RESUMO
Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.
Assuntos
Antibacterianos , Bacteriófagos , Antibacterianos/farmacologia , Staphylococcus aureus/metabolismo , Lisostafina , N-Acetil-Muramil-L-Alanina Amidase , Bacteriófagos/metabolismoRESUMO
Methyl p-coumarate (methyl p-hydroxycinnamate) (MH) is a natural compound found in a variety of plants. In the present study, we evaluated the ameliorative effects of MH on airway inflammation in an experimental model of allergic asthma (AA). In this in vitro study, MH was found to exert anti-inflammatory activity on PMA-stimulated A549 airway epithelial cells by suppressing the secretion of IL-6, IL-8, MCP-1, and ICAM-1. In addition, MH exerted an inhibitory effect not only on NF-κB (p-NF-κB and p-IκB) and AP-1 (p-c-Fos and p-c-Jun) activation but also on A549 cell and EOL-1 cell (eosinophil cell lines) adhesion. In LPS-stimulated RAW264.7 macrophages, MH had an inhibitory effect on TNF-α, IL-1ß, IL-6, and MCP-1. The results from in vivo study revealed that the increases in eosinophils/Th2 cytokines/MCP-1 in the bronchoalveolar lavage fluid (BALF) and IgE in the serum of OVA-induced mice with AA were effectively inhibited by MH administration. MH also exerted a reductive effect on the immune cell influx, mucus secretion, and iNOS/COX-2 expression in the lungs of mice with AA. The effects of MH were accompanied by the inactivation of NF-κB. Collectively, the findings of the present study indicated that MH attenuates airway inflammation in mice with AA, suggesting its potential as an adjuvant in asthma therapy.
Assuntos
Asma , NF-kappa B , Animais , Camundongos , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6 , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , OvalbuminaRESUMO
Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.
Assuntos
Anticorpos Neutralizantes/farmacologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Complicações do Diabetes/tratamento farmacológico , Disfunção Erétil/tratamento farmacológico , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/antagonistas & inibidores , Regeneração Nervosa/fisiologia , Ereção Peniana/fisiologia , Análise de Variância , Angiopoietina-1/metabolismo , Animais , Western Blotting , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/imunologia , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Regeneração Nervosa/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ereção Peniana/efeitos dos fármacos , Receptor TIE-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: We examined whether and how Sac-1004, a vascular leakage blocker, would restore erectile function in an animal model of diabetic erectile dysfunction. MATERIALS AND METHODS: Eight-week-old C57BL/6J mice were used. Diabetes was induced by intraperitoneal injection of streptozotocin. Eight weeks after diabetes induction the animals were divided into 6 groups, including controls, diabetic mice that received repeat intracavernous injections of phosphate buffered saline (20 µl) on days -3 and 0, and diabetic mice that received repeat intracavernous injections of Sac-1004 on days -3 and 0 (1, 2, 5 and 10 µg, respectively, in 20 µl phosphate buffered saline). One week after injection erectile function was measured by cavernous nerve stimulation. The penis was then harvested for histological examinations and Western blot analysis. RESULTS: Local delivery of Sac-1004 in the corpus cavernosum restored erectile function in diabetic mice. The highest erectile response was noted at a dose of 5 µg with a response comparable to that in the control group. Sac-1004 significantly increased cavernous endothelial and smooth muscle contents, and induced endothelial nitric oxide synthase phosphorylation (Ser1177). Sac-1004 decreased extravasation of oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins and pericyte content. Sac-1004 also promoted tube formation in primary cultured mouse cavernous endothelial cells in vitro. Sac-1004 mediated cavernous angiogenesis and erectile function recovery was abolished by inhibiting angiopoietin-1-Tie2 signaling with soluble Tie2 antibody. CONCLUSIONS: With the effects of angiogenesis and antipermeability Sac-1004 reestablishes structural and functional cavernous sinusoids. This is highly promising for future treatment of erectile dysfunction from vascular causes.
Assuntos
Indutores da Angiogênese/uso terapêutico , Diabetes Mellitus Experimental/complicações , Disfunção Erétil/tratamento farmacológico , Saponinas/uso terapêutico , Indutores da Angiogênese/farmacologia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Disfunção Erétil/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Saponinas/farmacologia , Estreptozocina , Resultado do TratamentoRESUMO
INTRODUCTION: Diabetic erectile dysfunction is a disease mostly of vascular origin and men with diabetic erectile dysfunction respond poorly to oral phosphodiesterase-5 inhibitors. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and angiogenesis. AIM: To determine the effectiveness of recombinant human (rh)-HGF in restoring erectile function in diabetic mice. METHODS: Four groups of mice were used: control non-diabetic mice and streptozotocin-induced diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days -3 and 0), a single intracavernous injection of rh-HGF (day 0), or two successive intracavernous injections of rh-HGF (days -3 and 0). We also examined the effect of rh-HGF in primary cultured mouse cavernous endothelial cells and in major pelvic ganglion culture in vitro, which was incubated under a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition. MAIN OUTCOME MEASURES: Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve and the penis was harvested for histologic studies. RESULTS: Repeated intracavernous injections of rh-HGF protein induced significant restoration of erectile function in diabetic mice (89-100% of control values), whereas a single intracavernous injection of rh-HGF protein elicited modest improvement. Rh-HGF significantly induced phosphorylation of its receptor c-Met, increased the content of endothelial cells and smooth muscle cells, and decreased the generation of reactive oxygen species (superoxide anion and peroxynitrite) and extravasation of oxidized low-density lipoprotein in diabetic mice. Under the high-glucose condition, rh-HGF protein also promoted tube formation in mouse cavernous endothelial cells and enhanced neurite sprouting in major pelvic ganglion culture in vitro. CONCLUSION: The dual angiogenic and neurotrophic effects of HGF could open a new avenue through which diabetic erectile dysfunction can be treated.
Assuntos
Disfunção Erétil/tratamento farmacológico , Fator de Crescimento de Hepatócito/farmacologia , Ereção Peniana/efeitos dos fármacos , Animais , Proliferação de Células/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Endotélio/metabolismo , Disfunção Erétil/fisiopatologia , Humanos , Injeções Intralesionais , Lipoproteínas LDL/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/irrigação sanguínea , Inibidores da Fosfodiesterase 5/farmacologia , Fosforilação/fisiologia , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Regeneração/fisiologiaRESUMO
OBJECTIVES: To examine the therapeutic effect of adenovirus encoding histone deacetylase 2 (HDAC2) small hairpin RNA (Ad-HDAC2 shRNA) in a rat model of Peyronie's disease (PD) and to determine the mechanisms by which HDAC2 knockdown ameliorates fibrotic responses in primary fibroblasts derived from human PD plaque. MATERIALS AND METHODS: Rats were distributed into four groups (n = 6 per group): age-matched controls without treatment; rats in which PD has been induced (PD rats) without treatment; PD rats receiving a single injection of control adenovirus encoding scrambled small hairpin RNA (Ad-shRNA) (day 15; 1 × 10(8) pfu/0.1 mL phosphate-buffered saline [PBS]); and PD rats receiving a single injection of Ad-HDAC2 shRNA (day 15; 1 × 10(8) pfu/0.1 mL PBS) into the lesion. PD-like plaque was induced by repeated intratunical injections of 100 µL each of human fibrin and thrombin solutions on days 0 and 5. On day 30, the penis was harvested for histological examination. Fibroblasts isolated from human PD plaque were pretreated with HDAC2 small interfering (si)RNA (100 pmoL) and then stimulated with transforming growth factor (TGF)-ß1 (10 ng/mL) to determine hydroxyproline levels, procollagen mRNA, apoptosis and protein expression of poly(ADP-ribose) polymerase 1 (PARP1) and cyclin D1. RESULTS: We observed that Ad-HDAC2 shRNA decreased inflammatory cell infiltration, reduced transnuclear expression of phospho-Smad3 and regressed fibrotic plaque of the tunica albuginea in PD rats in vivo. siRNA-mediated silencing of HDAC2 significantly decreased the TGF-ß1-induced transdifferentiation of fibroblasts into myofibroblasts and collagen production, and induced apoptosis by downregulating the expression of PARP1, and decreased the expression of cyclin D1 (a positive cell-cycle regulator) in primary cultured fibroblasts derived from human PD plaque in vitro. CONCLUSION: Specific inhibition of HDAC2 with RNA interference may represent a novel targeted therapy for PD.
Assuntos
Histona Desacetilase 2/genética , Induração Peniana/genética , Induração Peniana/fisiopatologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Animais , Apoptose/genética , Células Cultivadas , Modelos Animais de Doenças , Histona Desacetilase 2/metabolismo , Humanos , Hidroxiprolina/metabolismo , Inflamação , Masculino , Induração Peniana/terapia , Ratos , Proteína Smad3/metabolismo , Transfecção/métodosRESUMO
INTRODUCTION: Men with erectile dysfunction (ED) respond poorly to oral phosphodiesterase-5 inhibitors following radical prostatectomy. Recent studies have reported that up-regulation of transforming growth factor-ß1 (TGF-ß1) and activation of the Smad signaling pathway play important roles in cavernous fibrosis and in the deterioration of erectile function in a mouse model of cavernous nerve injury (CNI) and in patients with spinal cord injury. The mothers against decapentaplegic homolog 7 (Smad7) is known to inhibit the phosphorylation of Smad2 and Smad3. AIM: To investigate the effectiveness of adenoviruses encoding Smad7 gene (Ad-Smad7) on erectile function in a mouse model of CNI. METHODS: Twelve-week-old C57BL/6J mice were used and distributed into 7 groups: sham operation group, untreated CNI group, and CNI groups receiving a single intracavernous injection of adenovirus encoding LacZ (1 × 10(8) virus particles [vp]/20 µL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 10(7), 1 × 10(8), 2 × 10(8), or 1 × 10(9) vp/20 µL). MAIN OUTCOME MEASURES: Two weeks after bilateral cavernous nerve crushing and treatment, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS: The highest erectile response was noted in CNI mice treated with Ad-Smad7 at a dose of 1 × 10(8) vp, which reached up to 82-85% of sham control values. Local delivery of Ad-Smad7 significantly decreased endothelial cell apoptosis and the production of extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV, and induced endothelial nitric oxide synthase phosphorylation in the corpus cavernosum tissue of CNI mice. CONCLUSION: The adenovirus-mediated gene transfer of Smad7 successfully restored erectile function by enhancing endothelial cell function and through antifibrotic effects. These findings suggest that inhibition of the TGF-ß signaling pathway by use of Smad7 may represent a promising therapeutic strategy for ED induced by radical prostatectomy.
Assuntos
Disfunção Erétil/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Traumatismos dos Nervos Periféricos/terapia , Proteína Smad7/genética , Adenoviridae , Animais , Apoptose/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Disfunção Erétil/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compressão Nervosa , Óxido Nítrico Sintase Tipo III/metabolismo , Ereção Peniana , Pênis/inervação , Pênis/patologia , Pênis/cirurgia , Traumatismos dos Nervos Periféricos/complicações , Fosforilação , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Regulação para CimaRESUMO
INTRODUCTION: Erectile dysfunction (ED) is a major complication of radical prostatectomy. Men with radical prostatectomy-induced ED respond less positively to oral phosphodiesterase-5 inhibitors. AIM: The study aims to examine whether and how stromal vascular fraction (SVF) restores erectile function in mice with cavernous nerve injury (CNI). METHODS: Twelve-week-old male C57BL/6J mice were used and the animals were distributed into five groups: sham operation group and CNI group receiving a single intracavernous injection of phosphate-buffered saline (PBS) or SVF (1 × 10(4) , 1 × 10(5) , or 3 × 10(5) cells/20 µL, respectively). SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. MAIN OUTCOME MEASURES: Two weeks after injection, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to platelet/endothelial cell adhesion molecule-1, phosphohistone H3, and phosphorylated endothelial nitric oxide synthase (phospho-eNOS). We also performed Western blot for angiopoietin-1 (Ang-1), vascular endothelial growth factor-A, hepatocyte growth factor, phospho-eNOS, and eNOS in the corpus cavernosum tissue. RESULTS: Local delivery of SVF restored erectile function in a dose-dependent manner in CNI mice. The highest erectile response was noted at a dose of 3 × 10(5) cells, for which the response was comparable with that in the sham operation group. Local delivery of SVF significantly increased the expression of angiogenic factor proteins and induced cavernous endothelial cell proliferation and eNOS phosphorylation compared with that in the PBS-treated CNI group. SVF-induced promotion of cavernous angiogenesis and erectile function was diminished in the presence of soluble antibody to Tie2, a receptor tyrosine kinase of Ang-1. CONCLUSION: Secretion of angiogenic factors from SVF is an important mechanism by which SVF induces cavernous endothelial regeneration and restores erectile function. These findings suggest that cavernous endothelial regeneration by using SVF may represent a promising treatment strategy for radical prostatectomy-induced ED.
Assuntos
Disfunção Erétil/terapia , Células Estromais/transplante , Traumatismos do Sistema Nervoso/fisiopatologia , Tecido Adiposo/citologia , Indutores da Angiogênese/metabolismo , Angiopoietina-1/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Disfunção Erétil/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/irrigação sanguínea , Pênis/inervação , Fosforilação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regeneração , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Erectile dysfunction is often a harbinger of cardiovascular disease. We sought to gain mechanistic insight at the cellular and molecular levels into why erectile dysfunction precedes the clinical consequences of cardiovascular disease. MATERIALS AND METHODS: Diabetes was induced by intraperitoneal streptozotocin injection in 8-week-old C57BL/6J mice. At 8 weeks after diabetes induction, we determined the expression of endothelial cell-cell junction proteins and vascular endothelial permeability in the penis, heart and hind limb by systemic injection of various vascular space markers (350 Da to 2,000 kDa) or by immunohistochemical staining with antibody to oxidized low density lipoprotein. We also investigated the effect of recombinant Ang1 protein on cavernous endothelial permeability. RESULTS: Alterations in the integrity of the endothelial cell-cell junction, including a decrease in endothelial cell-cell junction proteins and an increase in vascular permeability to fluorescent tracers or oxidized low density lipoprotein, were prominent in the cavernous tissue of diabetic mice. In contrast, no significant changes in endothelial cell-cell junction proteins or vascular permeability were noted in heart or hind limb tissue according to the diabetic condition. Intracavernous injection of Ang1 protein, an anti-permeability factor, significantly decreased cavernous endothelial permeability to oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins in diabetic mice. CONCLUSIONS: The incompetent cavernous endothelial cell-cell junction in the diabetic condition provides an important clue to why erectile dysfunction is highly prevalent and often precedes other systemic vascular diseases.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/fisiopatologia , Disfunção Erétil/fisiopatologia , Junções Intercelulares/fisiologia , Análise de Variância , Angiopoietina-1/farmacologia , Animais , Western Blotting , Circulação Coronária , Membro Posterior/irrigação sanguínea , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pênis/irrigação sanguínea , Estatísticas não ParamétricasRESUMO
INTRODUCTION: Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI) but also result in structural changes in the cavernous tissues. Nerve injury-induced protein 1, Ninjurin-1 (Ninj1), is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. AIM: The study aims to determine whether and how Ninj1 neutralizing antibody (Ninj1-Ab) restores erectile function in mice with CNI. METHODS: Twelve-week-old C57BL/6J mice were used and distributed into four groups: sham operation group and CNI groups receiving a single intracavernous injection of immunoglobulin G (IgG) control antibody, low-dose Ninj1-Ab (1.0 µg/20 µL), or high-dose Ninj1-Ab (2.5 µg/20 µL). MAIN OUTCOME MEASURES: One week after bilateral cavernous nerve crush, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS: The cavernous expression of Ninj1 protein was upregulated up to 7 days after CNI and returned to baseline levels thereafter. Local delivery of Ninj1-Ab significantly increased penile neuronal nitric oxide synthase and neurofilament contents, induced cavernous endothelial proliferation and phosphorylation of Akt and endothelial nitric oxide synthase, and decreased endothelial cell apoptosis in the CNI mice by upregulating angiopoietin-1 and downregulating angiopoietin-2. High-dose Ninj1-Ab induced profound restoration of erectile function in the CNI mice (91% of sham control values), whereas low-dose Ninj1-Ab elicited partial improvement. CONCLUSION: The dual neurotrophic and angiogenic effects of Ninj1 blockade may provide a good opportunity for treating erectile dysfunction resulting from radical prostatectomy.
Assuntos
Anticorpos Neutralizantes/farmacologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Disfunção Erétil/tratamento farmacológico , Fatores de Crescimento Neural/antagonistas & inibidores , Ereção Peniana/efeitos dos fármacos , Pênis/efeitos dos fármacos , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Anticorpos Neutralizantes/administração & dosagem , Moléculas de Adesão Celular Neuronais/imunologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Estimulação Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Disfunção Erétil/metabolismo , Fibrose , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Compressão Nervosa , Fatores de Crescimento Neural/imunologia , Regeneração Nervosa/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/inervação , Pênis/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Hyposalivation is a common complaint among the elderly, but no established treatment prevents age-induced hyposalivation. Platelet derivatives such as platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and plasma rich in growth factor (PRGF), are used widely in different areas of regenerative medicine to enhance the wound healing processes. This study examined whether the local injection of the supernatant of activated PRP (saPRP) into the salivary gland (SG) could help prevent aging-induced SG dysfunction and explored the mechanisms responsible for the protective effects on the SG hypofunction. The platelets were separated from the blood of male SD rats (220 ± 20 g). saPRP was manufactured by removing the fibrin clot after activating platelet with calcium ionophore 10 µM (A23187). The total protein and TGF-ß1 levels were significantly higher in saPRP than in PRP. Human salivary gland epithelial cell(hSGEC) was treated with saPRP or PRP after senescence through irradiation. The significant proliferation of hSGEC was observed in saPRP treated group compared to irradiation only group and irradiation + PRP group. Cellular senescence, apoptosis, and inflammation significantly reduced in saPRP group. The SG function and structural tissue remodeling by the saPRP were investigated with naturally aged mice. The mice were divided into three groups: 3 months old (3 M), 22 months old (22 M), and 22 months old treated with saPRP (22 M + saPRP). Salivary flow rate and lag time were significantly improved in 22 M + saPRP group compared to 22 M group. The histologic examinations showed the significant proliferation of acinar cell in 22 M + saPRP group. The decrease of senescence, apoptosis, and inflammation observed by western blot in 22 M + saPRP group. The saPRP induced the proliferation of hSGECs, leading to a significant decrease in cellular senescence via decrease inflammation and apoptosis, in vitro. Moreover, the acini cells of the salivary gland were regenerated, and the salivary function increased in aged mice. These results showed that saPRP could be a treatment agent against aging-induced SG dysfunction.
Assuntos
Plasma Rico em Plaquetas , Xerostomia , Masculino , Humanos , Camundongos , Ratos , Animais , Idoso , Lactente , Proliferação de Células , Células Cultivadas , Ratos Sprague-Dawley , Plasma Rico em Plaquetas/metabolismo , Envelhecimento , Xerostomia/metabolismo , Inflamação/metabolismoRESUMO
Dry mouth is frequently observed in the elderly, and enhanced lipid accumulation plays a critical role in cellular senescence in the salivary gland (SG). We investigated the mechanisms that mediate lipogenesis-associated SG senescence. Adult (28.6 ± 6.6 y.o. and 43.3 ± 1.5 y.o.) and aged (82.0 ± 4.3 y.o. and 88.0 ± 4.3 y.o.) human parotid and submandibular glands were compared with respect to histologic findings, 8-OHdG (8-hydroxy 2 deoxyguanosine) expression patterns, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SA-ß-gal (senescence-associated ß-galactosidase) assay results. Also, microarray analysis was performed on RNA extracted from adult and aged SG to identify DEGs (differentially expressed genes). The effects of silencing ADIPOQ (Adiponectin) were evaluated by quantifying cell proliferation, immunohistochemical staining for cellular senescence and inflammation-associated proteins, SA-ß-gal assays, RT-PCR, and western blot. Histological findings demonstrated the presence of more lipocytes, chronic inflammation, fibrosis, and lymphocytic infiltration in old SG. In addition, old tissues demonstrated higher expressions of SA-ß-gal, more apoptotic cells in TUNEL assays, and higher oxidative stress by 8-OHdG immunostaining. Microarray analysis showed lipogenesis was significantly upregulated in old tissues. Silencing of ADIPOQ (a lipogenesis-related gene) reduced inflammation and SA-ß-gal levels and increased cell proliferation and the expressions of amylase and aquaporin 5 in human SG epithelial cells. The study shows ADIPOQ is a potential target molecule for the modulation of lipogenesis associated with SG senescence.
Assuntos
Adiponectina , Glândulas Salivares , Idoso , Humanos , Adiponectina/genética , Senescência Celular/genética , beta-Galactosidase/metabolismo , Inflamação , LipídeosRESUMO
Hyperproduction of immune cell-derived inflammatory molecules and recruitment of immune cells promote the development of allergic asthma (AA). Aromadendrin (ARO) has various biological properties including anti-inflammatory effects. In this study, we evaluated the ameliorative effects of ARO on the development of AA in vitro and in vivo. Phorbol 12-myristate 13-acetate (PMA, 100 nM) was used to induce inflammation in A549 airway epithelial cells. The cohesion of A549 and eosinophil EOL-1 cells was studied. Ovalbumin (30 or 60 µg)/Alum (3 mg) mixture was adapted for AA induction in mice. ARO (5 or 10 mg/kg, p. o.) was administered to mice to investigate its ameliorative effect on AA development. Enzyme-linked immunosorbent assay, western blotting, and hematoxylin and eosin/periodic acid Schiff staining were performed to study the ameliorative effect of ARO on bronchial inflammation. In PMA-stimulated A549 cells, the upregulation of cytokines (interleukin [IL]-1ß/IL-6/tumor necrosis factor alpha [TNF-α]/monocyte chemoattractant protein [MCP]-1]) and nuclear factor kappa B (NF-κB) activation was effectively reduced by ARO pretreatment. ARO suppressed the adhesion of A549 cells and eosinophils. In ovalbumin-induced AA mice, the levels of cells, such as eosinophils, Th2 cytokines, MCP-1 in bronchoalveolar lavage fluid, IgE in serum, and inducible nitric oxide synthase/cyclooxygenase-2 expression in the lung tissue were upregulated, which were all suppressed by ARO. In addition, the increase in cell inflow and mucus formation in the lungs of AA mice was reversed by ARO as per histological analysis. ARO also modulated NF-κB activation in the lungs of AA mice. Overall, the anti-inflammatory properties of ARO in vitro/in vivo studies of AA were notable. Thus, ARO has a modulatory effect on bronchial inflammation and may be a potential adjuvant for AA treatment.
RESUMO
Leukotriene B4 (LTB4) is a potent chemoattractant that can recruit and activate immune cells such as neutrophils, eosinophils, and monocytes to sites of inflammation. Excessive production of LTB4 has been linked to acute and chronic inflammatory diseases, including asthma, rheumatoid arthritis, and psoriasis. Inhibiting the binding of LTB4 to its receptors, BLT1 and BLT2, is a potential strategy for treating these conditions. While several BLT1 antagonists have been developed for clinical trials, most have failed due to efficacy and safety issues. Therefore, discovering selective BLT2 antagonists could improve our understanding of the distinct functions of BLT1 and BLT2 receptors and their pharmacological implications. In this study, we aimed to discover novel BLT2 antagonists by synthesizing a series of biphenyl analogues based on a BLT2 selective agonist, CAY10583. Among the synthesized compounds, 15b was found to selectively inhibit the chemotaxis of CHO-BLT2 cells with an IC50 value of 224 nM without inhibiting the chemotaxis of CHO-BLT1 cells. 15b also inhibited the binding of LTB4 and BLT2 with a Ki value of 132 nM. Furthermore, 15b had good metabolic stability in liver microsomes and moderate bioavailability (F = 34%) in in vivo PK studies. 15b also showed in vivo efficacy in a mouse model of asthma, reducing airway hyperresponsiveness by 59% and decreasing Th2 cytokines by up to 46%. Our study provides a promising lead for the development of selective BLT2 antagonists as potential therapeutics for inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.
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Artrite Reumatoide , Asma , Camundongos , Cricetinae , Animais , Leucotrieno B4 , Asma/tratamento farmacológico , Inflamação , Células CHO , Receptores do Leucotrieno B4/metabolismoRESUMO
Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.
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Introduction: Salivary gland (SG) damage is commonly caused by aging, irradiation, and some medications, and currently, no damage modifying agent is available. However, cell therapy based on mesenchymal stem cells (MSCs) has been proposed as a therapeutic modality for irradiated SGs. Therefore, we administered cell-derived vesicles (CDVs) of adipose-derived mesenchymal stem cells (ADMSCs) to irradiated SG cells to investigate their radioprotective effects in vitro. Methods: The artificial CDVs were obtained from ADMSC by tangential flow filtration (TFF) purification and ultracentrifugation. Cultured human SG epithelial cells were exposed to 2, 5 or 15 Gy of 4 MV X-rays produced by a linear accelerator. The effects of ADMSC-CDVs on SG epithelial cells damaged by irradiation were tested by proliferation activity, transepithelial electrical resistance (TEER), and amylase activity. Results: Exposure to penetrating radiation inhibited the proliferation of SG epithelial cells, but the radiation intensity required to reduce the proliferation of human submandibular gland epithelial cells (hSMGECs) was greater than required for other SG cells. ADMSC-CDVs restored the proliferative ability of SG epithelial cells reduced by irradiation, and the proliferation capacities of irradiated human parotid gland epithelial cells (hPGECs) and human sublingual gland epithelial cells (hSLGECs) were increased by administering ADMSC-CDVs to non-irradiated SG epithelial cells. Furthermore, amylase activity in irradiated hPGECs, hSMGECs, and hSLGECs was lower than in non-irradiated controls. However, amylase ability was restored in all by ADMSC-CDV treatment. Also, TEER was diminished by irradiation in hPGECs, hSMGECs, and hSLGECs and restored by ADMSC-CDV administration. Conclusion: Overall, our findings demonstrate that ADMSC-CDVs have potent radioprotective effects on irradiated SG cells.
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Methyl phydroxycinnamate (MH), an esterified derivative of pCoumaric acid exerts antiinflammatory effects on lipopolysaccharide (LPS)stimulated RAW264.7 macrophages. Based on these effects, the present study investigated the protective role of MH in a mouse model of LPSinduced acute respiratory distress syndrome (ARDS). The results demonstrated that administration of LPS (5 mg/kg intranasally) markedly increased the neutrophil/macrophage numbers and levels of inflammatory molecules (TNFα, IL6, IL1ß and reactive oxygen species) in the bronchoalveolar lavage ï¬uid (BALF) of mice. On histological examination, the presence of inflammatory cells was observed in the lungs of mice administered LPS. LPS also notably upregulated the secretion of monocyte chemoattractant protein1 and protein content in BALF as well as expression of inducible nitric oxide synthase in the lungs of mice; it also caused activation of p38 mitogenactivated protein kinase (MAPK) and NFκB signaling. However, MH treatment significantly suppressed LPSinduced upregulation of inflammatory cell recruitment, inflammatory molecule levels and p38MAPK/NFκB activation, and also led to upregulation of heme oxygenase1 (HO1) expression in the lungs of mice. In addition, the ability of MH to induce HO1 expression was confirmed in RAW264.7 macrophages. Taken together, the findings of the present study indicated that MH may exert protective effects against airway inflammation in ARDS mice by inhibiting inflammatory cell recruitment and the production of inflammatory molecules.
Assuntos
Anti-Inflamatórios/farmacologia , Cinamatos/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , Síndrome do Desconforto Respiratório/tratamento farmacológico , Animais , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Transdução de SinaisRESUMO
Vocal cord paralysis caused by recurrent laryngeal nerve (RLN) injury during thyroidectomy results in hoarseness, aspiration, and dyspnea. We evaluated the usefulness of nerve guidance conduits (NGCs) constructed from an asymmetric polycaprolactone (PCL)/Pluronic F127 porous membrane and filled with platelet-rich plasma (PRP) for functional RLN regeneration. We evaluated the proliferation and migration of Schwann cells (SCs) after PRP treatment in vitro. For the in vivo study, rabbits were divided into a non-loaded NGC group and a PRP-loaded NGC group. The left RLNs were resected and interposed with the NGCs. Functional and histological examinations of the vocal cords were performed. SC proliferation and migration increased in a PRP dose-dependent manner, with the PRP increasing the levels of neurotrophic factors, myelin-associated glycoprotein, and ERK. In vivo, the PRP group showed significantly better vocal cord mobility and less vocalis muscle atrophy than the non-loaded NGC group. Histologically, the ingrowth of nerve endings occurred more rapidly in the PRP group, and acetylcholinesterase, neurofilament, and S-100 expression in neural endings were significantly higher in the PRP group. Furthermore, transmission electron microscopy showed that myelinated axons were more tightly packed in the PRP group. This study shows that PRP-loaded NGCs provide a favorable environment for neural regeneration and suggests that this technique has therapeutic potential for promoting RLN recovery.
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BACKGROUND: We examined the regenerative efficacy of the activated platelet-rich plasma (PRP) concentrate administered by local injection in an animal model mimicking partial glossectomy for tongue cancer. METHODS: Four-week-old mice were randomized to four groups; (1) a treatment-naïve control group, (2) a PRP group, (3) a hemiglossectomy group, and (4) a hemiglossectomy + PRP group. The activated PRP concentrate was injected into the deep layer of resected surfaces of mouse tongues immediately after excision, and tongue widths and lengths were measured on postoperative days (POD) 5 and 12. Gross tongue morphologies and microscopic findings were investigated. Inflammation and fibrous tissue areas were also measured, and immunohistochemical analysis was performed for c-kit, neurofilament, and S-100. RESULTS: The activated PRP concentrate reduced wound scar contracture, promoted wound healing, and reduced inflammation and wound fibrosis. On POD 12, histologic findings in the hemiglossectomy + PRP group were similar to those in the normal control group, and the intensity of stem cell factor receptor c-kit expression was also significantly greater in the PRP group than in the hemiglossectomy group on POD 12. Immunohistochemical staining revealed S100 and neurofilament expressions in the hemiglossectomy + PRP group were significantly more intense than in the hemiglossectomy group. CONCLUSION: Intralesional activated PRP concentrate injection has potential use for tongue regeneration, wound healing, and neural regeneration with minimal scarring after partial glossectomy.
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Glossectomia , Plasma Rico em Plaquetas , Regeneração , Língua , Animais , Modelos Animais de Doenças , Inflamação , Camundongos , Língua/cirurgia , CicatrizaçãoRESUMO
The occurrence of shivering during the induction period of targeted temperature management (TTM) remains a therapeutic obstacle, which delays the achievement of target temperature. The aim of this study was to identify risk factors leading to shivering during the induction period. We analyzed a prospective cohort of adult out-of-hospital cardiac arrest (OHCA) survivors treated with TTM from January 2015 to June 2017. Patients who developed shivering during the induction period were compared to those who did not. Multivariable analysis was performed to determine risk factors of shivering. Among 80 patients treated with TTM, shivering occurred in 22 patients (27.5%). In the shivering group, the time to achieve target temperature was significantly delayed (245 minutes vs. 151 minutes, p = 0.005). Multivariable analysis showed that being underweight (OR, 18.40; 95% CI, 1.89-179.19) or overweight (OR, 8.65; 95% CI, 1.60-46.80), age <65 years (OR, 5.54; 95% CI, 1.25-16.12), and duration of cardiac arrest <20 minutes (OR, 4.50; 95% CI, 1.25-16.12) were predictors for the occurrence of shivering. OHCA patients with abnormal body weight, age <65 years, and duration of cardiac arrest <20 minutes should be monitored thoroughly for early recognition of shivering.