, invasion and promoted apoptosis, which coupled with the down-regulation of p21, Bcl-2, MMP2, RhoA, p-ROCK1, Vimentin and the up-regulations of CyclinD1, Bax, cleaved caspases. miR-99a was found to be highly expressed in response to Salidroside treatment. Besides, the inhibition of MAPK/ERK and PI3K/AKT signaling induced by Salidroside was attenuated by miR-99a silence and in this process, IGF1R worked as a target of miR-99a. The anti-GC effect of Salidroside was also confirmed in a mouse model of GC. The promoting effect of Salidroside on miR-99a expression was also verified in vivo. Furthermore, Salidroside promoted the cisplatin-sensitivity of SGC7901/DDP cells. In conclusion, this study demonstrated that Salidroside possessed anti-GC effects through regulating miR-99a/IGF1R axis and inhibiting MAPK/ERK and PI3K/AKT pathways.


Assuntos
Antineoplásicos/farmacologia , Glucosídeos/farmacologia , MicroRNAs/genética , Fenóis/farmacologia , Neoplasias Gástricas/patologia , Regulação para Cima/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Vasc Med ; 24(5): 395-404, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451089

RESUMO

Peripheral artery disease (PAD) is caused by atherosclerotic occlusions of vessels outside the heart, particularly those of the lower extremities. Angiogenesis is one critical physiological response to vessel occlusion in PAD, but our understanding of the molecular mechanisms involved in angiogenesis is incomplete. Dual specificity phosphatase 5 (DUSP5) has been shown to play a key role in embryonic vascular development, but its role in post-ischemic angiogenesis is not known. We induced hind limb ischemia in mice and found robust upregulation of Dusp5 expression in ischemic hind limbs. Moreover, in vivo knockdown of Dusp5 resulted in impaired perfusion recovery in ischemic limbs and was associated with increased limb necrosis. In vitro studies showed upregulation of DUSP5 in human endothelial cells exposed to ischemia, and knockdown of DUSP5 in these ischemic endothelial cells resulted in impaired endothelial cell proliferation and angiogenesis, but did not alter apoptosis. Finally, we show that these effects of DUSP5 on post-ischemic angiogenesis are a result of DUSP5-dependent decrease in ERK1/2 phosphorylation and p21 protein expression. Thus, we have identified a role of DUSP5 in post-ischemic angiogenesis and implicated a DUSP5-ERK-p21 pathway that may serve as a therapeutic target for the modulation of post-ischemic angiogenesis in PAD.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/enzimologia , Neovascularização Fisiológica , Doença Arterial Periférica/enzimologia , Animais , Linhagem Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Doença Arterial Periférica/genética , Doença Arterial Periférica/fisiopatologia , Fosforilação , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Transdução de Sinais
20.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438541

RESUMO

Oxidative stress has been implicated in the pathogenesis of many diseases including chronic liver diseases. Nrf2 is a master transcriptional factor regulating the induction of cellular antioxidant defense systems. Here, the Nrf2-activating effect of the crude methanol extract of dried leaves of Pogostemon cablin Bentham was demonstrated by measuring the antioxidant response element (ARE)-driven luciferase activity and pachypodol, 4',5-dihydroxy-3,3',7-trimethoxyflavone, was isolated by bioactivity-guided fractionation and further separation using chromatographic techniques. To our knowledge, this is the first study to evaluate the antioxidant and cytoprotective effects of pachypodol in HepG2 cells as well as the underlying molecular mechanisms. Indeed, pachypodol protected HepG2 cells from cell death caused by tert-butylhydroperoxide-induced oxidative stress and also attenuated ROS production. The ability of pachypodol to activate Nrf2/ARE pathway was further confirmed by observing Nrf2 expression in nuclear fraction, mRNA levels of Nrf2 target antioxidants, and cellular glutathione content in HepG2 cells. Extracellular signal-regulated kinase (ERK) is one of the important kinases involved in Nrf2 activation. Pachypodol increased ERK phosphorylation and ERK inhibition by PD98059 totally abrogated the increase in ARE luciferase activity, nuclear Nrf2 accumulation and mRNA levels of antioxidant enzymes by pachypodol. In conclusion, pachypodol isolated from P. cablin can protect hepatocytes from oxidative injury, possibly mediated by enhancing endogenous antioxidant defense system through ERK-dependent Nrf2 activation.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Pogostemon/química , Quercetina/análogos & derivados , Antioxidantes/química , Antioxidantes/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Quercetina/química , Quercetina/farmacologia
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