OBJECTIVETo investigate whether or not lipopolysaccharide (LPS) and ovalbumin (OA) prime rat peripheral leukocytes, the effect of sensitization on priming and the role of phospholipase D in priming.

METHODSThe peripheral leukocytes were separated and purified from sensitized or unsensitized rats. LPS or OA was used as a priming agent and formylmethionylphenylalanine (fMLP) as an activating agent. Degradation of leukocyte was determined by measurement of elastase release and myeloperoxidase (MPO) activity. Phospholipase D (PLD) activity was assayed by the generation of choline,which was measured by choline-oxidase-catalyzed formation of H(2)O(2) and Trinder reaction.

RESULTCompared with cells treated by fMLP alone,leukocytes from unsensitized rat challenged with fMLP after incubated with LPS released more elastase and MPO (P<0.05). But there was no significant difference between leukocytes challenged with fMLP after incubated with OA and fMLP treated alone. In sensitized rat,there was no difference between leukocytes challenged with fMLP after incubated with LPS and fMLP treated alone. But leukocytes challenged with fMLP after incubated with OA released significantly more elastase and MPO than fMLP treated alone (P<0.05). A significant correlation was obtained between the release of elastase and PLD activity (r(s)=0.51,P<0.01), and also between the release of MPO and PLD activity (r(s)=0.73,P<0.01) in unsensitized rat. In sensitized rat, it was 0.48 (P<0.01) and 0.37 (P<0.05) respectively.

CONCLUSION(1) LPS primes peripheral leukocytes from unsensitized rats; (2) OA primes peripheral leukocytes from actively sensitized rats; (3) PLD plays a role in priming of rat peripheral leukocytes.

Animals ; Leukocyte Elastase ; secretion ; Leukocytes ; drug effects ; enzymology ; Lipopolysaccharides ; pharmacology ; Male ; N-Formylmethionine Leucyl-Phenylalanine ; pharmacology ; Ovalbumin ; immunology ; Peroxidase ; blood ; Phospholipase D ; physiology ; Rats ; Rats, Sprague-Dawley

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.
Anti-Inflammatory Agents, Non-Steroidal/pharmacology* ; Chromatography, Affinity ; Computer Simulation ; Enzyme Inhibitors/pharmacology ; Human ; Isoenzymes/isolation & purification ; Isoenzymes/antagonists & inhibitors ; Ketoprofen/pharmacology ; Leukocyte Elastase/isolation & purification ; Leukocyte Elastase/antagonists & inhibitors* ; Models, Molecular ; Naproxen/pharmacology ; Phenylbutazone/analogs & derivatives ; Salicylates/pharmacology ; Spectrum Analysis, Raman

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

OBJECTIVETo study and compare the effect of neutrophil elastase inhibitors (GW311616A and sivelestat) on the proliferation and apoptosis of U937 cells.

METHODSInhibitory effects of GW311616A and sivelestat on the proliferation of U937 cells were assayed by MTT assay. The morphologic changes of U937 cells were detected by transmission electron microscope, and apoptosis was observed by AnnexinV-FITC/PI staining. The changes of cell cycle and apoptosis were detected by flow cytometry. The expression of NE in U937 cells was observed by indirect immunofluorescence, the variations of content and activity of NE in U937 cells were measured through ELISA assay and colorimetric method.

RESULTSMTT showed that both NE inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. The IC50 of GW311616A and sivelestat were 150 and 214 μmol/L respectively. The inhibition effect of GW311616A was significantly higher than of sivelestat (P<0.01). Typical apoptosis morphological changes of U937 cells was observed through electron microscope. AnnexinV-FITC/PI staining showed that U937 cells could be induced to undergo apoptosis by the two inhibitors, the apoptosis ratio of 150μmol/L GW311616A group (13.60%) was significantly higher than that of 150μmol/L sivelestat group (3.69%)(P<0.01). The result of flow cytometry indicated that the apoptosis ratio of 150 μmol/L GW311616A group was 14.61%, U937 cell cycle was mainly blocked in G2/M phase; meanwhile 150 μmol/L sivelestat group as 4.25% with cell cycle in S phase. The fluorescence intensity of GW311616A group obviously decreased than of sivelestat group. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than of sivelestat (P<0.01).

CONCLUSIONGW311616A and sivelestat could inhibit the proliferation and cause apoptosis of U937 cells. Furthermore, GW311616A was more effective and harmful to cells than sivelestat.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Glycine ; analogs & derivatives ; pharmacology ; Humans ; Leukocyte Elastase ; antagonists & inhibitors ; Piperidines ; pharmacology ; Proteinase Inhibitory Proteins, Secretory ; pharmacology ; Sulfonamides ; pharmacology ; U937 Cells

Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.
Animals ; Enzyme Inhibitors/*pharmacology ; Female ; Fibrin Tissue Adhesive/pharmacology ; Invertebrate Hormones/analysis/pharmacokinetics/*pharmacology ; Leukocyte Elastase/*antagonists & inhibitors ; Macrophages/immunology ; Mice ; Research Support, Non-U.S. Gov't ; Skin/drug effects/*injuries/pathology ; Tongue/drug effects/*injuries/pathology ; Wound Healing/*drug effects

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

To study the coumarins of Anemone raddeana Regel, the compounds were separated by silica gel column chromatography and HPLC. Their structures were identified by their physicochemical property and spectral analysis. Two new compounds were isolated and identified as 4, 7-dimethoxyl-5-methyl-6-hydroxy coumarin (1) and 4, 7-dimethoxyl-5-formyl-6-hydroxycoumarin (2). The bioassays indicated that compounds 1 and 2 could significantly inhibit the proliferation of cancer cell, and showed the agonist effect on the transactivity of retinoic acid receptor-alpha (RARalpha). In addition, the two compounds had inhibitory effect against human leukocyte elastase (HLE).
Anemone ; chemistry ; Antineoplastic Agents, Phytogenic ; chemistry ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coumarins ; chemistry ; isolation & purification ; pharmacology ; Humans ; Inhibitory Concentration 50 ; Leukocyte Elastase ; metabolism ; Molecular Structure ; Plants, Medicinal ; chemistry ; Receptors, Retinoic Acid ; genetics ; metabolism ; Retinoic Acid Receptor alpha ; Rhizome ; chemistry ; Transcriptional Activation

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

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