Neutrophil elastase:From mechanisms to therapeutic potential / 药物分析学报

Neutrophil elastase(NE),a major protease in the primary granules of neutrophils,is involved in microbicidal activity.NE is an important factor promoting inflammation,has bactericidal effects,and shortens the inflammatory process.NE also regulates tumor growth by promoting metastasis and tumor microenvironment remodeling.However,NE plays a role in killing tumors under certain conditions and promotes other diseases such as pulmonary ventilation dysfunction.Additionally,it plays a complex role in various physiological processes and mediates several diseases.Sivelestat,a specific NE inhibitor,has strong potential for clinical application,particularly in the treatment of coronavirus disease 2019(COVID-19).This review discusses the pathophysiological processes associated with NE and the potential clinical applications of sivelestat.

Research progress on the development of human neutrophil elastase inhibitors / 药学学报

Human neutrophil elastase (hNE) is a serine proteolytic enzyme mainly distributed in neutrophils. When the balance between anti-hNE protein and hNE is broken, excessive release of hNE can cause the occurrence of various diseases. Therefore, inhibition of hNE is a promising therapeutic strategy. In this paper, the structure, action mechanism, physiological function of hNE and the development of hNE inhibitors were briefly summarized, in order to provide information for the related research.

Inhibition of lead-induced hepatic inflammation by inhibiting neutrophil granule protein in mice / 中国职业医学

Objective To explore the beneficial effects and mechanisms of neutrophil elastase (NE) and myeloperoxidase (MPO) on lead-induced hepatic inflammation in mice. Methods The specific pathogen free male C57BL/6 mice were randomly divided into four groups： control group, lead-exposed group, NE inhibitor group, and MPO inhibitor group, with three mice in each group. The mice in lead-exposed group, NE inhibitor group, and MPO inhibitor group were intraperitoneally injected with a dose of 10 mg/kg body mass of lead acetate solution, while the mice of control group received an equal volume of 0.9% saline three times per week for four weeks. In the last seven days, mice in both inhibitor groups were intraperitoneally injected with a dose of 40 mg/kg NE inhibitor sivelestat sodium or MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) once per day. Mouse body weight and liver histopathological changes were observed. The mRNA expression of genes associated with inflammation, such as tumor necrosis factor-α (Tnfa), interleukin-1β (Il1b), interleukin-6 (Il6), and nucleotide-binding oligomerization domain-like receptor protein 3(Nlrp3), apoptosis-associated speck-like protein (Asc) and cysteinyl aspartate specific proteinase (Caspase1) in the mouse liver tissues was detected by real-time quantitative polymerase chain reaction. The protein expression of NLRP3, ASC, and CASPASE-1 was detected using Western blotting. Results The activities of mice in all four groups were generally normal, and there was no significant difference in body weight (P>0.05). The results of hematoxylin-eosin staining showed that the cell size of hepatocytes varied in the lead-exposed mice, with indistinct cell boundaries, indicating early inflammatory responses in liver tissues. After intervention with NE or MPO inhibitors, the early inflammatory responses improved in the liver tissues of the mice in both inhibitor groups, with a better improvement observed in MPO inhibitor group compared with the NE inhibitor group. The mRNA expression of Tnfa, Il1b, Il6, Nlrp3, Asc, and Caspase1, as well as the protein expression of ASC, and CASPASE-1 in the livers of mice in the lead-exposed group was higher compared with those in the control group (all P<0.05). Compared with the lead-exposed group, the relative mRNA expression of Tnfa, Il1b, Il6, Nlrp3 and Asc was decreased in the liver tissues of mice in the NE inhibitor group (all P<0.05), while the relative expression of mRNA of Tnfa, Il1b, Il6, Caspase1 and the protein expression of ASC and CASPASE-1 were decreased in the liver tissues of mice in the MPO inhibitor group (all P<0.05). Conclusion Lead induce hepatic inflammation in mice by activating NLRP3 inflammasome. The inhibition of NE or MPO improve the lead-induced hepatic inflammatory responses in mice by alleviating NLRP3 inflammasome activation.

Selective decrease in alpha1-antitrypsin levels in diabetic retinopathy: Could the levels of it be playing a role in the pathophysiology of diabetic retinopathy?

Background & objectives: Type 2 diabetes mellitus (T2DM) is known to induce inflammation and activation of neutrophils causing the release of neutrophil elastase (NE), a pro-inflammatory proteinase. The activity of NE is regulated by endogenous inhibitors alpha1-antitrypsin (?1-AT) and alpha2- macroglobulin (?2-MG). Disrupted proteolytic homeostasis in T2DM patients is one of the causes for vascular complications. This study was carried out for evaluating the levels of plasma NE, ?1-AT, ?2-MG and NE-?1-AT complex to understand their roles in the pathophysiology of diabetic nephropathy (DN) and diabetic retinopathy (DR). Methods: A total of 240 participants (Control, n=60; T2DM, n=60; DN, n=60; and DR, n=60) were recruited after recording history, clinical examination and laboratory investigations. Retinopathy was confirmed by fundoscopy and nephropathy by urinary albumin excretion and serum creatinine levels. NE was measured using STANA. ?1-AT, ?2-MG and NE-?1-AT complex were estimated by ELISA. Results: Baseline clinical and laboratory findings were confirmatory to the study groups. The mean elastase activity was higher (P<0.0005) in diabetes groups (T2DM=0.73±0.31, DN=0.87±0.35, DR=0.76±0.41) than controls (0.35±0.20). The levels of ?1-AT were lower in DR (8.77±2.85) than DN (26.26±6.16) and T2DM (41.13±14.06) when juxtaposed with controls (122.95±25.71). The approximate fold decrease of ?1-AT levels was 15 for DR and four for DN compared to controls. The levels of ?2-MG were lowered in T2DM (167.29±30.45), DN (144.66±13.72), and DR (104.67±11.47) than controls (208.87±31.16). The NE-?1-AT complex levels were: controls (215.83±13.61), T2DM (98.85±23.85), DN (129.26±20.40) and DR (153.25±17.11). Interpretation & conclusions: Homeostasis of NE, ?1-AT and ?2-MG is disrupted in T2DM, DN and DR. Strikingly reduced levels of ?1-AT observed in DR are indicative of its possible role in the pathophysiology of retinopathy and emphasizes ?1-AT as a plausible therapeutic target.

Mutational Spectrum of the CFTR Gene in the Kazakhstan Population

Objective: To study the frequency and spectrum of CFTR gene variants in different ethnic groups of Kazakhstan. Methods: We reviewed the records of 58 patients with cystic fibrosis. All the patients underwent molecular genetic analysis to reveal genotype-phenotype correlations. Results: The median (IQR) age of the patients was 5.4 year (7 months, 18 year); 40% were diagnosed at the age of 5-10 year. The study identified 28 specific variants: p.Phe508del, the variant most common in the European population, was detected in 30 patients (51.7%). Variants other than p.Phe508del were revealed in 31% (21 patients). Conclusions: We found a number of specific variants characteristic of the Kazakhstani population. A pronounced regression of disease symptoms was detected in patients with mild mutations; whereas in patients with severe mutations, therapy produced very little effect.

Research progress of effect of neutrophil elastase and its inhibitors in sepsis / 中华危重病急救医学

Sepsis is a clinical syndrome of life-threatening organ dysfunction caused by infection. When an infection occurs, as the first line of defense of the body's immune system, neutrophils are first recruited to the site of infection to capture and kill pathogens by releasing neutrophil elastase (NE). However, a large amount of NE release will injury the surrounding normal tissues and induce organ dysfunction or failure. NE inhibitors can inhibit NE activity and reduce inflammatory response, which may be a promising drug for the treatment of sepsis. Currently, a variety of NE inhibitors have been developed and reported, but there is no systematic overview of their characteristics, and the role and underlying mechanisms of NE and related inhibitors in sepsis have not been thoroughly discussed. This article will make a review in this regard, in order to elucidate the effect of NE and its inhibitors in sepsis.

Efficacy of minocycline hydrochloride ointment combined with metronidazole film in the treatment of periodontitis and their effects on C-reactive protein and elastase levels in gingival crevicular fluid / 中国基层医药

Objective:To investigate the efficacy of minocycline hydrochloride ointment combined with metronidazole film in the treatment of periodontitis and their effects on C-reactive protein and elastase levels in the gingival crevicular fluid.Methods:76 patients with periodontitis who received treatment in Jiaxing Hospital of Traditional Chinese Medicine from May 2019 to January 2020 were included in this study. They were randomly allocated to undergo treatment with metronidazole film (control group, n = 38) or minocycline hydrochloride ointment plus metronidazole film (observation group, n = 38) for 4 weeks. We compared clinical efficacy, periodontal system examination indexes (gingival index, periodontal probing pocket depth, gingival bleeding index, plaque index, loss of attachment), gingival crevicular fluid biochemical markers (C-reactive protein, elastase in the pellet, elastase in the supernatant) measured before and after treatment, the incidence of adverse reactions, and the recurrence rate within half a year after treatment between the two groups. Results:The total response rate was significantly higher in the observation group than in the control group [97.37% (37/38) vs. 78.95% (30/38), χ2 = 6.17 , P < 0.05]. Gingival index, periodontal probing pocket depth, gingival bleeding index, plaque index, and loss of attachment measured after treatment were significantly lower in the observation group than in the control group (all P < 0.001). C-reactive protein, elastase in the pellet, and elastase in the supernatant measured after treatment were (5.31 ± 1.19) μg/L, (0.70 ± 0.20) Abs/mL, (0.48 ± 0.19) Abs/mL respectively in the observation group, which were significantly lower than those in the control group [(7.92 ± 1.27) μg/L, (1.15 ± 0.52) Abs/mL, (1.12 ± 0.31) Abs/mL, t = 9.24, 4.97, 10.85, all P < 0.001]. The recurrence rate within half a year in the observation group was significantly lower than that in the control group [2.63% (1/38) vs. 20% (6/38), χ2 = 3.93, P < 0.05]. There was no significant difference in the incidence of adverse reactions between the two groups ( P > 0.05). Conclusion:Minocycline hydrochloride ointment combined with metronidazole film is safe and effective in the treatment of periodontitis. The combined therapy help downregulate the levels of C-reactive protein, elastase in the pellet, elastase in the supernatant of the gingival crevicular fluid, alleviate inflammation, improve the periodontal status, and reduce the recurrence rate.

Impact of angiotensin-converting enzyme inhibition on hemodynamic and autonomic profile of elastase-2 knockout mice

Elastase-2 (ELA-2) is an angiotensin II-generating enzyme that participates in the cardiovascular system. ELA-2 is involved in hemodynamic and autonomic control and is upregulated in myocardial infarction and hypertension. The inhibition of angiotensin-converting enzyme (ACE) increased ELA-2 expression in the carotid arteries and heart of spontaneously hypertensive rats. In this study, we sought to investigate the role of ACE inhibition in hemodynamic and autonomic balance in elastase-2 knockout (ELA-2 KO) mice. Male ELA-2 KO and C57BL/6 mice were treated with the ACE inhibitor enalapril or saline for 10 days. After treatment, mice underwent surgery for cannulation of the femoral artery and arterial pressure recordings were made five days later in awake animals. The variability of systolic blood pressure (SBP) and pulse interval (PI) was evaluated in the time and frequency domain. Spontaneous baroreflex was assessed by the sequencing method. ACE inhibition caused a significant decrease in mean arterial pressure (117±2.2 vs 100±2.8 mmHg) and an increase in heart rate (570±32 vs 655±15 bpm) in ELA-2 KO mice. Despite a tendency towards reduction in the overall heart rate variability (standard deviation of successive values: 7.6±1.1 vs 4.7±0.6 ms, P=0.08), no changes were found in the root of the mean sum of squares or in the power of the high-frequency band. ACE inhibition did not change the spontaneous baroreflex indices (gain and baroreflex effectiveness index) in ELA-2 KO mice. Altogether, this data suggested that ACE played a role in the maintenance of hemodynamic function in ELA-2 KO mice.

Efficacy and safety of sivelestat in patients with acute lung injury in the intensive care unit / 中华急诊医学杂志

Objective:To explore the efficacy and safety of sivelestat, a neutrophil elastase (NE) inhibitor, in the treatment of acute lung injury (ALI) in the intensive care unit (ICU).Methods:A retrospective analysis was performed on 171 patients with ALI in the ICU of the First Affiliated Hospital of Zhengzhou University from June 2020 to June 2021, including 77 patients in the sivelestat group and 94 patients in the conventional treatment group. Acute physiology and chronic health evaluation (APACHE) Ⅱ score, Murray lung injury score, oxygenation index (PaO 2/FiO 2 ratio), inflammatory cytokines (IL-6, IL-10, TNF-α), ventilator-free days (VFD), the length of ICU stay, and the 28-day mortality were collected to assess the efficacy of sivelestat. At the same time, adverse reactions and laboratory test results within 30 days after the use of sivelestat were recorded to assess the safety. Results:Compared with conventional treatment, oxygenation index, Murray lung injury scores, IL-6, IL-10, and TNF-α were significantly improved after 7 days of sivelestat treatment. Compared with the conventional treatment group, the VFD was significantly longer ( P = 0.0119) and the length of ICU stay was significantly shorter ( P = 0.0269) in the sivelestat group. The mortality was 14.29% in the sivelestat group and 22.34% in the conventional treatment group and, with no statistically significant. In the meantime, sivelestat did not increase adverse reactions within 30 days after treatment. Conclusions:Sivelestat treatment is safe and more effective than conventional treatment for ALI patients in the ICU.

The study of the mechanism related to abdominal aortic aneurysm formation in elastase-induced aneurysm mice model / 中华胸心血管外科杂志

Objective:To establish a mouse model of the abdominal aortic aneurysm by elastase perfusion and to provide a reference for the study of the mechanism related to abdominal aortic aneurysm formation.Methods:AAAs were induced by porcine pancreatic elastase (PPE) infusion in male C57BL/6 mice. The control group was perfused with normal saline (Saline). The changes in abdominal aortic diameter were compared at 14 days after perfusion. The diameter of the abdominal aorta stained with HE was measured. The destruction of the elastic plate in the abdominal aortic wall was observed by elastic plate staining. TUNEL assay was used to evaluate the apoptosis in aneurysm tissues.Results:Porcine pancreatic elastase (PPE) perfusion successfully established the mouse model of abdominal aortic aneurysm, in which an aneurysm formation rate was 100% at 14 days after the operation. After modelling, the abdominal aorta diameter in the mouse was significantly increased, higher than that in the control group perfused with normal saline ( P<0.05). In the PPE group, the elastic plate of the aortic wall was straightened and thinned, and interrupted. The proportion of TUNEL-positive cells in the PPE group was significantly higher than that in the control group perfused with normal saline ( P<0.05). Conclusion:Elastase perfusion can stably establish the abdominal aortic aneurysm model, and we observe the destruction of the elastic plate in the medial layer of the abdominal aortic wall and the up-regulation of the apoptosis process in the model. It provides a reference to study the pathogenesis of abdominal aortic aneurysm further.

Elastase injection into the cerebellomedullary cistern to induce a model of vertebrobasilar dolichoectasia in mice and evaluation of magnetic resonance angiography / 国际脑血管病杂志

Objective:To investigate the effect of magnetic resonance angiography (MRA) in evaluating the mouse model of vertebrobasilar dolichoectasia (VBD) induced by injection of elastase into cerebellomedullary cistern.Methods:Twenty-four male C57/BL6 mice were selected. The mice in the elastase group ( n=12) were injected in the cerebellomedullary cistern with 2.5 μl of phosphate buffer containing 25 mU elastase, and the mice in the saline control group ( n=12) were injected with the same volume of normal saline. MRA examination of the brains of living mice was performed 2 weeks after modeling. Successful modeling was defined as the basilar artery bending angle ≤170°, or the basilar artery bending length accounts for ≥10%, or the basilar artery deviated from the midline by more than 1 grade, or the percentage increase in artery diameter was ≥25%. Results:In the elastase group and the saline control group, 2 mice and 1 mouse did not wake up normally or died, respectively. The 11 surviving mice in the saline control group had no obvious vertebral artery and basilar artery abnormalities. The success rate of modeling in the 10 surviving mice in the elastase group was 80%, and the difference in the success rate between the two groups was statistically significant ( P<0.05). There were significant differences in mean basilar artery diameter (0.30 mm vs. 0.22 mm; P<0.05), mean basilar artery bending angle (115° vs. 170°; P<0.05), and proportion of mean basilar artery bending length (31% vs. 5%; P<0.05) of the surviving mice between the elastase group and the saline control group. Conclusion:MRA can better evaluate the mouse VBD model induced by elastase injection in the cerebellomedullary cistern.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis / 中国天然药物

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (K

An aqueous pomegranate peel extract (Punica granatum) protect against Elastase-induced pulmonary emphysema in Sprague Dawley rats model

We investigated the effect of Punica granatum peel aqueous extract (PGE), on pulmonary inflammation and alveolar degradation induced by intratracheal administration of Elastase in Sprague Dawley rats. Lung inflammation was induced in rats by intratracheal instillation of Elastase. On day 1 and 2, animals received an intraperitoneal injection of PGE (200 mg/mL), three hours later, they were intratracheally instilled with 25U/kg pancreatic porcine Elastase. Animals were sacrificed 7 days later. Bronchoalveolar lavage (BAL) were collected and cellularity, histology and mRNA expression of Monocyte chemotactic protein 1(MCP-1), Tumor Necrosis Factor-Alpha (TNF-α), Interleukin 6 (IL-6), and Matrix Metalloproteinase-2 (MMP-2) were studied. In addition, activity of TNF- α, IL-6 and MCP-1 on BAL were also analyzed by ELISA Kit. Elastase administration increased: BAL cellularity, neutrophils recruitment and BAL MCP1, IL-6 expressions. It also increased lung TNF-α, MCP-1, MMP-2 expressions, platelets recruitment, histological parameters at 7th day of elastase treatment. Intraperitoneal injection of 200 mg/kg of PGE reduced, significantly, BAL cellularity, and neutrophils recruitment. However, in animal treated with PGE, MCP-1, MMP-2 and IL-6 on day 7, were similar to the Sham group. Treatment with PGE (200 mg/ kg) also significantly reduced lung TNF-α, and MCP-1 expression. This study reveals that PGE Punica granatum protects against elastase lung inflammation and alveolar degradation induced in rats

Frey’s procedure: short term follow up results in terms of pain, endocrine and exocrine status, a tertiary care centre experience

Background: Chronic pancreatitis, a debilitating disease which affects the patient to an extent to render him bedridden sometimes and often make him/her unable to perform daily work, leading to poor quality of life, severe emotional stress and financial burden. Frey’s procedure one of the most commonly performed surgery in patient of chronic pancreatitis with stones in pancreatic duct and small inflammatory head mass. This procedure had shown promising results in terms of pain control with unclear outcome with respect to exocrine and endocrine status.Methods: Study was conducted in Government Kilpauk Medical College, Chennai during March 2016 to December 2019. Total 21 patients were followed up for 18 months and Post-operative pain was analysed using pain scoring system used by Bloechle et al. Exocrine status in terms of diarrhea, bloating sensation and Fecal Elastase level(preoperative and postoperative) were analysed. Patient glycaemic status were also analysed and compared. Patient Nutritional status was analysed in terms of weight gain.Results: In this study there was significant improvement in pain after surgery in terms of VAS pain Score (Preop Mean-82.14, Post-op Mean- 9.76, p<0.001) , frequency of pain (Preop Mean-78.57, Post-op Mean- 14.29, p<0.001), analgesia required (Preop Mean-15, Post-op Mean- 3.10, p<0.001), and inability to work due to pain (Preop Mean-80.95, Post-op Mean- 11.90, p<0.001). Overall total Pain score was significantly improved after surgery (Preop Mean-256.67, Post-op Mean- 39.05, p<0.001). There was significant weight gain and significant improvement in Fecal Elastase level in patients following surgery, however there was no significant difference in pancreatic exocrine status on the basis of symptoms.Conclusions: Frey’s procedure is very effective in pain control in properly selected patients of chronic calcific pancreatitis, and it also improves nutritional status of patient. Frey’s procedure might improve patient exocrine and endocrine status but further  multicentre studies are needed to come to a conclusion.

Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population

PURPOSE: Various immune cells, including eosinophils and neutrophils, are known to contribute to the development of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the current understanding of the role of neutrophils in the development of CRSwNP still remains unclear. Therefore, we investigated risk factors for refractoriness of CRSwNP in an Asian population. METHODS: Protein levels of 17 neutrophil-related mediators in nasal polyps (NPs) were determined by multiplex immunoassay, and exploratory factor analysis using principal component analysis was performed. Immunofluorescence analysis was conducted to detect human neutrophil elastase (HNE) or myeloperoxidase (MPO)-positive cells. Tissue eosinophilic nasal polyp (ENP) and tissue neutrophilia (Neu(high)) were defined as greater than 70 eosinophils and 20 HNE-positive cells, otherwise was classified into non-eosinophilic nasal polyp (NENP) and absence of tissue neutrophilia (Neu(low)). RESULTS: In terms of disease control status, NENP-Neu(low) patients showed the higher rate of disease control than NENP-Neu(high) and ENP-Neu(high) patients. Linear by linear association demonstrated the trend in refractoriness from NENP-Neu(low) to NENP-Neu(high) or ENP-Neu(low) to ENP-Neu(high). When multiple logistic regression was performed, tissue neutrophilia (hazard ratio, 4.38; 95% confidence interval, 1.76-10.85) was found as the strongest risk factor for CRSwNP refractoriness. Additionally, exploratory factor analysis revealed that interleukin (IL)-18, interferon-γ, IL-1Ra, tumor necrosis factor-α, oncostatin M, and MPO were associated with good disease control status, whereas IL-36α and IL-1α were associated with refractory disease control status. In subgroup analysis, HNE-positive cells and IL-36α were significantly upregulated in the refractory group (P = 0.0132 and P = 0.0395, respectively), whereas MPO and IL-18 showed higher expression in the controlled group (P = 0.0002 and P = 0.0009, respectively). Moreover, immunofluorescence analysis revealed that IL-36R⁺HNE⁺-double positive cells were significantly increased in the refractory group compared to the control group. We also found that the ratio of HNE-positive cells to α1 anti-trypsin was increased in the refractory group. CONCLUSIONS: Tissue neutrophilia had an influence on treatment outcomes in the Asian CRSwNP patients. HNE-positive cells and IL-36α may be biomarkers for predicting refractoriness in Asians with CRSwNP. Additionally, imbalances in HNE and α1 anti-trypsin may be associated with pathophysiology of neutrophilic chronic rhinosinusitis.

Establishment of a novel mouse mode of elastase-induced chronic obstructive pulmonary disease related osteoporosis / 中国骨伤

OBJECTIVE@#To establish and evaluate the model of chronic obstructive pulmonary disease (COPD) with osteoporosis induced by elastase in mice.@*METHODS@#Twenty four healthy female 8-week-old C57BL / 6 mice (weighing about 18 g) were randomly divided into three groups. The control group was given intratracheal drip of normal saline, the experimental group 1 and the experimental group 2 were given intratracheal drip of elastase, the control group and the experimental group 1 were kept for 8 weeks and then killed, the experimental group 2 was kept for 12 weeks and then killed. HE staining was used to evaluate the histopathological changes of lung and tibia in the control and experimental groups. The levels of serum inflammatory factors and broncho alveolar lavage factors (BALF) were detected by ELISA. Micro CT was used to detect the bone mass related parameters of mouse femur. The expression of osteoclastic and osteogenic genes was detected by real-time fluorescence quantitative PCR.@*RESULTS@#Lung histopathology showed that the structure of alveoli in the experimental group was disordered, the walls of alveoli became thin or broken, and the alveoli cavity expanded. IL-6 and TNF-α in BALF were significantly higher than those in control group (<0.001), while IL-1β and TNF-α in serum inflammatory factors were significantly higher than those in control group (<0.001). BV / TV(bone volume fraction), TB.Th(average bone trabecular thickness) and TB.N(average bone trabecular number) in the experimental group were significantly lower than those in the control group (<0.05), TB.Sp (average bone trabecular separation) and BS / BV (bone surface area fraction) in the experimental group were significantly higher than those in the control group (<0.01). Compared with the control group, the expression of osteoclast related marker genes increased in the experimental group (<0.05), but decreased in the experimental group(<0.05). The results of experiment 1 and experiment 2 were time-dependent.@*CONCLUSION@#In this study, elastase was used to construct a COPD model with osteoporosis successfully, which provides a suitable animal model for the future study of the pathogenesis of COPD with osteoporosis.

Relação entre genótipo e insuficiência pancreática em crianças e adolescentes com fibrose cística de um centro de referência no Brasil / Relationship between genotype and pancreatic insufficiency in children and adolescents with cystic fibrosis in a referral center in Brazil

Introdução: fibrose cística (FC) é uma doença genética que culmina em alterações na proteína transmembrana CFTR, resultando na produção de muco mais espesso em diversos órgãos, destacando-se os tratos respiratório e digestório. A insuficiência pancreática (IP) acomete até 95% dos pacientes com FC. Objetivos: determinar a frequência de IP através da dosagem de elastase fecal-1 (EF-1) e compará-la com o genótipo de pacientes com FC assistidos em um centro de referência. Metodologia: foi realizado um estudo transversal, incluindo-se pacientes com FC de 0 a 20 anos. Após a inclusão dos sujeitos à pesquisa, foi realizada consulta ao prontuário para a obtenção de dados clinicos e demograficos e amostras de fezes foram obtidas para dosagem da (EF-1). Os pacientes foram submetidos à análise molecular das mutações por métodos convencionais, através da extração do DNA em sangue periférico. Quando duas mutações patológicas não foram identificadas, o sequenciamento de nova geração com utilização da plataforma Illumina HiSeq foi realizado em amostras da mucosa oral. Resultados: foram incluídos 31 pacientes, 17 (54,8%) do sexo feminino, mediana de idade de 10 anos, e apenas um paciente foi classificado como branco. Vinte e dois (70,9%) pacientes apresentaram dosagem de EF-1 inferior a 200 µg/g, compatível com o diagnóstico de IP. Destes, 21 (95,4%) apresentaram dosagem de EF-1 menor ou igual a 15µg/g, caracteristica de IP grave. Todos os pacientes com IP apresentavam duas mutações de classes I a III. Conclusão: a IP foi identificada em 70% dos pacientes, ocorrendo em todos os pacientes com duas mutações de classe I-III.

Introduction: cystic fibrosis (CF) is a genetic disease that culminates in alterations in the CFTR transmembrane protein, resulting in the production of thicker mucus in various organs, especially the respiratory and digestive tract. Pancreatic insufficiency (PI) affects up to 95% of CF patients. Objectives: To determine the frequency of PI by measuring fecal elastase-1 (FE-1) and comparing it with the genotype of CF patients assisted at a referral Center. Methodology: a cross-sectional study was conducted, including patients with CF from 0 to 20 years. After the inclusion of the subjects to the research, medical records were consulted to obtain clinical and demographic data and stool samples were obtained for the measurement of (FE-1). Patients were submitted to molecular analysis of mutations by conventional methods by DNA extraction in peripheral blood. When two pathological mutations were not identified, next-generation sequencing using the Illumina HiSeq platform was performed on oral mucosa samples. Results: thirty one patients were included, 17 (54.8%) female, median age 10 years, and only one patient was classified as white. Twenty-two (70.9%) patients had an FE-1 dosage of less than 200 µg / g, compatible with the diagnosis of pancreatic insufficiency (PI). Of these, 21 (95,4%) had an EF-1 dosage less than or equal to 15µg / g, characteristic of severe PI. All patients with two mutations class I to III were PI. Conclusion: PI was identified in 70% of patients, occurring in all patients with class I-III mutations.

Isolation of a putative virulence agent, cytotoxic serine-elastase,from a newly isolated Pseudomonas aeruginosa ZuhP13

A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39%recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and40C. It showed stability at pH 6–9 for 1 h and up to 60C for 30 min with midpoint temperature (Tm) at 61.3Cand isoelectric value (pI) at 5.6±0.2. Its Km and catalytic efficiency (Kcat/Km) for the substrate azocasein were 1.3 mg/mLand 4.629107 M-1s-1, respectively. On contrary to most P. aeruginosa proteases, Zn2?, EDTA, 2,20-bipyridine ando-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitorII) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Takentogether, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic andhemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformedhepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.

Effect of Tangnaikang on Imbalance Between NE and α1-AT in Spontaneous Type 2 Diabetic Mouse / 中国实验方剂学杂志

Objective: To observe the regulatory effect of Tangnaikang (TNK) on imbalance between neutrophil elastase (NE) and α1-antitrypsin (α1-AT) in ob/ob mice with type 2 diabetes mellitus (T2DM). Method: Thirty-two male SPF ob/ob mice were randomly divided into model group (DM, normal saline) and high-dose TNK group (TNKH, TNK solution 16.04 g·kg-1), middle-dose TNK group (TNKM, TNK solution 8.02 g·kg-1) and low-dose TNK group (TNKL, TNK solution 4.01 g·kg-1). Another 8 C57BL/6J mice were included in normal group (Con, saline). The experiment lasted for four weeks. The general state, body weight (BW) and fasting blood glucose (FBG) of the mice were recorded weekly, the oral glucose tolerance (OGTT) test was performed on the 25th day, the insulin tolerance (ITT) test was performed on the 27th day, and the area under the curve (AUC) was calculated. After the end of the experiment, serum was used to detect the level of fasting insulin (Fins), insulin resistance index (HOMA-IR), total triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), NE and α1-AT. Adipose tissue was used to detect the expressions of NE, α1-AT, phosphor-insulin receptor substrate 1 antibody (p-IRS1) and glucose transporter 4 (GLUT4) proteins. Result: Compared with the Con group, the BW of the ob/ob mice of the model group increased significantly, the glucose and lipid metabolism indexes showed diabetes, the serum and adipose tissue NE increased significantly (Pα1-AT decreased significantly (PPPogtt and AUCITT were significantly decreased (PPα1-AT increased significantly (PPConclusion: TNK can reduce the BW of ob/ob mice, improve glycolipid metabolism, increase α1-AT level, decrease NE level, and regulate IRS1-GLUT4 signaling pathway, which may be one of its mechanisms in improving IR of adipose tissue mediated by neutrophil.

Transrectal ultrasound combined with seminal plasma elastase in diagnosis of male infertility caused by chronic seminal vesiculitis / 中国医学影像技术

Objective: To investigate the value of transrectal ultrasound combined with seminal plasma elastase (SPE) in diagnosis of male infertility caused by chronic seminal vesiculitis (CSV). Methods Transrectal ultrasonography was performed in 106 patients with male infertility caused by CSV (infertility group) and 106 healthy men (control group), and SPE was detected in two groups. The differences of seminal vesicle ultrasound parameters and SPE were compared between two groups. The correlations between SPE and seminal vesicle parameters were analyzed. ROC curves were used to evaluate the diagnostic efficiency of SPE combined with seminal vesicle ultrasound parameters on male infertility caused by CSV. Results: The short diameter of seminal vesicle, inner diameter of gland tube, the peak systolic velocity (PSV), resistance index (RI) and SPE level in infertility group were higher than those in control group (all P<0.01). The short diameter of seminal vesicle, inner diameter of gland tube, PSV, RI and SPE were impact factors of male infertility caused by CSV (all P<0.05). SPE was positively correlated with PSV, RI and short diameter of seminal vesicle (r=0.732, P<0.001; r=0.647, P=0.004; r=0.526, P=0.007). SPE combined with PSV, RI and short diameter of seminal vesicle could be used to diagnose male infertility caused by CVS (all P<0.05), among which SPE combined with PSV had the most effective diagnostic value, the area under ROC was 0.73, the sensitivity and specificity was 0.73 and 0.70, respectively. Conclusion: Transrectal ultrasound combined with SPE have certain clinical diagnostic value for male infertility induced by CSV.