Role of cathepsin B in mechanical ventilator-induced lung injury in rats: relationship with NLRP3 inflammasomes / 中华麻醉学杂志

Objective:To evaluate the role of cathepsin B (CTSB) in mechanical ventilator-induced lung injury (VILI) in rats and the relationship with NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome.Methods:Thirty-six SPF-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 220-300 g, were divided into 3 groups ( n=12 each) by the random number table method: control group (group C), VILI group (group V) and VILI + CA074-me group (group Me). CA074-me 5 mg/kg was intraperitoneally injected in group Me, while the equal volume of normal saline was given instead in group C and group V. Group C kept spontaneous breathing for 4 h, and the animals were mechanically ventilated (tidal volume 20 ml/kg, respiratory rate 80 breaths/min, fraction of inspired oxygen 21%, PEEP 0 cmH 2O). Blood samples from femoral artery were collected for arterial blood gas analysis before tracheal intubation and after spontaneous breathing or ventilation, and PaO 2 was recorded.Rats were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected and lung tissues were collected for determination of the wet/dry lung weight ratio (W/D ratio), serum interleukin-1beta (IL-1β) and IL-18 concentrations in BALF (by enzyme-linked immunosorbent assay), expression of CTSB, NLRP3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) and caspase-1 mRNA in lung tissues (quantitative real-time polymerase chain reaction), and expression of CTSB, NLRP3, ASC and caspase-1 in lung tissues (by Western blot) and for microscopic examination of the pathological changes (using HE staining). Lung injury was assessed and scored. Results:Compared with group C, PaO 2 was significantly decreased after the end of ventilation, the lung injury score, W/D ratio and concentrations of IL-1β and IL-18 in serum and BALF were increased, and the expression of CTSB, NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was up-regulated in group V and group Me ( P<0.01). Compared with group V, PaO 2 was significantly increased after the end of ventilation, the lung injury score, W/D ratio and concentrations of IL-1β and IL-18 in serum and BALF were decreased, and the expression of CTSB, NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was down-regulated in group Me ( P<0.01). Conclusions:CTSB is involved in VILI in the rats, and the mechanism may be related to activation of NLRP3 inflammasomes.

Selección de blanco terapéutico en la catepsina B para el desarrollo de fármacos contra el cáncer de mama / Therapeutic targeting of Cathepsin B for the development of drugs for breast cancer

RESUMEN Introducción: el cáncer de mama se ha incrementado en un 50 % en las dos últimas décadas. La catepsina B es una proteasa que participa en el proceso de tumurogénesis. Uno de los problemas actuales es la aparición de resistencias a fármacos. La búsqueda de nuevas alternativas terapéuticas puede reducir su morbimortalidad. Objetivo: caracterizar in silico estructural y funcionalmente la región conservada en la catepsina B como blanco terapéutico potencial en el tratamiento del cáncer de mama Métodos: con el uso de la herramienta ENTREZ del NCBI se obtuvieron 2 485 secuencias de la catepsina B. Las secuencias son sometidas a un alineamiento múltiple empleando Clustall Omega 1.2.4. Se realiza la caracterización estructural y funcional de la proteasa en estudio a partir de las bases de datos InterPro, Prosite, Uniprot y UniprotKB. Con el empleo del visualizador Jalview se seleccionó la mayor zona conservada de las especies de catepsina B. Resultados: la proteasa participa en la regulación de la actividad catalítica, proteólisis, regulación negativa de la muerte celular, procesos catabólicos del colágeno y posee actividad hidrolasa. El análisis múltiple permitió la visualización de las características aminoacídicas del sitio activo de la catepsina B y la selección de la región proteica más conservada. Conclusiones: la zona conservada de la catepsina B constituye un blanco potencial en el desarrollo de inhibidores como fármacos contra el cáncer de mama. Los análisis in silico reducen costo de las investigaciones actuales y contribuyen a la bioseguridad farmacológica.

ABSTRACT Introduction: breast cancer has increased by 50% in the last two decades. Cathepsin B is a protease involved in the process of tumorigenesis. One of the current problems is the emergence of drug resistance. The search for new therapeutic alternatives can reduce its morbidity and mortality. Objective: in-silico structural and functional characterization of the conserved region in Cathepsin B as a potential therapeutic target in the treatment of breast cancer. Methods: using the NCBI ENTREZ tool 2,485 Cathepsin B sequences were obtained. The sequences were subjected to multiple alignments using Clustall Omega 1.2.4. Structural and functional characterization of the protease under study was performed using the InterPro, Prosite, Uniprot and UniprotKB databases. Using the Jalview viewer, the largest conserved area of Cathepsin B species was chosen. Results: the protease is involved in the regulation of catalytic activity, proteolysis, negative regulation of cell death, collagen catabolic processes and possesses hydrolase activity. The multiple analyses allowed the visualization of the aminoacid characteristics of the active site of Cathepsin B and the selection of the most conserved protein region. Conclusions: the conserved region of Cathepsin B constitutes a potential target in the development of inhibitors as drugs against breast cancer. In-silico analysis reduces the cost of current research and contributes to pharmacological biosafety.

Improvement of cognitive function in older adults based on near-infrared spectroscopy / 中国组织工程研究

BACKGROUND: Cognitive aging is an age-related cognitive degeneration that can develop into a cognitive dysfunction in the end stage. There is no clear therapeutic principle in clinical practice, but it is generally recognized that exercises can delay cognitive aging. OBJECTIVE: To summarize the current progress and shortcomings of exercise-delayed cognitive aging based on near-infrared spectroscopy. METHODS: In line with the PRISMA guidelines, the first author searched PubMed, WOS, CNKI, and WanFang using the keywords of “exercise, near-infrared spectroscopy, cognition, elderly or older adults, cathepsin B, brain-derived neurotrophic factor” in Chinese and English, respectively. Literature addressing senile cognitive function based on the near-infrared spectroscopy technique was retrieved, and 37 eligible articles were retained for further analysis. RESULTS AND CONCLUSION: We performed a comprehensive analysis of exercise-delayed cognitive aging based on near-infrared spectroscopy and confirmed that exercise improves the activation of cortex in different brain regions and thus improves cognitive function. Long-term aerobic exercise has better effects in the improvement of cognitive function than short-term exercise, which is more conducive to delaying cognitive aging. The underlying physiological mechanism may be that exercise improves blood flow in the brain, stimulates the secretion of neurotrophic factors from the skeletal muscle, and promotes the growth, survival and proliferation of neurons. However, there is no uniform standard for the interventional strategies. There are many links to be further improved, such as integration of individual differences and indexes in different brain regions, procedures for testing physical conditions (cardiovascular and lung diseases), to enhance the reliability of relevant parameters.

Avaliação da resistência de formas mutantes da enzima L-asparaginase a proteases séricas humanas / Evaluation of L-asparaginase mutant forms resistance to human serum proteases

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações

The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications

Ortho-nitrobenzyl derivatives as potential anti-schistosomal agents

ABSTRACT In the search for new anti-schistosomal agents, a series of fifteen ortho-nitrobenzyl derivatives was assayed in vitro against both the schistosomulum (somule) and adult forms of Schistosoma mansoni. Compounds 8 and 12 showed significant activity against somules at low micromolar concentrations, but none was active against adults. The SAR demonstrated that the compounds most active against the parasite were mutagenic to the human cell line RKO-AS45-1 only at concentrations 10- to 40-fold higher than the worm-killing dose. Given their electrophilicity, compounds were also screened as inhibitors of the S. mansoni cysteine protease (cathepsin B1) in vitro. Amides 5 and 15 exhibited a modest inhibition activity with values of 55.7 and 50.6 % at 100 µM, respectively. The nitrobenzyl compounds evaluated in this work can be regarded as hits in the search for more active and safe anti-schistosomal agents.

Cathepsin B in hepatic Kupffer cells regulates activation of TLR4-independent inflammatory pathways in mice with lipopolysaccharide-induced sepsis / 南方医科大学学报

OBJECTIVE@#To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis.@*METHODS@#Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1β, TNF-α and IL-18 were detected.@*RESULTS@#Compared with the WT mice,TLR4mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines.@*CONCLUSIONS@#Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.

Effect of Cathepsin B and vascular endothelial growth factor on the retinal neovascularization induced by hyperoxia / 中华实验眼科杂志

Background Vascular endothelial growth factor (VEGF) plays a key role in the vascular development and neovascularization.Studies have proved that Cathepsin B is related to the formation of neovascularization,but its mechanism is unclear.Objective This study was to investigate the expression of Cathepsin B and VEGF in the retinal neovascularization induced by hyperoxia and the relationship between them.Methods Forty-four 7-day-old C57BL/6J mice were randomly assigned into 4 groups:normal control group,hyperoxia-induced group,normal control (NC)-green fluorescent protein (GFP)-lentivirus (Lv) group and Cathepsin B-RNA interference (RNAi)-Lv group,with 11 mice 22 eyes for each group.The mice in normal control group were survival in natural environment.The other three groups of 7-day-old mice were put in a sealed box of oxygen volume fraction (75±2)％ for 5 days and then sent back to normal environment.The mice of hyperoxia-induced group did not have any drug intervention,while the 12-day-old mice of NC-GFP-Lv group and Cathepsin B-RNAi-Lv group received intravitreal injection of NC-GFP-Lv 1 μl or Cathepsin B-RNAi-Lv 1 μl.All 17-day-old mice were sacrificed and the retinas were collected.The mRNA expression levels of Cathepsin B and VEGF were performed by real-time PCR;the protein expression levels of Cathepsin B and VEGF were detected by Western blot.Results Fluorescence microscope results showed that the layer and branch of retinal neovascularization were less in the Cathepsin B-RNAi-Lv group than those in the NC-GFP-Lv group and hyperoxia-induced group.The relative expression levels of Cathepsin B and VEGF mRNA in each group were significantly different (F =444.89,P =0.00;F =519.78,P =0.00).The relative expression levels of Cathepsin B and VEGF mRNA in hyperoxia-induced group,NC-GFP-Lv group and Cathepsin B-RNAi-Lv group were higher than those in normal control group,and the relative expression levels of Cathepsin B and VEGF mRNA in Cathepsin B-RNAi-Lv group were lower than those in hyperoxia-induced group and NC-GFP-Lv group,with significant differences between them (all at P ＜ 0.05).The relative expression levels of Cathepsin B and VEGF protein in each group were significantly different (F =54.37,P =0.00;F =79.65,P =0.00).The relative expression levels of Cathepsin B and VEGF protein in hyperoxia-induced group,NC-GFP-Lv group and Cathepsin B-RNAi-Lv group were higher than those in normal control group,and the relative expression levels of Cathepsin B and VEGF protein in Cathepsin B-RNAi-Lv group were lower than those in hyperoxia-induced group and NC-GFP-Lv group,with significant differences between them (all at P＜0.05).Conclusions The expression levels of Cathepsin B and VEGF in retinal neovascularization of hyperoxia-induced group were significantly higher than those in normal control group,Cathepsin B-RNAi-Lv can inhibit the mRNA and protein expression of Cathepsin B and VEGF,the expression of VEGF may be influenced by the expression of Cathepsin B.

Dynamic changes of cathepsin B expression in the ipsilateral thalamus after cerebral cortical infarction in rats / 国际脑血管病杂志

ObjectiveTo investigate the expression change of cathepsin B (CathB) in the ventroposterior nucleus (VPN) of the ipsilateral thalamus after cortical infarction in rats.MethodsThe adult male Sprague-Dawley rats were randomly divided into either a sham operation group or a model group.The latter was further divided into postoperative 1-, 2-, 3-, 4-, and 8-week groups.A model of cerebral cortical infarction was induced by electrocoagulation the cortical branch of middle cerebral artery.Immunohistochemical staining and immunofluorescence were used to detect the protein expression and cellular localization of CathB in the VPN at each time point.ResultsThe expression level of VPN CathB in thalamus increased gradually after cerebral cortical infarction.It reached the peak at 4 weeks, and decreased at 8 weeks, however it was still higher than the control group (all P<0.05).The release of CathB from the lysosomes into the cytoplasm were found.In addition, the expression level of CathB in the activated astrocytes was significantly increased at 3 weeks after cerebral cortical infarction.ConclusionsDuring 1-8 week after cerebral cortex infarction, CathB in the VPN of the ipsilateral thalamus maintained higher expression level, suggesting that it might play a certain role in secondary degeneration in the thalamus after cerebral cortical infarction.

Mechanism of NLRP3 inflammasome activation caused by albumin in renal tubulointerstitial cells / 临床与实验病理学杂志

Purpose To study the mechanism of NLRP3 inflammasome activation caused by albumin in renal tubulointerstitial cells.Methods Cathepsin B was detected by immunohistochemistry in renal biopsy tissue of 30 membranous nephropathy patients which had different levels of proteinuria.HK-2 cells were stimulated by albumin,and then were treated by high concentration KCl,CA 074 Me and DPI,which was Cathepsin B inhibitor and ROS inhibitor.Finally,IL-1β and IL-18 were detected by Western blot and real time PCR,respectively.Results The expression of Cathepsin B in tubulointerstitial cells was significantly higher in patients with severe proteinuria than that in patients with mild proteinuria (P ＜ 0.05).CA 074 Me and DPI significantly reduced IL-1β and IL-18 secretion in HK-2 cells stimulated by albumin (P ＜ 0.05),but high concentration KCl did not result in this change (P ＞ 0.05).Conclusion NLRP3 inflammasome is activated via Cathepsin B release and increases ROS production caused by proteinuria,but not via K + efflux.

Effect of sufentanil postconditioning on level of cathepsin B in myocardium during ischemia-reperfusion in rats / 中华麻醉学杂志

Objective To evaluate the effect of sufentanil postconditioning on the level of cathepsin B in the myocardium during ischemia-reperfusion (I/R) in the rats.Methods Eighteen healthy adult male Sprague-Dawley rats,weighing 250-300 g,were divided into 3 groups (n=6 each) using a random nunber table:shamn operation group (group S),group I/R and sufentanil postconditioning group (group SP).The rats were anesthetized with intraperitoneal 20％ urethane 5 ml/kg.Myocardial I/R was induced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.At 5 min before reperfusion,sufentanil 1.0 μg/kg was intravenously injected in group SP,and the equal volume of normal saline was given instead in S and I/R groups.At the end of reperfusion,blood samples from the abdominal aorta were collected for determination of serum cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) concentrations,and myocardial specimnens were obtained for examination of the ultrastructure of cardiomyocytes (using transmission electron microscopy) and for determination of Beclin1,microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and cathepsin B expression (by Western blot) and cathepsin B activity (by fluorometric assay).Results Compared with group S,the serum cTnI and CK-MB concentrations were significantly increased,the expression of Beclin-1,LC3 Ⅱ and cathepsin B was up-regulated,and the activity of cathepsin B was enhanced in I/R and SP groups (P＜0.05).Compared with group I/R,the serum cTnI and CK-MB concentrations were significantly decreased,the expression of Beclin-1 and LC3 Ⅱ was down-regulated,the expression of cathepsin B was up-regulated,and the activity of cathepsin B was enhanced (P＜0.05),the pathological changes of myocardial tissues were signifieantly attenuated,and autophagosomes were reduced in group SP.Conclusion The mechanism by which sufentanil postconditioning inhibits cardiomyocyte autophagy during myocardial I/R is probably related to increased expression and activity of cathepsin B in rats.

Study on the role of Cathepsin B and JNK signaling pathway in the development of cerebral aneurysm

Objective To investigate the correlation between JNK signal and the apoptosis of VSMC as well as the expression of Cathepsin B and to explore the role of JNK signal in the development of cerebral aneurysm. Methods Rat models of cerebral aneurysm were established and histopathologic changes of cerebral aneurysm and the apoptosis of VSMC were analyzed. Rat models were respectively subject to subcutaneous injection of Cathepsin B siRNA and JNK inhibitor SP600125. Western blot technique was used to detect the expression of proteins like Cathepsin B, Caspase-3, and p-JNK. Spearman's rho was used to examine the correlation between p-JNK and Cathepsin B, as well as the expression of relevant proteins. Results The success rate of modeling rats with cerebral aneurysm was 88.75%. After the respective injection of Cathepsin B siRNA, SP600125 and their combination, the cell densities of VSMC of rats with cerebral aneurysm all increased significantly (P < 0.05 or P < 0.01), but the apoptosis rate of VSMC decreased significantly (P < 0.01). Compared with normal rats, the expression of Cathepsin B, Caspase-3 and p-JNK in Cerebral aneurysm models increased significantly. Effectively intervening Cathepsin B genes with Cathepsin B siRNA could significantly inhibit the expression of Cathepsin B and Caspase-3, but hardly influence the expression of p-JNK. JNK inhibitor SP600125 had no influence on the expression of Cathepsin B and Caspase-3, but effectively inhibited the expression of p-JNK. In cerebral aneurysm tissues, positive correlation was observed between the expression of p-JNK and Cathepsin B, the correlation coefficient was r = 0.640. Conclusion After the attack of cerebral aneurysm, proteins like Cathepsin B, Caspase-3 and p-JNK are all involved in the apoptosis of VSMCs. This process may be realized by Cathepsin B which activates the apoptosis mechanism of Caspase-3 and mediate the apoptosis of VSMC through the JNK signaling pathway. Therefore, silencing Cathepsin B gene or inhibiting the conduction through JNK signaling pathway can mitigate the apoptosis of vascular smooth muscle cells in cerebral aneurysm.

Study on the role of Cathepsin B and JNK signaling pathway in the development of cerebral aneurysm

OBJECTIVE@#To investigate the correlation between JNK signal and the apoptosis of VSMC as well as the expression of Cathepsin B and to explore the role of JNK signal in the development of cerebral aneurysm.@*METHODS@#Rat models of cerebral aneurysm were established and histopathologic changes of cerebral aneurysm and the apoptosis of VSMC were analyzed. Rat models were respectively subject to subcutaneous injection of Cathepsin B siRNA and JNK inhibitor SP600125. Western blot technique was used to detect the expression of proteins like Cathepsin B, Caspase-3, and p-JNK. Spearman's rho was used to examine the correlation between p-JNK and Cathepsin B, as well as the expression of relevant proteins.@*RESULTS@#The success rate of modeling rats with cerebral aneurysm was 88.75%. After the respective injection of Cathepsin B siRNA, SP600125 and their combination, the cell densities of VSMC of rats with cerebral aneurysm all increased significantly (P < 0.05 or P < 0.01), but the apoptosis rate of VSMC decreased significantly (P < 0.01). Compared with normal rats, the expression of Cathepsin B, Caspase-3 and p-JNK in Cerebral aneurysm models increased significantly. Effectively intervening Cathepsin B genes with Cathepsin B siRNA could significantly inhibit the expression of Cathepsin B and Caspase-3, but hardly influence the expression of p-JNK. JNK inhibitor SP600125 had no influence on the expression of Cathepsin B and Caspase-3, but effectively inhibited the expression of p-JNK. In cerebral aneurysm tissues, positive correlation was observed between the expression of p-JNK and Cathepsin B, the correlation coefficient was r = 0.640.@*CONCLUSION@#After the attack of cerebral aneurysm, proteins like Cathepsin B, Caspase-3 and p-JNK are all involved in the apoptosis of VSMCs. This process may be realized by Cathepsin B which activates the apoptosis mechanism of Caspase-3 and mediate the apoptosis of VSMC through the JNK signaling pathway. Therefore, silencing Cathepsin B gene or inhibiting the conduction through JNK signaling pathway can mitigate the apoptosis of vascular smooth muscle cells in cerebral aneurysm.

Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study / Análises das isoformas de heparanase e da catepsina B em plasma de pacientes com carcinomas gastrointestinais: estudo transversal analítico

CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group). DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the ...

CONTEXTO E OBJETIVO: A heparanase-1 degrada heparam sulfato e está relacionada à progressão de tumor. Apesar de a isoforma heparanase-2 não possuir atividade catalítica, parece ser importante para modular a atividade da heparanase-1. A catepsina B é uma proteinase envolvida na metástase de tumores. O objetivo deste estudo foi analisar a expressão das isoformas de heparanase e atividade da catepsina B em amostras de plasma de pacientes com carcinomas gastrointestinais, comparando-se com indivíduos saudáveis (grupo controle). TIPO DE ESTUDO E LOCAL: Este é um estudo transversal analítico. Foram coletadas amostras de sangue periférico, em hospital público brasileiro, de 21 pacientes com diagnóstico histopatológico de carcinoma gastrointestinal e 43 indivíduos saudáveis. As análises foram realizadas em duas faculdades de medicina brasileiras. MÉTODOS: As isoformas da heparanase foram identificadas e quantificadas em amostras de plasma por Western blot. As atividades enzimáticas de heparanase-1 e catepsina B foram também mensuradas. RESULTADOS: Os resultados demonstraram que as expressões das isoformas de heparanase foram significativamente maiores nas amostras de plasma de pacientes com carcinoma gastrointestinal em comparação com ...

Expression of cathepsin B, cathepsin L and uPA mRNA levels in non-Hodgkin lymphoma and their significance / 白血病·淋巴瘤

Objective To investigate mRNA expression levels of cathepsin B,cathepsin L and uPA in non-Hodgkin lymphoma (NHL) samples,and analyze the relationship between three genes and clinicopathological factors.Methods Real-time fluorescence quantitative RT-PCR was used to quantify the relative expression levels of cathepsin B,cathepsin L and uPA mRNA in 39 NHL patients without chemotherapy or radiotherapy and 15 patients with lymph node reactive hyperplasia.Relationship between their expression and clinico-pathological factors was analyzed.Results mRNA expression levels of cathepsin B,cathepsin L and uPA in NHL group were significantly higher than those in the control group (0.403 vs 0.128,0.209 vs 0.057,0.459 vs 0.031,all P ＜ 0.05).The mRNA expression levels of cathepsin B,cathepsin L and uPA were correlated with each other in NHL.The mRNA expression levels of cathepsin B,cathepsin L and uPA in advanced staged NHL were markedly higher than those in early staged NHL (0.453 vs 0.341,0.204 vs 0.085,0.372 vs 0.196,all P ＜ 0.05).mRNA levels of cathepsin B,cathepsin L and uPA were higher in NHL patients sensitive to chemotherapy than in those resistant to chemotherapy(0.401 vs 0.556,0.085 vs 0.205,0.316 vs 0.499,all P ＜ 0.05).Conclusions The high expression of cathepsin B,cathepsin L and uPA could be detected in NHL,and their expression has a close relationship with the biological behavior characters of NHL.The dynamic examination of cathepsin B,cathepsin L and uPA may be used as clinical auxiliary diagnosis and prognosis of NHL.

Expression of cathepsin B in acutely photodamaged fibroblasts and its significance / 中华皮肤科杂志

Objective To investigate the changes in cathepsin B (CatB) expression in acutely photodamaged human dermal fibroblasts (HDFs) and their significance.Methods HDFs were isolated from the foreskin of children,and subjected to primary culture and subculture.The fourth-to eighth-passage HDFs were used in the following experiment.HDFs were divided into two groups to receive irradiation with different doses of ultraviolet A (UVA) for different durations (acutely photodamaged group) or remain unirradiated (control group).Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferative activity of HDFs after irradiation with UVA at 5,10,15,20 and 25 J/cm2 respectively.Western blot and quantitative real-time reverse transcription PCR were performed to measure the protein and mRNA expressions of CatB respectively in HDFs at 24,48 and 72 hours after exposure to UVA at 10 J/cm2,and at 48 hours after exposure to UVA at 10,15,20 and 25 J/cm2.Statistical analysis was carried out by analysis of variance and least significant difference (LSD) test using the SPSS 13.0 software.Results UVA radiation induced a decrease in the proliferative activity of HDFs.When the dose of UVA was ≤ 10 J/cm2,the survival rate of HDFs maintained higher than 85％,and significant differences were observed in cell survival rate between unirradiated and irradiated HDFs at 24,48 and 72 hours (all P ＜ 0.05).Western blot showed that the gray value of CatB protein in the acutely photodamaged group irradiated with 10 J/cm2 UVA was significantly higher than that in the control group at 24 hours (0.76 ± 0.14 vs.0.35 ± 0.01,P ＜ 0.05),48 hours (1.34 ± 0.38 vs.0.45 ± 0.12,P＜ 0.05) and 72 hours (0.82 ± 0.09 vs.0.61 ± 0.06,P＜ 0.05).Increased mRNA expressions of CatB were also observed in the acutely photodamaged group compared with the control group at 24 hours (0.149 ± 0.009 vs.0.089 ± 0.015,P ＜ 0.05),48 hous (0.173 ± 0.009 vs.0.091 ± 0.010,P ＜ 0.05) and 72 hours (0.185 ± 0.158 vs.0.111 ± 0.017,P ＜ 0.05) after UVA radiation at 10 J/cm2.The gray value of CatB protein was 0.99 ± 0.07,1.49 ± 0.14,1.89 ± 0.08,2.07 ± 0.06 in HDFs at 48 hours after exposure to UVA of 10,15,20 and 25 J/cm2,respectively,significantly higher than that in the control group (0.60 ± 0.05,all P ＜ 0.05).Similarly,the mRNA expression of CatB was up-regulated in HDFs at 48 hours after UVA radiation at 10,15,20 and 25 J/cm2 compared with the unirradiated HDFs.Conclusion The protein and mRNA expressions of CatB are up-regulated in acutely photodamaged HDFs induced by UVA radiation.

Cloning,expression and bioinformatics analysis of cathepsin B of Echino-coccusgranulosus / 中国血吸虫病防治杂志

Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. Methods The total RNA of E. granulosus was extracted and reverse?ly transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In?Fusion PCR cloning method and expressed by a wheat germ cell?free system,and then the recombinant protein was identified by Western blotting. The signal peptide,transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online soft?ware SignalP 4.1,TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently,the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally,the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam,SMART,Predictprotein,Swiss?model,NetOGlyc 4.0 and NetNGlyc 1.0 approaches. Results The EgCatB gene was successfully amplified from cDNA of E. granulosus and ex?pressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a cata?lytic active center was formed through Gln106,Cys112,His282 and Asn302. It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. Conclusions The EgCatB gene is cloned and expressed successfully,and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful informa? tion for further functional study of the EgCatB protein.

Protective function of S jcb2 DNA vaccine in mice infected with Schistosoma j aponicum / 中国人兽共患病学报

The protective effect and mechanism of Schistosoma japonicum cathepsin B (Sjcb2) DNA vaccine in the mouse model of schistosomiasis were studied through construction pcDNA3 .1 (+ ) / Sjcb2 DNA recombinant vector ,which provided effective candidate antigen for anti-schistosome vaccine .The 6-week-old female BALB/c mice were randomly divided into pcDNA3 .1(+ )/Sjcb2 DNA vaccine group ,pcDNA3 .1(+ ) plasmid group and normal saline group ,respectively .Each group was composed of 35 mice ,and 100 μg of S jcb2 plasmid DNA was injected in the hind leg quadriceps of mice once every two weeks .PCR and immunohistochemistry assay were used to detect the expression and stability of Sjcb2 gene in mice .MTT assay was used for testing the specific proliferation response of mice spleen lymphocytes .The level of Sjcb2 antibodies in mouse serum and the IFN-γand IL-4 levels in mice spleen lymphocyte culture supernatant before and after schistosome infection were assayed by ELISA .At last ,we counted load of Schistosome adult worms in mouse and eggs in liver of mouse .The results showed that the Sjcb2 gene was detected in all mice of the Sjcb2 DNA vaccine group ,and Sjcb2 gene expression was positive in the muscle cells in Sjcb2 DNA immunized mice by IHC assay .MTT assay showed that T-cell proliferation rate was in-creased significantly in S jcb2 DNA vaccinated group .ELISA results showed that the IFN-γlevels were increased significantly in the vaccinated group ,while the IL-4 levels were significantly increased after Schistosoma japonicum infection in all mice of every group .The load of worms and eggs in Sjcb2 DNA vaccinated group was reduced significantly than that of control group (P<0 .05) ,the reduction rates of adult worms and eggs were 36 .32% and 60 .61% respectively .In conclusion ,the Sjcb2 gene was stably expressed in muscle cells of mice after injection of S jcb2 recombinant plasmid ,and S jcb2 produced protective effects of anti-schistosoma infection in mice possibly by mean of regulating Th1 cell subgroups through increasing the IFN-γ level and decreasing IL-4 levels .

Bone marrow mesenchymal stem cells repair vascular injury after cervical spondylotic vertebral arteriopathy / 中国组织工程研究

BACKGROUND:Inflammation is an important factor in cervical spondylotic vertebral arteriopathy, bone marrow mesenchymal stem cells have the potential to treat cervical spondylotic vertebral arteriopathy because of the immunomodulatory effects to inhibit inflammation. OBJECTIVE:To investigate the injury mechanism of vascular injury in the model of cervical spondylotic vertebral arteriopathy and to study the therapeutic effects of bone marrow mesenchymal stem cells on it. METHODS:Forty Japanese big ear rabbits were randomly divided into four groups:control group, model group, tanshinone group, and stem cellgroup. After modeling, the control and model groups were not given intervention, while the tanshinone and stem cellgroups were injected with tanshinone II A sodium sulfonate solution (10 mL) and bone marrow mesenchymal stem cellsuspension (10 mL) along the ear vein, respectively. After 2 weeks, the routine pathological examination was done to observe the vascular morphological changes, immunofluorescence staining was done to observe the cathepsin B expression in the vertebral artery, and ELISA was used to detect the expression of tumor necrosis factor-αand interleukin-6 in the vertebral artery. RESULTS AND CONCLUSION:Compared with the model group, the arterial smooth muscle cellhypertrophy and hyperplasia was obviously restrained in stem cellgroup, and vascular endothelial fold was in symmetry, while no significant difference was found between stem cellgroup and tanshinone group. Compared with the model group, expressions of cathepsin B, interleukin 6 and tumor necrosis factor-αexpression were reduced significantly in the stem cellgroup (P0.05). Inflammatory reaction may be one of mechanisms for vertebral artery damage, and bone marrow mesenchymal stem cells can effectively inhibit inflammation of the vertebral artery and improve vascular remodeling.

Inhibitory effect of cathepsin B-RNAi-lentivirus on mouse retinal neovascularization / 中华实验眼科杂志

Background Retinal neovascularization disease is a group of threatening-vision eye diseases.Researches showed that cathepsin B is involved in angiogenesis.Exploring a drug which inhibit retinal blood vessels will provide the basis for the molecular mechanism of these diseases.Objective This study was to investigate the inhibitory role of cathepsin B-RNAi-lentivirus on retinal angiogenesis.Methods Sixty 7-day-old C57BL/6J mice were raised together with maternal mice in the closed box with the oxygen concentration of (75-2)％ for 5 days to establish the retinal angiogenesis mouse models.The mice were then taken into the normal air environment for continuous raise and were randomized into 3 groups.NC-GFP-LV of 1 μl and the equal volume of cathepsin B-RNAi-lentivirus was intravitreously injected respectively in 40 eyes in the control group and the gene treatment group,and no drug was administered in the 40 eyes of the model group.The mice were sacrificed and retinas were obtained.Expression of cathepsin B protein in the retina was detected by Western blot assay (cathepsin B/β-actin).Real-time PCR was used to detect and compare the expression level of cathepsin B mRNA (2△△Ct).FITC-dextran was used to perform heart infusion for the retinal stretched preparation 5 days after intravitreously injection.Retinal neovascularization was examined by fluorescent angiography.Results The expression level (2-△△Ct) of cathepsin B mRNA was 0.74 ±0.12 in the gene treatment group,showing a significant decline in comparison with 1.66±0.17 and 1.58±0.29 in the model group and control group (q--0.746,1.588,P＜ 0.01).The expression level of cathepsin B protein (cathepsin B/β-actin) in the retina was 0.64±0.06,0.93±0.09 and 0.96±0.09 respectively in the gene treatment group,model group and control group,indicating a significant reduce in the gene treatment group (q =0.637,0.894,P＜0.01).Distorted vessels were seen in the mice retinas of the model group with more branches and vascular anastomosis,and fluorescine leakage was exhibited under the fluorescence microscope.However,the vessels were regular with less branches and angiogenesis.Conclusions Cathepsin B-RNAi-lentivirus can effectively inhibit oxygen-induced retinal angiogenesis in mouse.

Protective mechanisms of a cathepsin B inhibitor, CA-074 Me, on muscle fibers in coxsackievirus B1-induced polymyositis in guinea pigs: an experimental study / 中华皮肤科杂志

Objective To evaluate the effect of a specific cathepsin B inhibitor,CA-074 Me,on the expression of cathepsin B in coxsackievirus B1-induced polymyositis in guinea pigs,and to elucidate the protective mechanisms of CA-074 Me on muscle fibers.Methods Polymyositis model was established in 32 guinea pigs by infection with coxsackievirus B1,which were then equally divided into 4 groups: γ-interferon group treated with intraperitoneal γ-interferon (150 000 IU per kilogram per day) from week 5 to week 8,polymyositis model group receiving no treatment,pseudo-intervention group treated with intraperitoneal sodium chloride physiological solution,CA-074 Me group treated with intraperitoneal CA-074 Me (4 mg per kilogram per day) for 7 days,after the infection with coxsackievirus B1.Eight guinea pigs receiving no infection or treatment served as the healthy control group.Blood samples and muscle tissue samples were obtained from the guinea pigs in the γ-interferon group on week 8 and in the other 4 groups on week 5.The serum level of muscle enzymes including creatine kinase (CK),CK-MM,aspartic transaminase (AST) and l-lactate dehydrogenase (LDH) were determined.Muscle tissue samples were studied by hematoxylin and eosin (HE) staining,and Envision two-step method was used to quantify the expression of cathepsin B and to numerate CD8+ T cells.The apoptosis in muscle cells was detected by terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling (TUNEL).One-way analysis of variance (ANOVA) was conducted to compare the serum level of muscle enzymes,inflammation score of muscle and apoptosis index of muscular cells,and Pearson chi-square test to compare the count of CD8 + T cells and cathepsin B expression,among these groups.Results Polymyositis model was successfully established by infection with coxsackievirus B1 in the 32 guinea pigs with a marked increase in the serum level of the tested enzymes,especially in that of CK.In detail,the serum level of CK was (410.7 ±167.9) U/L in the healthy control group,significantly lower than that in the polymyositis model group ((3537.3 ± 2141.6) U/L,P ＜ 0.05),pseudo-intervention group ((2222.0 ± 226.9) U/L,P ＜ 0.05),γ-interferon group ((973.8 ± 423.2) U/L,P＜ 0.05) and CA-074 Me group ((814.0 ± 268.4) U/L,P＜ 0.05).Compared with the pseudo-intervention group,the γ-interferon group and CA-074 Me group showed a slight increase in the serum level of all the four enzymes (all P ＜ 0.05).There was a significant elevation in the inflammation score of skeletal muscles in the polymyositis model group,pseudo-intervention group,γ-interferon group and CA-074 Me group compared with the healthy control group (1.75 ± 0.50,1.40 ± 0.55,2.38 ± 0.74 and 1.20 ± 0.45 vs.0.00 ± 0.00,all P ＜ 0.05),with the most intense infiltration of inflammatory cells observed in the γ-interferon group.Moreover,the number of CD8 + T cells,cathepsin B expression and muscular cell apoptosis index were all significantly higher in the polymyositis model group,pseudo-intervention group,γ-interferon group and CA-074 Me group than in the healthy control group (all P ＜ 0.05).Compared with the pseudo-intervention group,the CA-074 Me group showed less CD8+ T cells (42.3 ± 27.4 vs.68.0 ± 13.2,P ＜ 0.05) and lower expression of cathepsin B (31.3 ± 6.7 vs.37.5 ± 9.2,P ＜ 0.05),whereas the γ-interferon group exhibited elevated cathepsin B expression (49.3 ± 17.0 vs.37.5 ± 9.2,P＜ 0.05) and apoptosis index (40.1 ± 6.7 vs.25.4 ± 5.0,P＜ 0.05).Conclusions Cathepsin B is highly expressed in the guinea pig model of polymyositis,while CA-074 Me may protect muscle tissue in this model by downregulating the expression of cathepsin B and attenuating the inflammation and apoptosis induced by cathepsin B.