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The main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) represents a promising target for antiviral drugs aimed at combating COVID-19. Consequently, the development of Mpro inhibitor is an ideal strategy for combating the virus. In this study, we identified twenty-two dithiocarbamates (1a-h), dithiocarbamate-Cu(II) complexes (2a-hCu) and disulfide derivatives (2a-e, 2i) as potent inhibitors of Mpro, with IC50 value range of 0.09-0.72, 0.9-24.7 and 15.1-111 µM, respectively, through FRET screening. The enzyme kinetics, inhibition mode, jump dilution, and DTT assay revealed that 1g may be a partial reversible inhibitor, while 2d and 2f-Cu are the irreversible and dose- and time-dependent inhibitors, potentially covalently binding to the target. Binding of 2d, 2f-Cu and 1g to Mpro was found to decrease the stability of the protein. Additionally, DTT assays and thermal shift assays indicated that 2f-Cu and 2d are the nonspecific and promiscuous cysteine protease inhibitor. ICP-MS implied that the inhibitory activity of 2f-Cu may stem from the uptake of Cu(II) by the enzyme. Cytotoxicity assays demonstrated that 2d and 1g exhibit low cytotoxicity, whereas 2f-Cu show certain cytotoxicity in L929 cells. Overall, this work presents two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.
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Non-small cell lung cancer (NSCLC) is a prominent etiology of cancer-related mortality. The heterogeneous nature of this disease impedes its accurate diagnosis and efficacious treatment. Consequently, constant advancements in research are imperative in order to comprehend its intricate nature. In addition to currently available therapies, the utilization of nanotechnology presents an opportunity to enhance the clinical outcomes of NSCLC patients. Notably, the burgeoning knowledge of the interaction between the immune system and cancer itself paves the way for developing novel, emerging immunotherapies for treating NSCLC in the early stages of the disease. It is believed that with the novel engineering avenues of nanomedicine, there is a possibility to overcome the inherent limitations derived from conventional and emerging treatments, such as off-site drug cytotoxicity, drug resistance, and administration methods. Combining nanotechnology with the convergence points of current therapies could open up new avenues for meeting the unmet needs of NSCLC treatment.
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Neuropathic pain (NP) is a type of chronic pain affecting 6-8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1ß. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.
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Neuralgia , Receptores Purinérgicos P2X4 , Ratos , Humanos , Animais , Ratos Sprague-Dawley , Receptores Purinérgicos P2X4/metabolismo , Neuroglia/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Gânglios Espinais/metabolismoRESUMO
The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (Mpro) of SARS-CoV-2 is promising target for antiviral drugs, which plays a vital role for viral duplication. Development of the inhibitor against Mpro is an ideal strategy to combat COVID-19. In this work, twenty-three hydroxamates 1a-i and thiosemicarbazones 2a-n were identified by FRET screening to be the potent inhibitors of Mpro, which exhibited more than 94% (except 1c) and more than 69% inhibition, and an IC50 value in the range of 0.12-31.51 and 2.43-34.22 µM, respectively. 1a and 2b were found to be the most effective inhibitors in the hydroxamates and thiosemicarbazones, with an IC50 of 0.12 and 2.43 µM, respectively. Enzyme kinetics, jump dilution and thermal shift assays revealed that 2b is a competitive inhibitor of Mpro, while 1a is a time-dependently inhibitor; 2b reversibly but 1a irreversibly bound to the target; the binding of 2b increased but 1a decreased stability of the target, and DTT assays indicate that 1a is the promiscuous cysteine protease inhibitor. Cytotoxicity assays showed that 1a has low, but 2b has certain cytotoxicity on the mouse fibroblast cells (L929). Docking studies revealed that the benzyloxycarbonyl carbon of 1a formed thioester with Cys145, while the phenolic hydroxyl oxygen of 2b formed H-bonds with Cys145 and Asn142. This work provided two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.
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Tratamento Farmacológico da COVID-19 , Tiossemicarbazonas , Animais , Antivirais/química , Proteases 3C de Coronavírus , Humanos , Camundongos , Simulação de Acoplamento Molecular , Pandemias , Inibidores de Proteases/química , SARS-CoV-2 , Tiossemicarbazonas/farmacologiaRESUMO
Bacterial resistance caused by metallo-ß-lactamases (MßLs) has become an emerging public health threat, and the development of MßLs inhibitor is an effective way to overcome the resistance. In this study, thirteen novel O-aryloxycarbonyl hydroxamates were constructed and assayed against MßLs. The obtained molecules specifically inhibited imipenemase-1 (IMP-1) and New Delhi metallo-ß-lactamase-1, exhibiting an IC50 value in the range of 0.10-18.42 and 0.23-22.33 µM, respectively. The hydroxamate 5 was found to be the most potent inhibitor, with an IC50 of 0.1 and 0.23 µM using meropenem and cefazolin as substrates. ICP-MS analysis showed that 5 did not coordinate to the Zn(II) ions at the active site of IMP-1, while the rapid dilution, thermal shift and MALDI-TOF assays revealed that the hydroxamate formed a covalent bond with the enzyme. Cytotoxicity assays indicated that the hydroxamates have low toxicity in MCF-7 cells. This work provided a potent scaffold for the development of MßLs inhibitors.
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Ácidos Hidroxâmicos/química , Inibidores de beta-Lactamases/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Humanos , Células MCF-7 , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/farmacologiaRESUMO
Following the publication of the above article, the authors have realized that, on p. 8, some of the supplementary data were cited incorrectly in the main text. In the right-hand column, second paragraph, the sentence beginning on line 5 should have read as follows (changed text is highlighted in bold): "Meanwhile, the expression of miR513b5p in tumor tissues was decreased and the expression of PRPF39 was increased in tumor tissues with knockout of circG004213 (Fig. S3D and E)." (i.e., the reference to Fig. S3C and D was incorrect.). The authors regret their oversight in failing to correct the inaccurate citation of the data in the paper, are grateful to the Editor for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 23: Article no. 421, 2021; DOI: 10.3892/mmr.2018.12060].
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BACKGROUND: The key for conducting thymectomy by thoracoscope is the treatment of thymic veins, as they are the most common source of bleeding. The traditional treatment is to cut off the distal and proximal vessels after Ham-Lock clamping. Our team found that it is safe and reliable to use ultrasonic energy to cut off thymic veins in previous open operations. This study aimed to investigate the feasibility and safety of thymic vein sealing with ultrasonic energy in video-assisted thoracoscopic surgery (VATS) thymectomy. METHODS: A total of 169 patients with or without thymic tumors who underwent thymectomy by thoracoscope were enrolled in the study. Among them, group A, with 89 patients, underwent thoracoscopic thymic vein resection by Ham-Lock, and group B, with 80 patients, did so by ultrasonic energy. The groups were compared in terms of the characteristics of patients, tumors, and perioperative period. RESULTS: There were no significant differences in patients' characteristics between the two groups (P>0.05). There was no significant difference between the two groups in blood transfusion, operation time, drainage, and hospitalization (P>0.05). There were no deaths, secondary operation and post-operative blood transfusion, and no serious complications leading to prolonged hospitalization. One patient in group A was converted to thoracotomy due to left anonymous venous hemorrhage, and one patient in group B had thymic venous hemorrhage, but the hemorrhage was successfully stopped under thoracoscope. CONCLUSIONS: In VATS thymectomy (with or without thymic tumors), there is no significant difference in the treatment of thymic veins between Ham-Lock clipping and ultrasonic energy in conversion to open surgery in regards to thymic venous hemorrhage, postoperative drainage, blood transfusion, operation time, drainage, and hospital stay. Use of ultrasonic energy is a safe and reliable method for thymic vein disconnection.
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Following the publication of the above article, the authors have realized that the Materials and methods section and Figs. 1, 3 and 4 contained certain errors. In the Materials and methods section, the high glucose concentration was reported incorrectly as being 25 mmol/l; this should have been stated as 15 mmol/l. Furthermore, the normal concentration of glucose should have been reported as 5 mmol/l rather than 5.5 mmol, and the glucose concentration gradient should have been written as 0, 5, 10, and 15 mmol/l, and not as 0, 5, 10, and 25 mmol/l. Secondly, the authors have corrected some miscalculations associated with the bar charts featured in Figs. 1, 3, and 4, and the revised versions of these figures are shown here. All these corrections were approved by all authors. The authors regret that these errors were included in the paper, even though they did not substantially alter any of the major conclusions reported in the study, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 17: 85368541, 2018; DOI: 10.3892/mmr.2018.8914].
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Over the past few decades, it has been demonstrated that hyperglycemia can promote lung carcinoma growth, potentially through significantly increased glucose metabolism; however, the underlying mechanism remains to be fully elucidated. In the present study, treatment with a high concentration of glucose (HG) significantly promoted the proliferation and migration of A549 cells. Receptor for advanced glycation endproducts (RAGE) has previously been demonstrated to be associated with diabetes mellitus and oxidative stress, and nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are considered to be initiating factors of oxidative stress. Therefore, an MTT assay, woundhealing assay, quantitative polymerase chain reaction and western blotting assays were used to analyze the RAGENOX4 pathway and to determine its potential involvement in glycometabolismassociated tumorigenesis. The present study demonstrated that HG could increase the protein expression of RAGE and NOX4, whereas the inhibitor of RAGE (antiRAGE antibody) could suppress this effect. Futhermore, the inhibitor of NOX [diphenyl iodonium chloride (DPI)] could reduce the protein expression of RAGE and NOX4. Furthermore, inhibition of RAGE led to the downregulation of vascular endothelial growth factor (VEGF) and hypoxiainducible factor1α (HIF1α), thus suggesting that HG may influence angiogenesis and tumor metabolism via the RAGENOXs pathway. The present study also demonstrated that the RAGEblocking antibody downregulated NOX4 and subsequently reduced the production of downstream inflammatory factors, whereas DPI did not affect the mRNA expression of RAGE but it did reduce the protein level of RAGE and then attenuate the inflammatory response. These results indicated that inhibition of RAGE or NOXs may promote the reduced expression of VEGF and HIF1α, and NOXs may be downstream targets of RAGE, thus indicating a HGRAGENOXsVEGF/HIF1α association. Furthermore, the results indicated that HG may serve a role in the development of lung adenocarcinoma, mediated by the RAGEoxidative stress pathway; therefore, the regulation of this glucoseassociated pathway may be a promising novel direction for oncotherapy. However, while certain antidiabetic agents have been verified to exert inhibitory effects on tumor growth, they can also have longterm adverse effects on the body, which may limit the value of these drugs as anticancer treatments. In conclusion, the present study suggested a novel attempt to suppress glucoseinduced tumor growth using a RAGE inhibitor such as soluble RAGE while avoiding the risk of glucose fluctuation.
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Adenocarcinoma/metabolismo , Glucose/metabolismo , Neoplasias Pulmonares/metabolismo , NADPH Oxidases/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , NADPH Oxidases/antagonistas & inibidores , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The major mite allergenic components of protease allergens (group 1,3) and non-protease allergens (group 2,7) derived from Dermatophagoides peronyssinus (Dp) and D. farinae (Df) are reported to be capable of sensitizing 80-90% of mite-allergic patients. Although protease and non-protease allergens have been demonstrated to trigger innate and adaptive immune responses through epithelium activation, the simultaneous or sequential effects of both groups of allergens has not been reported. Since all allergens are present in the mite crude extracts, it is important to determine whether these allergens can synergistically trigger the immune responses to cause airway inflammation. A total of 60 house dust mite (HDM)-allergic asthmatic patients were recruited to analyze their serum-specific IgE response to both groups of allergens. Recombinant protease allergen (Der p1 and Der p3) and non-protease allergens (Der p2 and Der p7) were used to activate the human airway epithelium cell (Beas-2B). The cells were analyzed for mRNA expression of IL-6/IL-8 and the culture supernatants were analyzed for neutrophil chemotactic activity (NCA). The results showed 48/60 (80%) HDM-allergic patients were sensitized to all allergenic components of Der p1, Der p2, Der f1, and Der f2. Most of the allergic patients were sensitized to both groups of allergens simultaneously. The associations of Der p1 with Der p2 were 83.3% (50/60) and Der f1 with Der f2 were 80% (48/60). When Beas-2B cells were cultured with Der p2 in conjunction with Der p1 and Der p3, the results showed that there was increased expression of IL-6/IL-8 in comparison with culture with allergen alone. There was only a trivial effect on IL-6/IL-8 expression when Der p2 was co-cultured with Der p7. Similar findings were obtained in the NCA measurement. When Beas-2B was cultured with Der p2 in conjunction with Der p1 and Der p3, there was increased NCA in comparison with culture with allergen alone. There were also trivial effects when Der p2 was co-cultured with Der p7. The allergens (Der p2 and Der p3)-induced IL-6/IL-8 expression and NCA released from Beas-2B could be downregulated by dexamethasone and transcription factor inhibitor SP600125. The allergenic components derived from Dp and Df can sensitize allergic patients simultaneously and activate epithelium through protease allergens (group 1, 3) and non-protease allergen (group 2) synergistically.
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Hipersensibilidade/imunologia , Neutrófilos/imunologia , Mucosa Respiratória/imunologia , Animais , Antracenos/farmacologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Células Cultivadas , Cisteína Endopeptidases/imunologia , Dermatophagoides farinae/imunologia , Dermatophagoides pteronyssinus/imunologia , Dexametasona/farmacologia , Regulação da Expressão Gênica , Humanos , Doenças do Sistema Imunitário , Imunidade Inata , Imunoglobulina E/sangue , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Transtornos Leucocíticos , Peptídeo Hidrolases/imunologia , Mucosa Respiratória/patologia , Serina Endopeptidases/imunologiaRESUMO
PURPOSE: House-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation. METHODS: Internalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Internalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion. CONCLUSIONS: Human airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.
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Platelet-derived growth factor (PDGF) is released from vascular smooth muscle cells (VSMCs), endothelial cells, or macrophages after percutaneous coronary intervention and is related with neointimal proliferation and restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting growth and endogenous PDGF synthesis in VSMCs after in vitro mechanical injury. We analyzed the effects of berberine on VSMC growth, migration, and signaling events after exogenous PDGF stimulation in vitro in order to mimic a post-angioplasty PDGF shedding condition. Pretreatment of VSMCs with berberine inhibited PDGF-induced proliferation. Berberine significantly suppressed PDGF-stimulated Cyclin D1/D3 and Cyclin-dependent kinase (Cdk) gene expression. Moreover, berberine increased the activity of AMP-activated protein kinase (AMPK), which led to phosphorylation activation of p53 and increased protein levels of the Cdk inhibitor p21(Cip1). Compound C, an AMPK inhibitor, partly but significantly attenuated berberine-elicited growth inhibition. In addition, stimulation of VSMCs with PDGF led to a transient increase in GTP-bound, active form of Ras, Cdc42 and Rac1, as well as VSMC migration. However, pretreatment with berberine significantly inhibited PDGF-induced Ras, Cdc42 and Rac1 activation and cell migration. Co-treatment with farnesyl pyrophosphate and geranylgeranyl pyrophosphate drastically reversed berberine-mediated anti-proliferative and migratory effects in VSMCs. Based on these findings, we conclude that berberine inhibited PDGF-induced VSMC growth via activation of AMPK/p53/p21(Cip1) signaling while inactivating Ras/Rac1/Cyclin D/Cdks and suppressing PDGF-stimulated migration via inhibition of Rac1 and Cdc42. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine.
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Berberina/farmacologia , Complexos Multienzimáticos/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Quinases Ativadas por AMP , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/análise , Ciclina D3 , Ciclinas/análise , MAP Quinase Quinase 1/metabolismo , Músculo Liso Vascular/citologia , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Our previous study demonstrated that norepinephrine (NE) induces endothelial apoptosis mainly through down-regulation of Bcl-2 protein and activation of the beta-adrenergic and caspase-2 pathways. However, whether reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) are involved in this signal transduction remains unknown. Endothelial cells cultured from neonatal rat heart were treated with 100 microM NE. Proteins of MAPKs and Bcl-2 family were assayed by Western blotting. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated nick end-labeling assay. ROS was analyzed with flow cytometry. Caspase activity was measured using specific fluorogenic substrates. Treatment with NE increased intracellular ROS level and extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 phosphorylation. Whereas the phosphorylated form of Akt was decreased. The NE-induced apoptosis was abrogated by SP600125 (a specific inhibitor of JNK). Antioxidants such as vitamin C and N-acetyl cysteine inhibited NE-induced ROS production, JNK phosphorylation, caspase activation and apoptosis. Exogenously added superoxide dismutase or catalase markedly diminished NE-induced ROS production and cell death. In conclusions, our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells via a ROS-dependent JNK activation pathway. Antioxidants may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.
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Apoptose/efeitos dos fármacos , Células Endoteliais/citologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Norepinefrina/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Animais Recém-Nascidos , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Regulação para Baixo , Células Endoteliais/metabolismo , Ativação Enzimática , Peróxido de Hidrogênio/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Miocárdio/citologia , NADPH Oxidases/metabolismo , Norepinefrina/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos/metabolismo , Transdução de SinaisRESUMO
Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and post-angioplasty restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting SMC contraction and proliferation, yet the exact mechanism is unknown. We therefore investigated the effect of berberine on SMC growth after mechanic injury in vitro. DNA synthesis and cell proliferation assay were performed to show that berberine inhibited serum-stimulated rat aortic SMC growth in a concentration-dependent manner. Mechanical injury with sterile pipette tip stimulated the regrowth of SMCs. Treatment with berberine prevented the regrowth and migration of SMCs into the denuded trauma zone. Western blot analysis showed that activation of the MEK1/2 (mitogen-activated protein kinase kinase 1/2), extracellular signal-regulated kinase (ERK), and up-regulation of early growth response gene (Egr-1), c-Fos and Cyclin D1 were observed sequentially after mechanic injury in vitro. Semi-quantitative reverse-transcription PCR assay further confirmed the increase of Egr-1, c-Fos, platelet-derived growth factor (PDGF) and Cyclin D1 expression in a transcriptional level. However, berberine significantly attenuated MEK/ERK activation and downstream target (Egr-1, c-Fos, Cyclin D1 and PDGF-A) expression after mechanic injury in vitro. Our study showed that berberine blocked injury-induced SMC regrowth by inactivation of ERK/Egr-1 signaling pathway thereby preventing early signaling induced by injury in vitro. The anti-proliferative properties of berberine may be useful in treating disorders due to inappropriate SMC growth.
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Berberina/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Aorta Torácica , DNA/biossíntese , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas , Proteína 1 de Resposta de Crescimento Precoce/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Norepinephrine (NE)-induced apoptosis in cardiomyocytes is an important cause of heart failure. Previous studies revealed that reactive oxygen species (ROS) are involved in apoptosis. Tumor necrosis factor-alpha (TNF), a well-known mediator that stimulates apoptosis, is not only produced by macrophages but also by cardiomyocytes. Until now, the role of TNF and its relationship to ROS in NE-induced apoptosis of cardiomyocytes have never been investigated. METHODS: Neonatal rat cardiomyocytes were treated with various concentrations of NE. Apoptosis of cardiomyocytes was determined using the TUNEL assay. The level of secreted TNF was measured by ELISA and TNF mRNA expression was determined by semiquantitative reverse transcriptional polymerase chain reaction. Caspase activity was measured by a fluorogenic protease assay kit. Anti-TNF antibodies, caspase inhibitors and antioxidants (N-acetyl-L-cysteine or vitamin C) were added to determine if they could inhibit the apoptotic effect of NE. RESULTS: NE induced apoptosis of cardiomyocytes in a dose- and time-dependent manner. NE up-regulated TNF mRNA expression and increased TNF secretion and caspase-2,-3,-6, and -9 activities. A neutralizing anti-TNF antibody and caspase-2 and -3 inhibitors significantly attenuated NE-induced apoptosis. Antioxidants completely abrogated NE-induced TNF secretion, caspase activation, and apoptotic death. CONCLUSION: NE induced apoptosis in neonatal rat cardiomyocytes through a ROS-TNF-caspase signaling pathway.
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Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Norepinefrina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Antioxidantes/farmacologia , Apoptose , Caspases/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: Heart failure is associated with increased plasma norepinephrine (NE) and endothelial apoptosis. Recent reports have suggested that endothelial dysfunction is an important target for future therapies of heart failure. However, whether NE can induce endothelial apoptosis and its mechanism remains unknown. METHODS: Endothelial cells from neonatal rat heart were treated with various concentrations of NE for different durations. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and DNA fragmentation assays. Caspase activity was measured using specific fluorogenic substrates. Proteins of Bcl-2 family and cytochrome c were assayed by Western blotting. RESULTS: NE induced endothelial apoptosis in a dose- and time-dependent manner. After treatment for 48 h, increasing NE concentration from 5, 10, 50, 100 to 200 microM resulted in 6+/-3%, 14+/-5%, 43+/-4%, 66+/-5%, and 89+/-6% apoptotic cells, respectively. The apoptosis was accompanied by down-regulation of Bcl-2 protein synthesis but not by cytosolic cytochrome c translocation. Caspase-2, -3, -6 and -9 were activated during apoptosis and caspase-2 inhibitor (Z-VDVAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) significantly attenuated the apoptosis. Overexpression of Bcl-2 inhibited caspase activity and decreased the apoptosis. Moreover, the NE-mediated apoptotic effect was attenuated by beta- (beta2>beta>beta1) adrenergic antagonists (ICI-118,551>propranolol>atenolol) but was not affected by alpha1- or alpha2-adrenergic antagonists (prazosin or yohimbine). CONCLUSION: Our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells mainly through down-regulation of Bcl-2 protein and activation of the beta-adrenergic (beta2>beta1) and caspase-2 pathways. beta-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.
Assuntos
Caspases/metabolismo , Células Endoteliais/metabolismo , Genes bcl-2 , Miocárdio/metabolismo , Norepinefrina/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Atenolol/farmacologia , Western Blotting/métodos , Caspase 2 , Caspase 3 , Caspase 6 , Caspase 9 , Inibidores de Caspase , Células Cultivadas , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/patologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/farmacologia , Propanolaminas , Propranolol/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 microg/ml) enhanced the expression of Cdc25C and p21(Cip1/Waf1), increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27(Kip1). Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X(L) but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21(Cip1/Waf1) and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21(Cip1/Waf1), phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis.