RESUMO
Emphysema is one of the major components of chronic obstructive pulmonary disease (COPD), which is characterised by the destruction and enlargement of air spaces, leading to airflow limitation and dyspnoea, finally progressing to oxygen dependency. The alveolar wall destruction is due to chronic inflammation, oxidative stress, apoptosis, and proteinase/anti-proteinase imbalance. So far, there has been no effective therapy for patients with COPD. We evaluated the therapeutic efficacy of tannic acid (TA), a naturally occurring plant-derived polyphenol in the murine emphysema model. In C57BL/6 J mice, we established emphysema by intratracheal instillation of elastase (EL). Then, mice were treated with TA and evaluated 1 and 21 days post-EL instillation. After 24 h, TA treatment significantly reduced EL-induced histopathological alterations, infiltrating leukocytes, and gene expression of markers of inflammation and apoptosis. Similarly, after 21 days, TA treatment suppressed the mean linear intercept, gene expression of proteinases, and increased elastic fiber contents in the lungs when compared to the EL-alone group. Furthermore, EL induced the activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NF-kB) p65 pathways in the lungs was suppressed by TA treatment. In summary, TA has the potential to mitigate EL-induced inflammation, apoptosis, proteinase/anti-proteinase imbalance, and subsequent emphysema in mice.
Assuntos
Enfisema , Pneumonia , Polifenóis , Doença Pulmonar Obstrutiva Crônica , Humanos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática , Pneumonia/induzido quimicamente , Pneumonia/tratamento farmacológico , Inflamação/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Peptídeo HidrolasesRESUMO
During ejaculation, cauda epididymal spermatozoa are suspended in a protein-rich solution of seminal plasma, which is composed of proteins mostly secreted from the seminal vesicle. These seminal proteins interact with the sperm cells and bring about changes in their physiology, so that they can become capacitated in order for the fertilization to take place. Sulfhydryl oxidase (SOX) is a member of the QSOX family and its expression is found to be high in the seminal vesicle secretion (SVS) of mouse. Previously, it has been reported to cross-link thiol-containing amino acids among major SVS proteins. However, its role in male reproduction is unclear. In this study, we determined the role of SOX on epididymal sperm maturation and also disclosed the binding effect of SOX on the sperm fertilizing ability in vitro. In order to achieve the above two objectives, we constructed a Sox clone (1.7 kb) using a pET-30a vector. His-tagged recombinant Sox was overexpressed in Shuffle Escherichia coli cells and purified using His-Trap column affinity chromatography along with hydrophobic interaction chromatography. The purified SOX was confirmed by western blot analysis and by its activity with DTT as a substrate. Results obtained from immunocytochemical staining clearly indicated that SOX possesses a binding site on the sperm acrosome. The influence of SOX on oxidation of sperm sulfhydryl to disulfides during epididymal sperm maturation was evaluated by a thiol-labeling agent, mBBr. The SOX protein binds onto the sperm cells and increases their progressive motility. The effect of SOX binding on reducing the [Ca2+]i concentration in the sperm head was determined using a calcium probe, Fluo-3 AM. The inhibitory influence of SOX on the sperm acrosome reaction was shown by using calcium ionophore A32187 to induce the acrosome reaction. The acrosome-reacted sperm were examined by staining with FITC-conjugated Arachis hypogaea (peanut) lectin. Furthermore, immunocytochemical analysis revealed that SOX remains bound to the sperm cells in the uterus but disappears in the oviduct during their transit in the female reproductive tract. The results from the above experiment revealed that SOX binding onto the sperm acrosome prevents sperm capacitation by affecting the [Ca2+]i concentration in the sperm head and the ionophore-induced acrosome reaction. Thus, the binding of SOX onto the sperm acrosome may possibly serve as a decapacitation factor in the uterus to prevent premature capacitation and acrosome reaction, thus preserving their fertilizing ability.
Assuntos
Oxirredutases , Capacitação Espermática , Espermatozoides , Reação Acrossômica/fisiologia , Animais , Cálcio/metabolismo , Feminino , Masculino , Camundongos , Oxirredutases/metabolismo , Sêmen/metabolismo , Glândulas Seminais/enzimologia , Espermatozoides/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Serine protease inhibitor Kazal type 3 (SPINK3) from mouse seminal vesicles is a Kazal-type trypsin inhibitor. It has been shown to bind to the sperm acrosome and modify sperm activity by influencing the sub-cellular Ca2+ influx. Previously, SPINK3 was reported to suppress in vitro sperm capacitation. However, under natural coitus, SPINK3 is removed from the mouse acrosome in the female reproductive tract, leading to successful fertilisation. Identification of the SPINK3 binding partner becomes essential to develop a contraceptive that works by prolonging the binding of SPINK3 to the sperm acrosome. We identified the SPINK3 receptor by using recombinant SPINK3 (rSPINK3). Testicular serine protease 1 (TESP1) was identified as the receptor for SPINK3 by 2D gel electrophoresis coupled with western blot analysis. To authenticate TESP1 as the receptor for SPINK3, sperm cells were incubated with TESP1 peptide antibody followed by determining the intracellular [Ca2+]i concentration by flow cytometry using Fluo-3 AM as a calcium probe. Furthermore, the 3D structures of SPINK3 and TESP1 were predicted by homology modelling (Schrodinger suite) using the crystal structure of pancreatic secretory trypsin inhibitor (PDB ID-1TGS) and human prostasin (PDB ID-3DFJ) as templates. The modelled protein structures were validated and subjected to molecular dynamics simulation (MDS) using GROMACS v5.0.5. Protein-protein docking was performed using HDOCK and the complex was validated by MDS. The results predicted that SPINK3 and TESP1 had strong binding affinity, with a dock score of -430.70 and 14 hydrogen bonds as key active site residues. If the binding affinity between SPINK3 and TESP1 could be increased, the SPINK3-TESP1 association will be prolonged, which will be helpful in the development of a male contraceptive.
Assuntos
Reação Acrossômica , Acrossomo/enzimologia , Glicoproteínas/metabolismo , Proteínas Secretadas pela Próstata/metabolismo , Serina Endopeptidases/metabolismo , Inibidor da Tripsina Pancreática de Kazal/metabolismo , Animais , Cálcio/metabolismo , Glicoproteínas/genética , Ligação de Hidrogênio , Masculino , Camundongos , Simulação de Acoplamento Molecular , Proteínas Secretadas pela Próstata/genética , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Inibidor da Tripsina Pancreática de Kazal/genéticaRESUMO
Asthma is a chronic inflammatory disease of the airways, which is characterized by infiltration of inflammatory cells, airway hyperresponsiveness (AHR), and airway remodeling. This study aimed to explore the role and mechanism of tannic acid (TA), a naturally occurring plant-derived polyphenol, in murine asthma model. BALB/c mice were given ovalbumin (OVA) to establish an allergic asthma model. The results revealed that TA treatment significantly decreased OVA-induced AHR, inflammatory cells infiltration, and the expression of various inflammatory mediators (Th2 and Th1 cytokines, eotaxin, and total IgE). Additionally, TA treatment also attenuated increases in mucins (Muc5ac and Muc5b) expression, mucus production in airway goblet cells, mast cells infiltration, and airway remodeling induced by OVA exposure. Furthermore, OVA-induced NF-κB (nuclear factor- kappa B) activation and cell adhesion molecules expression in the lungs was suppressed by TA treatment. In conclusion, TA effectively attenuated AHR, inflammatory response, and airway remodeling in OVA-challenged asthmatic mice. Therefore, TA may be a potential therapeutic option against allergic asthma in clinical settings.
Assuntos
Asma/tratamento farmacológico , Hipersensibilidade/tratamento farmacológico , Taninos/uso terapêutico , Remodelação das Vias Aéreas , Alérgenos/imunologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Mucinas/metabolismo , Nozes/imunologia , Hipersensibilidade Respiratória , Células Th1 , Células Th2RESUMO
Pulmonary fibrosis (PF) is a chronic and irreversible scarring disease in the lung with limited treatment options. Therefore, it is critical to identify new therapeutic options. This study was undertaken to identify the effects of tannic acid (TA), a naturally occurring dietary polyphenol, in a mouse model of PF. Bleomycin (BLM) was intratracheally administered to induce PF. Administration of TA significantly reduced BLM-induced histological alterations, inflammatory cell infiltration and the levels of various inflammatory mediators (nitric oxide, leukotriene B4 and cytokines). Additionally, treatment with TA also impaired BLM-mediated increases in pro-fibrotic (transforming growth factor-ß1) and fibrotic markers (alpha-smooth muscle actin, vimentin, collagen 1 alpha and fibronectin) expression. Further investigation indicated that BLM-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinases 1 and 2) in lungs was suppressed by TA treatment. Findings of this study suggest that TA has the potential to mitigate PF through inhibiting the inflammatory response and fibrotic process in lungs and that TA might be useful for the treatment of PF in clinical practice.
Assuntos
Anti-Inflamatórios/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Taninos/farmacologia , Animais , Bleomicina , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/patologiaRESUMO
Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high-molecular-weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)-dependent Sox was purified from mouse SVS using ion exchange and high-performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The Km and Vmax of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross-linking activity among SVS I-III monomers to form HMWCs in SVS.