Human leukocyte elastase is an important selection target of inflammation and cancer. In this paper, a high throughput screening model was established for screening human leukocyte elastase inhibitors from thousands of strains of actinomycetes. As a result, a strain, N01WA-735 with potent suppression activity was isolated. Firstly, the strain N01WA-735 was identified as Streptomyces according to morphology and biochemical analysis. The Streptomyces N01WA-735 was processed by solvent extraction, silica column chromatography, Sephadex LH-20 column chromatography and crystallization to get a pure active compound named N01WA-735E. Its chemical structure was elucidated as the same as that of the compound named BE-52440A by physicochemical properties and spectral data of UV, MS, 1H-NMR and 13C-NMR respectively. The compound showed a strong inhibitory activity against human leukocyte elastase with IC50 of 3.02 micromol/L. The compound is reported as a human leukocyte elastase inhibitor for the first time.
Humans ; Leukocyte Elastase ; antagonists & inhibitors ; Protease Inhibitors ; isolation & purification ; metabolism ; Streptomyces ; isolation & purification ; metabolism

OBJECTIVETo investigate the effects of advanced glycation end products (AGE) on the biological behavior of neutrophils in vitro, to look for the relationship between accumulation of AGE and abnormal inflammation in wound healing in diabetic mellitus patients.

METHODSNeutrophils were isolated from SD rats and incubated in vitro. The cells were divided into four groups according to different concentrations of AGE in cell suspension: control group (C, with treatment of RPMI - 1640), A group (with treatment of 0.315 mg/mL AGE + RPMI - 1640), B group (with treatment of 0.625 mg/mL AGE + RPMI - 1640), D group (with treatment of 1.250 mg/mL AGE + RPMI - 1640). Activity of neutrophils were determined by MTT colorimetric assay. Selectin-L mRNA expressions were analyzed by reversible transcription polymerase chain reaction (RT -PCR) technique. The levels of reactive oxygen species (ROS) in neutrophils were measured with DCFH-DA method. The protein concentration of neutrophil elastase (NE) was assayed by ELISA.

RESULTSThe activity of neutrophils were obviously increased in A, B, and D groups when compared with that in C group [(0.170 +/- 0.040) in C group, (0.320 +/- 0.030) in A group, (0.380 +/- 0.020) in B group, (0.290 +/- 0.010) in D group, P <0. 05]. The expression of Selectin-L mRNA in A, B, D groups were significantly higher than that in C group (0.95 +/- 0.08, 1.36 +/- 0.27, 0.50 +/- 0.26.vs.0.36 +/- 0.26, P < 0.05. respectively). The ROS levels in A, B, D groups was markedly higher than that in C group (1.64 +/- 0.20, 2.16 +/- 0.26, 3.26 +/- 0.75. vs. 0.72 +/- 0.15, P <0.05, respectively). The levels of NE in A, B, D groups were significantly increased when compared with that in C group(1.98 +/- 0.43, 2.50 +/- 0.43, 2.01 +/- 0.18 vs 0.91 +/- 0. 21, P <0.05, respectively).

CONCLUSIONAGE can enhance the activity of neutrophil, with change in cellular biological behaviors, which may be one of main reasons for abnormal inflammation in wounds of diabetes mellitus patients.

Animals ; Cells, Cultured ; Glycation End Products, Advanced ; metabolism ; pharmacology ; L-Selectin ; metabolism ; Leukocyte Elastase ; metabolism ; Male ; Neutrophil Activation ; Neutrophils ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

OBJECTIVE: To explore the role in the pathogenesis of nasal polyp (NP) by comparison of eosinophil major basic protein (MBP), and neutrophil elastase (NE) in nasal polyps (NP) epithelium, stroma and the secretion of expression. METHOD: Immunohistochemical detection of 30 cases of patients with chronic sinusitis (CRS) NP epithelium NE stroma and the expression and secretion of MBP. RESULT: 1. There were significant differences of the expression of NE and MBP in epithelial tissue, stroma and secretion compared with the control group (P < 0.05); 2. There was not significant difference of the expression of NE and MBP between epithelial tissue and stroma (P > 0.05), while there was significant difference between epithelial tissue and the secretion (P < 0.05); 3. There were significant differences of the average positive expression of MBP and NE among epithelial tissue, stroma and secretion (P < 0.05); 4. MBP and NE were usually degranulated in secretion, while usually located in eosinophils (Eos) and neutrophils (Neu) in epithelial and mesenchymal; 5. There were abundant expression of MBP and NE in epithelial shedding regional, while small amounts of expression in stroma and integrated epithelial; 6. Electron microscopy could show the characteristics of electron density of MBP and NE particles. CONCLUSION MBP and NE collaborated to cause pathological effects on the occurrence of NP.
Adolescent ; Adult ; Aged ; Blood Proteins ; metabolism ; Bodily Secretions ; metabolism ; Eosinophil Major Basic Protein ; Eosinophils ; metabolism ; Female ; Humans ; Leukocyte Count ; Leukocyte Elastase ; metabolism ; Male ; Middle Aged ; Nasal Polyps ; metabolism ; pathology ; Proteoglycans ; metabolism ; Sinusitis ; metabolism ; pathology ; Young Adult

OBJECTIVETo observe the protective effect of ischemic postconditioning on ischemic reperfusion injury of rat liver graft and to investigate the possible mechanism.

METHODSMale Sprague Dawley rats were used as donors and recipients of orthotopic liver transplantation, and the period of cold preservation and anhepatic phase were 100 min and 18 min, respectively. Sixty rats were randomly divided into three groups, twelve rats in control group, twenty-four rats in ischemic reperfusion injury group and ischemic postconditioning group respectively. Control group is sham operation group, only the ligaments around liver were cut off; donor livers in ischemic reperfusion injury group were infused through portal vein with heparinized saline before harvested; ischemic postconditioning group: at very onset of reperfusion after donor liver was implanted, several brief reperfusion-ischemia were given before persistent reperfusion of portal vein. Half recipients of ischemic reperfusion injury group and ischemic postconditioning group were taken blood samples and hepatic tissue samples after 2 hours of reperfusion of liver graft. Rest recipients were taken samples of hepatic tissue after 6 hours of reperfusion. Recipients of control group were taken blood and hepatic tissue samples at corresponding time after abdomen was sutured.

RESULTSCompared with ischemic reperfusion injury group, liver functional parameters, cytokines and peroxidized products contents were lower in ischemic postconditioning group (P < 0.05); meanwhile, the antioxidases contents of hepatic tissue were higher in ischemic postconditioning group than those in ischemic reperfusion injury group (P < 0.05).

CONCLUSIONSIschemic postconditioning could relieve the ischemic reperfusion injury of rat liver graft. Through improving antioxidation capability and cutting down cytokines contents, ischemic postconditioning could apply its protective effect.

Animals ; Glutathione Peroxidase ; metabolism ; Leukocyte Elastase ; blood ; Liver ; blood supply ; metabolism ; Liver Transplantation ; Male ; Malondialdehyde ; metabolism ; Peroxidase ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; prevention & control ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUND: Neutrophil elastase (NE) was found to increase the respiratory mucin gene, MUC5AC, although the molecular mechanisms of this process remain unknown. We attempted to determine the signal transduction pathway through which NE induces MUC5AC gene expression in bronchial epithelial cells. METHODS: A fragment of 1.3 Kb MUC5AC promoter which had been cloned into the pGL3-Basic luciferase vector was transfected to the A549 cells. By measuring the luciferase activity, we were able to evaluate the MUC5AC promoter activity in A549 cells. The involvement of mitogen-activated protein kinases (MAPK) was confirmed by Western blotting. To confirm the involvement of nuclear factor kappaB (NF-kB), we used site-directed mutagenesis and electrophoretic mobility shift assay (EMSA) autoradiogram. The MUC5AC mRNA expression was confirmed by RT-PCR. RESULTS: NE increased the transcriptional activity of the MUC5AC promoter in A549 cells. The increased transcriptional activity of the MUC5AC promoter by NE was found to be associated with increased NF-kB activity. Site-directed mutagenesis showed that the transfection of the mutated NF-kB binding sites from the PGL3-MUC5AC-3752 promoter luciferase reporter plasmid decreased the luciferase activity after NE stimulation. Among the MAPKs, only extracellular signal-regulated kinases (ERK) were involved in this NE-induced MUC5AC mucin expression. RT-PCR also showed that NE increased MUC5AC mRNA. An EMSA autoradiogram revealed that NE induced NF-kB: DNA binding. CONCLUSIONS: These results indicate that human NE induces MUC5AC mucin through the epidermal growth factor receptor (EGF-R), ERK, and NF-kB pathways in A549 cells.
Transcription, Genetic ; Signal Transduction ; Receptor, Epidermal Growth Factor/*metabolism ; NF-kappa B/*metabolism ; Mucins/*biosynthesis/genetics ; Leukocyte Elastase/*metabolism ; Humans ; Gene Expression Regulation ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Epithelial Cells ; Cell Line, Tumor ; Bronchi/cytology

AIMTo investigate age-related inflammatory events in the male genital tract.

METHODSIn a total of 4265 randomly collected patients attending the andrological outpatient clinic of the Center for Dermatology and Andrology, University of Giessen, Germany, ejaculate volume, pH-value, sperm concentration, total and progressive sperm motility, concentration of polymorphonuclear (PMN) elastase, number of peroxidase-positive cells and fructose were measured and correlated with patient's age.

RESULTSWhile ejaculate volume, motility and fructose all correlated negatively with age, sperm concentration, PMN elastase and the pH-value showed a positive correlation. The prevalence of male genital tract inflammation (as defined by PMN elastase > 250 ng/mL) and its severity increased significantly. PMN elastase did not correlate with sperm motility. Fructose as a marker of seminal vesicle function showed a significant negative relationship with the PMN elastase levels, the number of peroxidase-positive cells and sperm motility.

CONCLUSIONThe significant increases of PMN-elastase levels as marker of male genital tract inflammation in older men appear to be indicative of age-related changes in local immunoregulatory mechanisms. Because there is no association of PMN elastase with sperm motility, a direct inhibitory effect of this enzyme can be excluded.

Adolescent ; Adult ; Aged ; Aging ; physiology ; Biomarkers ; metabolism ; Ejaculation ; Genital Diseases, Male ; enzymology ; pathology ; Humans ; Infertility, Male ; enzymology ; pathology ; Inflammation ; enzymology ; pathology ; Leukocyte Elastase ; metabolism ; Male ; Middle Aged ; Retrospective Studies ; Semen ; cytology ; enzymology ; physiology ; Sperm Count

OBJECTIVETo investigate the effect of Ulinastatin on the management of early myocardial injury and its mechanisms.

METHODSThirty-four severe burn patients with TBSA exceeding 50% were admitted into our hospital within 24 hrs after burns, and they were divided into burn group (n=17) and ulinastatin-treated group (n=17, UTI group). All patients received conventional treatment. The patients in UTI group were given 100,000 U ulinastatin intravenously immediately after admission, 3 times a day for a week. The plasma content of troponin I (cTnI) , creatine kinase (CK-MB) and PMN elastase were determined on 2, 4 and 7 postburn days (PBD), and the correlate relationship among these three indices were analyzed.

RESULTS(1) The plasma content of cTnI and PMN elastase at 2, 4, 7 PBDs were significantly higher than that of normal value (P < 0.01), but they were obviously lower in UTI group than that in burn group. (2) Compared with normal value, the plasma content of CK-MB in burn group was increased significantly on 2, 4 and 7 PBDs (P < 0.01), and it reached the peak on 4 PBD. Though it was obviously higher in UTS group on 2 and 4 PBDs compared with the normal value, but it was lower than that in burn group ( P < 0.05 or 0.01) , and it returned to normal value on 7 PBD. (3) There exhibited positive correlation among the PMN elastase content, cTnI content and CK-MB activity of the 34 patients. The correlation index of PMN elastase content and cTnI content was 0. 904, while that between cTnI content and CK-MB activity was 0.922, and that between PMN elastase content and CK-MB activity was 0.829 (P < 0.01).

CONCLUSIONUlinastatin is beneficial in alleviating myocardial injury after severe burns, and it reduces the release of PMN elastase.

Adult ; Burns ; blood ; complications ; drug therapy ; Creatine Kinase ; blood ; Female ; Glycoproteins ; therapeutic use ; Humans ; Leukocyte Elastase ; blood ; Male ; Myocardial Reperfusion Injury ; blood ; etiology ; prevention & control ; Myocardium ; metabolism ; pathology ; Troponin T ; blood

PURPOSE: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). METHODS: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. RESULTS: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. CONCLUSION Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways.
Animals ; Cadherins ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation Mediators ; metabolism ; Injections, Intralesional ; Injections, Intravenous ; Intestinal Diseases ; drug therapy ; etiology ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; Leukocyte Elastase ; metabolism ; Male ; Mucin-2 ; metabolism ; Rats, Wistar ; Sepsis ; complications ; Trypsin ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

Scutellarin is a flavonoid extracted from a traditional Chinese herb, Erigeron breviscapus. The present study investigated the effect of scutellarin on MUC5AC mucin production and the possible mechanism. Human bronchial epithelial 16 (HBE16) cells were pretreated with scutellarin for 60 min, and then exposed to human neutrophil elastase (HNE) or interleukin (IL)-13 for 12 hr. RT-PCR and ELISA were performed to measure the amount of MUC5AC mucin production. The results showed that scutellarin inhibited MUC5AC expression both in mRNA and protein level induced by HNE in a concentration-dependent manner. However, scutellarin failed to inhibit MUC5AC mucin production induced by IL-13. To investigate the intracellular mechanisms associated with the effect of scutellarin on MUC5AC mucin production, western blotting was carried out to examine the phosphorylation of protein kinase C (PKC), signal transducer and activator of transcription 6 (STAT6) and extracellular signal-regulated kinase 1/2 (ERK1/2). The phosphorylation of PKC and ERK1/2 was attenuated after treatment with scutellarin, whereas STAT6 was not significantly affected. Therefore, it is suggested that scutellarin down-regulates MUC5AC mucin production on HBE16 cells via ERK-dependent and PKC-dependent pathways.
Apigenin/chemistry/*pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Down-Regulation ; Epithelial Cells/*drug effects/metabolism ; Erigeron/chemistry ; Glucuronic Acids/chemistry/*pharmacology ; Humans ; Interleukin-13/*pharmacology ; Leukocyte Elastase/*pharmacology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Mucin 5AC/genetics/*metabolism ; Phosphorylation ; Protein Kinase C/metabolism ; Respiratory Mucosa/drug effects/*metabolism ; STAT6 Transcription Factor/metabolism ; Signal Transduction

OBJECTIVE: To investigate the effect of glycyrrhizin (Gly) on human neutrophil elastase (HNE)- induced mucin (MUC) 5AC overproduction in human bronchial epithelial cells (16HBE), and the potential signaling pathway involved in this process. METHODS: The cultured cells were divided into 3 groups: a control group, cultured in serum-free DMEM medium; an HNE group, pretreated with HNE alone; and a Gly group, incubated with HNE and Gly. After stimulation with a variety of Gly concentrations, the cytotoxicity was assessed by methyl thiazolyl tetrazolium method. The mRNA expressions of p38, nuclear factor κB (NF-κB) p65, inhibitory κBα (IκBα) and MUC5AC were detected by real-time PCR. The phosphorylation levels of p38 (p-p38), NF-κB p65 (p-NF-κB p65) and IκBα (p-IκBα) were measured by Western blot while the levels of MUC5AC protein were analyzed by emzyme-linked immunosorbent assay and immunofluorescence. RESULTS: Compared with the control group, the expression levels of MUC5AC mRNA and protein in the HNE group were both significantly increased. There was a significant increase in p-p38 and p-NF-κB p65, while the production of IκBα was much lower than that in the control group. Gly significantly inhibited the increase of MUC5AC, p38 and NF-κB p65, but increased the activity of IκBα. CONCLUSION Glycyrrhizin can inhibit MUC5AC overproduction via p38-NF-κB p65/IκBα signaling pathway.
Bronchi ; cytology ; Cell Line ; Epithelial Cells ; metabolism ; Glycyrrhizic Acid ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Leukocyte Elastase ; metabolism ; Mucin 5AC ; biosynthesis ; NF-KappaB Inhibitor alpha ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcription Factor RelA ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism