RESUMO
BACKGROUND AND AIMS: Inflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. METHODS: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. RESULTS: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. CONCLUSIONS: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.
Assuntos
Aterosclerose , Modelos Animais de Doenças , Progressão da Doença , Lipoproteínas HDL , Animais , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Aterosclerose/enzimologia , Aterosclerose/metabolismo , Aterosclerose/tratamento farmacológico , Lipoproteínas HDL/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos , Camundongos Knockout para ApoE , Placa Aterosclerótica , Masculino , Elastase Pancreática/metabolismo , Aorta/patologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Doenças da Aorta/patologia , Doenças da Aorta/enzimologia , Doenças da Aorta/metabolismo , Inibidores de Proteases/farmacologia , alfa 1-Antitripsina/farmacologia , alfa 1-Antitripsina/metabolismoRESUMO
The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with ß-aminopropionitrile (BAPN)-supplemented drinking water to reliably produce true infrarenal aneurysms with behaviors that mimic human AAAs. Topically applying elastase to the adventitia of the infrarenal aorta causes structural damage to the elastic layers of the aortic wall and initiates aneurysmal dilation. Co-administering BAPN, a lysyl oxidase inhibitor, promotes sustained wall degeneration by reducing collagen and elastin crosslinking. This combination results in large AAAs that progressively expand, form intraluminal thrombus, and are capable of rupture. Refining surgical techniques, such as circumferentially isolating the entire infrarenal aortic segment, can help standardize the procedure for a consistent and thorough application of porcine pancreatic elastase despite different operators and anatomic variations between mice. Therefore, the elastase/BAPN model is a refined approach to surgically inducing AAA in mice, which may better recapitulate human aneurysms and provide additional opportunities to study aneurysm growth and rupture risk.
Assuntos
Aminopropionitrilo , Aneurisma da Aorta Abdominal , Modelos Animais de Doenças , Elastase Pancreática , Animais , Elastase Pancreática/administração & dosagem , Aneurisma da Aorta Abdominal/patologia , Aminopropionitrilo/administração & dosagem , Camundongos , Administração Oral , Administração Tópica , MasculinoRESUMO
Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.
Assuntos
Aneurisma da Aorta Abdominal , Cloreto de Cálcio , Modelos Animais de Doenças , Elastase Pancreática , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Animais , Camundongos , Aorta Abdominal/patologia , Masculino , Camundongos Endogâmicos C57BL , SuínosRESUMO
BACKGROUND/AIMS: Pancreatic steatosis (PS) is a pathology associated with metabolic syndrome (MS), endocrin and exocrine disfunctions of the pancreas, and fatty liver. The data on the frequency of PS are very limited. We aimed to evaluate the frequency of PS detected by transabdominal ultrasonography (TAU) in gastroenterology clinics located in different geographical regions of Turkey and the factors associated with it. MATERIALS AND METHODS: Volunteers were evaluated by TAU for PS and hepatosteatosis (HS), and its degree. Pancreatic stiffness was evaluated by ultrasonographic shear wave elastography (SWE). All demographic, physical, and biochemical parametres were measured. RESULTS: A total of 1700 volunteers from 14 centers throughout Turkey were included in the study. Mean age was 48.03 ± 20.86 years (56.9% female). Prevalance of PS was detected in 68.9%. In the PS group, age, body mass index (BMI), waist circumference, systolic blood pressure, fasting blood glucose (FBG), lipid levels, insulin resistance, diabetes mellitus, hypertension, MS frequency, and pancreatic SWE score were increasing, and fecal elastase level was decreasing in correlation with the degree of PS. The frequency of HS was 55.5%. Hepatosteatosis [odds ratio (OR): 9.472], increased age (OR: 1.02), and BMI (OR: 1.089) were independent risk factors for the occurrence of PS. Lean-PS rate was 11.8%. The lean-PS group was predominantly female and younger than non-lean PS. Also it has lower blood pressure, FBG, liver enzymes, lipid levels, and HS rates. CONCLUSION: The frequency of PS was found 68.9% in Turkey. Its relationship was determined with age, BMI, HS, MS (and its components), pancreatic stiffness, and fecal elastase level.
Assuntos
Técnicas de Imagem por Elasticidade , Fígado Gorduroso , Síndrome Metabólica , Pancreatopatias , Humanos , Turquia/epidemiologia , Feminino , Pessoa de Meia-Idade , Masculino , Prevalência , Adulto , Fatores de Risco , Síndrome Metabólica/epidemiologia , Pancreatopatias/epidemiologia , Fígado Gorduroso/epidemiologia , Índice de Massa Corporal , Idoso , Pâncreas/diagnóstico por imagem , Elastase Pancreática/análise , Circunferência da Cintura , Resistência à Insulina , Glicemia/análise , Glicemia/metabolismoRESUMO
The existence of alpha-1 antitrypsin variants with apparently unremarkable phenotypes and serum concentrations, contrasting with a clinical picture suggestive of a severe deficiency, led us to investigate whether in these cases there was a reduction or even suppression of the capacity of alpha-1 antitrypsin to inhibit elastase. To this end, in two different laboratories, we adapted and validated a method for measuring the functional activity of alpha-1 antitrypsin, based on spectrophotometric kinetic analysis of the inhibition by serum alpha-1 antitrypsin of the hydrolytic activity of porcine pancreatic elastase on a chromogenic substrate. This method has proved to be robust, reproducible and transferable and made possible to define, on the basis of an analysis of a hospital population, a functionality index with a confidence interval comprised between 0.87 and 1.2, allowing to identify subjects likely to have a functional deficiency of alpha-1 antitrypsin, whether this deficiency being of a genetic origin without any quantitative or phenotypic translation, or whether being acquired under the effect of external agents (cigarette smoke or viruses).
Assuntos
Elastase Pancreática , Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/análise , Humanos , Deficiência de alfa 1-Antitripsina/diagnóstico , Deficiência de alfa 1-Antitripsina/sangue , Elastase Pancreática/análise , Elastase Pancreática/sangue , Reprodutibilidade dos Testes , Feminino , Masculino , Animais , Adulto , Suínos , Pessoa de Meia-Idade , Espectrofotometria/métodosRESUMO
Exocrine pancreatic insufficiency (EPI) is a condition caused by a deficiency of exocrine pancreatic enzymes, resulting in malabsorption of nutrients. Clinical manifestations of EPI may include steatorrhea, weight loss, diarrhea, and abdominal pain. Although direct testing is the most sensitive and specific for EPI, these tests are invasive, time consuming, expensive, and not well standardized. Fecal elastase (FE-1) has been shown to be an indirect marker of the exocrine secretory capacity of the pancreas and has become the most commonly employed indirect test for diagnosis of EPI. Measurement of fecal elastase consists of two main phases, a preanalytical phase and analytical phase. The preanalytical phase involves stool collection, storage and handling. The second phase is the analytical phase, which includes the actual assay processes and products used to produce a result. For FE-1 this includes sample extraction and measurement on an immunoassay. Each step in the process can influence the result and contribute to heterogeneity in FE-1 measurement, potentially impacting clinical diagnosis and management. Thus, this paper provides an overview of the preanalytical and analytical factors that can affect measurement and interpretation of FE-1 results.
Assuntos
Insuficiência Pancreática Exócrina , Fezes , Elastase Pancreática , Humanos , Fezes/química , Fezes/enzimologia , Elastase Pancreática/metabolismo , Elastase Pancreática/análise , Insuficiência Pancreática Exócrina/diagnóstico , Manejo de Espécimes/métodos , Biomarcadores/análise , Biomarcadores/metabolismo , Fase Pré-AnalíticaRESUMO
BACKGROUND: Endoscopic ultrasound (EUS) is the most sensitive method for diagnosing chronic pancreatitis (CP) in its early stages, and Rosemont Classification (RC) is used for its evaluation. Data on the correlation between EUS features and pancreatic exocrine insufficiency (PEI) are limited. We investigated the correlation between the EUS findings and PEI. METHODS: This was a retrospective, monocentric cohort study involving patients prospectively enrolled from 2018 to 2022, with definite or probable CP according to the M-ANNHEIM criteria. All the patients underwent EUS and exocrine function investigations within 12 months of diagnosis. PEI was diagnosed using fecal elastase (FE) or when overt steatorrhea was reversed by pancreatic enzyme replacement therapy. Logistic regression analyses, rank correlation, ROC curve, and area under the curve (AUROC) were performed to evaluate the association between EUS features and PEI, and the accuracy of RC in predicting PEI. RESULTS: Among 128 patients examined (63.3 % male; mean age, 47 years), 69.5 % were diagnosed with PEI. In multivariate logistic regression among all the RC criteria, only lithiasis in the main pancreatic duct (MPD) was associated with increased risk of PEI (OR 2.92, 95 % CI 1.29-6.61; p = 0.01). Rank analysis showed a weak inverse correlation between RC and FE (Spearman's rho = -0.02; p = 0.03). The accuracy of RC was moderate (AUROC 0.62, p = 0.014). CONCLUSIONS: Among RC EUS features, lithiasis in the MPD is helpful for predicting the risk of PEI, while other findings are of limited utility in evaluating exocrine function.
Assuntos
Endossonografia , Insuficiência Pancreática Exócrina , Pancreatite Crônica , Humanos , Pancreatite Crônica/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Feminino , Adulto , Insuficiência Pancreática Exócrina/diagnóstico por imagem , Estudos Retrospectivos , Idoso , Pâncreas Exócrino/diagnóstico por imagem , Pâncreas Exócrino/fisiopatologia , Testes de Função Pancreática , Estudos de Coortes , Elastase PancreáticaRESUMO
BACKGROUND: Inflammation is a key component in the development of abdominal aortic aneurysm (AAA), yet insights into the roles of immune cells and their interactions in this process are limited. METHODS: Using single-cell RNA transcriptomic analysis, we deconstructed the CD45+ cell population in elastase-induced murine AAA at the single-cell level. We isolated each group of immune cells from murine AAA tissue at different time points and divided them into several subtypes, listed the remarkable differentially expressed genes, explored the developmental trajectories of immune cells, and demonstrated the interactions among them. RESULTS: Our findings reveal significant differences in several immune cell subsets, including macrophages, dendritic cells, and T cells, within the AAA microenvironment compared with the normal aorta. Especially, conventional dendritic cell type 1 exclusively existed in the AAA tissue rather than the normal aortas. Via CellChat analysis, we identified several intercellular communication pathways like visfatin, which targets monocyte differentiation and neutrophil extracellular trap-mediated interaction between neutrophils and dendritic cells, which might contribute to AAA development. Some of these pathways were validated in human AAA. CONCLUSIONS: Despite the absence of external pathogenic stimuli, AAA tissues develop a complex inflammatory microenvironment involving numerous immune cells. In-depth studies of the inflammatory network shall provide new strategies for patients with AAA.
Assuntos
Aorta Abdominal , Aneurisma da Aorta Abdominal , Células Dendríticas , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Análise de Célula Única , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/metabolismo , Animais , Aorta Abdominal/patologia , Aorta Abdominal/metabolismo , Aorta Abdominal/imunologia , Camundongos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/imunologia , Masculino , Transcriptoma , RNA-Seq , Linfócitos T/imunologia , Linfócitos T/metabolismo , Perfilação da Expressão Gênica/métodos , Elastase Pancreática , Comunicação CelularRESUMO
We advance the quality of first-principles calculations of protein electronic circular dichroism (CD) through an amelioration of a key deficiency of a previous procedure that involved diabatization of electronic states on the amide chromophore (to obtain interamide couplings) in a ß-strand conformation of a diamide. This yields substantially improved calculated far-ultraviolet (far-UV) electronic circular dichroism (CD) spectra for ß-sheet conformations. The interamide couplings from the diabatization procedure for 13 secondary structural elements (13 diamide structures) are applied to compute the CD spectra for seven example proteins: myoglobin (α helix), jacalin (ß strand), concanavalin A (ß type I), elastase (ß type II), papain (α + ß), 310-helix bundle (310-helix) and snow flea antifreeze protein (polyproline). In all cases, except concanavalin A and papain, the CD spectra computed using the interamide couplings from the diabatization procedure yield improved agreement with experiment with respect to previous first-principles calculations.
Assuntos
Dicroísmo Circular , Concanavalina A , Concanavalina A/química , Mioglobina/química , Proteínas/química , Papaína/química , Papaína/metabolismo , Peptídeos/química , Elétrons , Elastase Pancreática/química , Elastase Pancreática/metabolismo , Estrutura Secundária de ProteínaRESUMO
Chronic obstructive pulmonary disease (COPD), an inflammatory lung disease, causes approximately 3 million deaths each year; however, its pathological mechanisms are not fully understood. In this study, we examined whether HX110B, a mixture of Taraxacum officinale, Dioscorea batatas, and Schizonepeta tenuifolia extracts, could suppress porcine pancreatic elastase (PPE)-induced emphysema in mice and its mechanism of action. The therapeutic efficacy of HX110B was tested using a PPE-induced emphysema mouse model and human bronchial epithelial cell line BEAS-2B. In vivo data showed that the alveolar wall and air space expansion damaged by PPE were improved by HX110B administration. HX110B also effectively suppresses the expression levels of pro-inflammatory mediators including IL-6, IL-1ß, MIP-2, and iNOS, while stimulating the expression of lung protective factors such as IL-10, CC16, SP-D, and sRAGE. Moreover, HX110B improved the impaired OXPHOS subunit gene expression. In vitro analysis revealed that HX110B exerted its effects by activating the PPAR-RXR signaling pathways. Overall, our data demonstrated that HX110B could be a promising therapeutic option for COPD treatment.
Assuntos
Elastase Pancreática , Extratos Vegetais , Transdução de Sinais , Animais , Transdução de Sinais/efeitos dos fármacos , Camundongos , Elastase Pancreática/metabolismo , Humanos , Extratos Vegetais/farmacologia , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/patologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Modelos Animais de Doenças , Linhagem Celular , Masculino , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Camundongos Endogâmicos C57BL , SuínosRESUMO
INTRODUCTION: Dissection or rupture of the aorta is accompanied by high mortality rates, and there is a pressing need for better prediction of these events for improved patient management and clinical outcomes. Biomechanically, these events represent a situation wherein the locally acting wall stress exceed the local tissue strength. Based on recent reports for polymers, we hypothesized that aortic tissue failure strength and stiffness are directly associated with tissue mass density. The objective of this work was to test this novel hypothesis for porcine thoracic aorta. METHODS: Three tissue specimens from freshly harvested porcine thoracic aorta were treated with either collagenase or elastase to selectively degrade structural proteins in the tissue, or with phosphate buffer saline (control). The tissue mass and volume of each specimen were measured before and after treatment to allow for density calculation, then mechanically tested to failure under uniaxial extension. RESULTS: Protease treatments resulted in statistically significant tissue density reduction (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.003), which in turn was significantly and directly correlated with both ultimate tensile strength (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.03) and tangent modulus (sham vs. collagenase p = 0.007 and sham vs elastase p = 0.03). CONCLUSIONS: This work demonstrates for the first time that tissue stiffness and tensile strength are directly correlated with tissue density in proteolytically-treated aorta. These findings constitute an important step towards understanding aortic tissue failure mechanisms and could potentially be leveraged for non-invasive aortic strength assessment through density measurements, which could have implications to clinical care.
Assuntos
Aorta Torácica , Resistência à Tração , Animais , Suínos , Aorta Torácica/fisiologia , Elastase Pancreática/metabolismo , Colagenases , Proteólise , Rigidez Vascular/fisiologia , Estresse MecânicoRESUMO
Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAßN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAßN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAßN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAßN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAßN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.
Assuntos
Antibacterianos , Proteínas de Bactérias , Dipeptídeos , Imipenem , Testes de Sensibilidade Microbiana , Elastase Pancreática , Infecções por Pseudomonas , Pseudomonas aeruginosa , Percepção de Quorum , Fatores de Virulência , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Antibacterianos/farmacologia , Humanos , Infecções por Pseudomonas/microbiologia , Dipeptídeos/farmacologia , Imipenem/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Farmacorresistência Bacteriana , MetaloendopeptidasesRESUMO
CLINICAL PROBLEM: Most abdominal aortic aneurysms (AAAs) are small with low rupture risk (<1%/y) when diagnosed but slowly expand to ≥55 mm and undergo surgical repair. Patients and clinicians require medications to limit AAA growth and rupture, but drugs effective in animal models have not translated to patients. RECOMMENDATIONS FOR INCREASING TRANSLATION FROM MOUSE MODELS: Use models that simulate human AAA tissue pathology, growth patterns, and rupture; focus on the clinically relevant outcomes of growth and rupture; design studies with the rigor required of human clinical trials; monitor AAA growth using reproducible ultrasound; and perform studies in both males and females. SUMMARY OF STRENGTHS AND WEAKNESSES OF MOUSE MODELS: The aortic adventitial elastase oral ß-aminopropionitrile model has many strengths including simulating human AAA pathology and modeling prolonged aneurysm growth. The Ang II (angiotensin II) model performed less well as it better simulates acute aortic syndrome than AAA. The elastase plus TGFß (transforming growth factor-ß) blocking antibody model displays a high rupture rate, making prolonged monitoring of AAA growth not feasible. The elastase perfusion and calcium chloride models both display limited AAA growth.
Assuntos
Aneurisma da Aorta Abdominal , Ruptura Aórtica , Modelos Animais de Doenças , Animais , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/patologia , Humanos , Ruptura Aórtica/prevenção & controle , Ruptura Aórtica/diagnóstico por imagem , Ruptura Aórtica/patologia , Elastase Pancreática , Camundongos , Aorta Abdominal/patologia , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/diagnóstico por imagem , Aorta Abdominal/metabolismo , Feminino , Progressão da Doença , MasculinoRESUMO
Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.
Assuntos
Sinalização do Cálcio , Hepatite Alcoólica , Hepatócitos , Neutrófilos , Elastase Pancreática , Humanos , Neutrófilos/metabolismo , Neutrófilos/patologia , Animais , Camundongos , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/patologia , Hepatite Alcoólica/genética , Elastase Pancreática/metabolismo , Masculino , Proliferação de Células , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , FemininoRESUMO
The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.
Assuntos
Barreira Hematoencefálica , Modelos Animais de Doenças , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Elastase Pancreática , Transtornos Cerebrovasculares , Cisterna Magna , Masculino , Demência VascularRESUMO
Pesticides, including fungicides, are one of the important groups of environmental toxins that affect human and animal health. Studies have shown that these compounds are considered chemical pollutants. Carbendazim is a systemic fungicide. Unfortunately, excessive use of carbendazim has caused environmental pollution all over the world. In this study, the effect of carbendazim on the enzyme elastase (secreted from the endocrine gland of the pancreas) has been investigated. In a study, the performance and reaction of carbendazim with elastase were investigated using spectroscopic techniques. The stability and structure of elastase enzymes were studied under the influence of carbendazim. The results of fluorescence emission and UV-visible absorption spectrum showed that in the presence of carbendazim, there is an increase in UV-Vis absorption and a decrease in the intensity of the intrinsic fluorescence emission in the protein spectrum. Additionally, a decrease in the thermal stability of elastase was observed in the presence of carbendazim. The stability and structure of elastase enzyme were investigated in the presence of carbendazim. The results revealed that the UV-Vis absorption increased due to the presence of carbendazim, as indicated by the hyperchromic spectrum at 220 and 280 nm peaks. Additionally, the intrinsic fluorescence emission in the protein spectrum decreased with increasing carbendazim concentration at three different temperatures (298, 303, and 313 K). Moreover, the study demonstrated that the TM decreased from 2.59 to 4.58 with the increase of carbendazim, suggesting a decrease in the stability of the elastase structure in response to the elevated carbendazim concentration. According to the results of the research, the interaction between elastase and carbendazim has occurred, and changes have been made in the enzyme under the influence of carbendazim. The formation of the complex between elastase and carbendazim was consistent with the results obtained from molecular simulation and confirmed the thermodynamic data.
Assuntos
Benzimidazóis , Carbamatos , Elastase Pancreática , Espectrometria de Fluorescência , Carbamatos/química , Carbamatos/metabolismo , Benzimidazóis/química , Elastase Pancreática/metabolismo , Simulação de Acoplamento Molecular , Espectrofotometria Ultravioleta , Animais , Termodinâmica , Estabilidade Enzimática/efeitos dos fármacos , Ligação Proteica , Simulação por Computador , Humanos , Fungicidas Industriais/químicaRESUMO
As a clinical anti-glioma agent, the therapeutic effect of carmustine (BCNU) was largely decreased because of the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and the blood-brain barrier (BBB). To overcome these obstacles, we synthesized a BCNU-loaded hypoxia/esterase dual stimulus-activated nanomicelle, abbreviated as T80-HACB/BCNU NPs. In this nano-system, Tween 80 acts as the functional coating on the surface of the micelle to facilitate transport across the BBB. Hyaluronic acid (HA) with active tumor-targeting capability was linked with the hypoxia-sensitive AGT inhibitors (O6-azobenzyloxycarbonyl group) via an esterase-activated ester bond. The obtained T80-HACB/BCNU NPs had an average particle size of 232.10 ± 10.66 nm, the zeta potential of -18.13 ± 0.91 mV, and it showed high drug loading capacity, eximious biocompatibility and dual activation of hypoxia/esterase drug release behavior. The obtained T80-HACB/BCNU NPs showed enhanced cytotoxicity against hypoxic T98G and SF763 cells with IC50 at 132.2 µM and 133.1 µM, respectively. T80 modification improved the transportation of the micelle across an in vitro BBB model. The transport rate of the T80-HACB/Cou6 NPs group was 12.37 %, which was 7.6-fold (p<0.001) higher than the micelle without T80 modification. T80-HACB/BCNU NPs will contribute to the development of novel CENUs chemotherapies with high efficacy.
Assuntos
Antineoplásicos Alquilantes , Carmustina , Hipóxia Celular , Nanopartículas , Elastase Pancreática , Polissorbatos , Polissorbatos/química , Micelas , Elastase Pancreática/química , Elastase Pancreática/metabolismo , Carmustina/síntese química , Carmustina/química , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Nanopartículas/química , Nanopartículas/toxicidade , Ácido Hialurônico/química , Humanos , Linhagem Celular Tumoral , Dextranos/química , Sistemas de Liberação de Medicamentos , Apoptose/efeitos dos fármacosRESUMO
Irreversible alveolar airspace enlargement is the main characteristic of pulmonary emphysema, which has been extensively studied using animal models. While the alterations in lung mechanics associated with these morphological changes have been documented in the literature, the study of the mechanical behavior of parenchymal tissue from emphysematous lungs has been poorly investigated. In this work, we characterize the mechanical and morphological properties of lung tissue in elastase-induced emphysema rat models under varying severity conditions. We analyze the non-linear tissue behavior using suitable hyperelastic constitutive models that enable to compare different non-linear responses in terms of hyperelastic material parameters. We further analyze the effect of the elastase dose on alveolar morphology and tissue material parameters and study their connection with respiratory-system mechanical parameters. Our results show that while the lung mechanical function is not significantly influenced by the elastase treatment, the tissue mechanical behavior and alveolar morphology are markedly affected by it. We further show a strong association between alveolar enlargement and tissue softening, not evidenced by respiratory-system compliance. Our findings highlight the importance of understanding tissue mechanics in emphysematous lungs, as changes in tissue properties could detect the early stages of emphysema remodeling. STATEMENT OF SIGNIFICANCE: Gas exchange is vital for life and strongly relies on the mechanical function of the lungs. Pulmonary emphysema is a prevalent respiratory disease where alveolar walls are damaged, causing alveolar enlargement that induces harmful changes in the mechanical response of the lungs. In this work, we study how the mechanical properties of lung tissue change during emphysema. Our results from animal models show that tissue properties are more sensitive to alveolar enlargement due to emphysema than other mechanical properties that describe the function of the whole respiratory system.
Assuntos
Elastase Pancreática , Enfisema Pulmonar , Animais , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologia , Pulmão/patologia , Ratos , Masculino , Alvéolos Pulmonares/patologia , Fenômenos BiomecânicosRESUMO
Five dihydrophenanthropyrans (1-5) were isolated from the pseudobulbs of Pholidota chinensis, among which 1,3-di(4'-hydroxybenzy)-imbricatin (3) was isolated from the nature for the first time. Their structures were elucidated and established through various spectroscopic methods. These compounds exhibited a potent inhibition effect on both N-formyl-methionyl-leucyl-phenylalanine (fMLF)-induced superoxide anion generation and elastase release with IC50 values ranging from 0.23 to 7.63 µM. Furthermore, dihydrophenanthropyrans (1-3) also demonstrated a dose-dependent reactive oxygen species (ROS) scavenging effect. In addition, dihydrophenanthropyrans (2-3) exhibited a dose-dependent reduction in the intracellular Ca2+ concentration ([Ca2+]i) in fMLF-activated human neutrophils. Moreover, dihydrophenanthropyrans (1-3) selectively inhibited the phosphorylation of c-Jun N-terminal kinases (JNKs) and p38, while only dihydrophenanthropyran (1) inhibited the phosphorylation of extracellular signal-regulated kinases (ERKs) in fMLF-activated human neutrophils. Notably, dihydrophenanthropyrans (1-3) did not affect protein kinase B (AKT) activity in these cells. These findings highlight the potent anti-inflammatory capabilities of dihydrophenanthropyrans, manifested through their ability to inhibit superoxide anion generation, suppress elastase release, and selectively modulate key signaling pathways in human neutrophils. This suggests that dihydrophenanthropyrans hold significant promise as therapeutic agents for conditions associated with neutrophil-mediated inflammation.
Assuntos
Cálcio , Neutrófilos , Superóxidos , Neutrófilos/efeitos dos fármacos , Humanos , Estrutura Molecular , Cálcio/metabolismo , Superóxidos/metabolismo , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/isolamento & purificação , Orchidaceae/química , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Inflamação/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , China , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND/OBJECTIVES: Autoimmune pancreatitis (AIP) is a steroid-responsive inflammatory disease of the pancreas. Few studies investigated pancreatic exocrine function (PEF) in patients suffering from AIP and no definitive data are available on the effect of steroids in PEF recovery. Aim of the study is the evaluation of severe pancreatic insufficiency (sPEI) prevalence in AIP at clinical onset and after steroid treatment. METHODS: 312 Patients with diagnosis of AIP between January 1st, 2010 and December 31st, 2020 were identified in our prospectively maintained register. Patients with a pre-steroid treatment dosage of fecal elastase-1 (FE-1) were included. Changes in PEF were evaluated in patients with available pre- and post-treatment FE (between 3 and 12 months after steroid). RESULTS: One-hundred-twenty-four patients were included, with a median FE-1 of 122 (Q1-Q3: 15-379) µg/g at baseline. Fifty-nine (47.6 %) had sPEI (FE-1<100 µg/g). Univariable analysis identified type 1 AIP, radiological involvement of the head of the pancreas (diffuse involvement of the pancreas or focal involvement of the head), weight loss, age and diabetes as associated with a greater risk of sPEI. However, at multivariable analysis, only the involvement of the head of the pancreas was identified as independent risk factor for sPEI. After steroids, mean FE-1 changed from 64 (15-340) to 202 (40-387) µg/g (P = 0.058) and head involvement was the only predictor of improvement of sPEI. CONCLUSION: The inflammatory involvement of the head of the pancreas is associated with PEF severity, as well as PEF improvement after treatment with steroids in patients with AIP.