Neutrophil elastase:From mechanisms to therapeutic potential

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.

Pterostilbene supresses inflammation-induced melanoma metastasis by impeding neutrophil elastase-mediated thrombospondin-1 degradation

Objective: Chronic inflammation plays a fatal role in tumor metastasis. Pterostilbene(PTE) is a natural dimethylated analogue of resveratrol with anticancer and anti-inflammatory activities. This study aimed to investigate the inhibitory effect of PTE on inflammation-associated metastasis and explore the underlying mechanisms.Methods: Lipopolysaccharide(LPS)-induced lung inflammation and melanoma metastasis models were established in mice. After PTE treatment for four weeks, the organ index, histological changes, proinflammatory cytokines, and the expression and activity of neutrophil elastase(NE), a biomarker of neutrophil influx in the lungs, were analysed. Additionally, direct effects of PTE on NE-induced B16 cell migration were explored in wound healing and Transwell assays, and the expression of thrombospondin-1(TSP-1) and epithelial-mesenchymal transition(EMT) markers were also detected.Results: PTE obviously attenuated the LPS-induced metastasis of circulatory B16 cells to lungs by reducing the number of metastatic nodules on the lung surfaces and the lung weight/body weight ratio. PTE treatment also significantly reduced LPS-activated increase levels of tumor necrosis factor(TNF)-a and interleukin(IL)-6 in the lungs of tumor-bearing mice. In addition, increased expression and enzyme activity of NE and decreased expression of TSP-1 were observed, and these were blocked by PTE. In vitro, PTE at concentrations without cytotoxicity also markedly suppressed NE-triggered B16 cell migration, prevented NE-induced TSP-1 proteolysis and reversed the expression of vimentin, N-cadherin and Ecadherin.Conclusion: PTE could block inflammation-enhanced tumor metastasis, and the underlying mechanism might be associated with the inhibition of NE-mediated TSP-1 degradation.

miRNA-141-5p Affects the Levels of Neutrophil Elastase in Preeclampsia by Regulating MAPK1

Objective:The objective of this study was to investigate the expression levels of microRNA-141-5p(miRNA-141-5p),MAPK1 and neutrophil elastase in patients with and without preeclampsia(PE),and the relationship between miRNA-141-5p and MAPK1 with respect to the secretion of elastase by neutrophils in patients with PE.Methods:Thirty patients with PE and 30 healthy pregnant(HP)women were recruited from The Second Hospital of Shanxi Medical University,Taiyuan,China,between February 2017 and July 2018.Neutrophils were isolated from 8 mL peripheral blood samples and cultured.We recorded neutrophil count and morphology during culture.Apoptosis was detected by flow cytometry in different groups at 0,24,and 48 h.The expression levels of elastase were detected in neutrophils by enzyme-linked immunosorbent assay,whereas the expression levels of miRNA-141-5p in peripheral blood neutrophils were detected by real-time polymerase chain reaction.We used TargetScanHuman Release 7.2 to analyze the target genes of miRNA-141-5p.The expression of MAPK1 in peripheral blood neutrophils was detected by western blotting.Data were analyzed by SPSS version 21.0 software,and comparisons between groups were carried out with the Studentt test.Results:There was no significant difference between the PE and HP groups(P>0.050)with regard to age or body mass index.The weight of newborns in the PE group(2846.00±600.00 g)was significantly lower than that in the HP group(3055.00±230.68 g).The number of neutrophilic granulocytes(NGs)in blood samples from the PE group was significantly higher than that in the HP group(P=0.003).There was no significant difference between the groups with regard to morphology.Apoptosis in the PE group was delayed when compared with the HP group at different time points.TheP value of apoptosis in the PE and HP groups were respectively 0.790,<0.001 and 0.030 at 0 h,24 h and 48 h.The expression levels of miRNA-141-5p in the PE group were significantly lower than those in the HP group(P<0.050).The expression levels of MAPK1 in neutrophils from the PE group were significantly higher than those in the HP group(P<0.050)by western blot.The expression levels of elastase in neutrophils from the PE group were significantly higher than those in the HP group(P<0.050).Furthermore,the number of NGs in peripheral blood from the PE group was higher than that of the HP group;however,the levels of apoptosis were lower.The expression levels of miRNA-141-5p in NGs decreased,the expression of MAPK1 increased,and the secretion of neutrophil elastase in the NG medium increased in the PE group than those in the HP group.Conclusion:Collectively,our analysis suggested that miRNA-141-5p may be involved in the pathogenesis of PE by regulating the MAPK1 signaling pathway to activate neutrophils and increase the secretion of elastase.

Neutrophils disrupt B-1a cell homeostasis by targeting Siglec-G to exacerbate sepsis

B-1a cells,an innate-like cell population,are crucial for pathogen defense and the regulation of inflammation through their release of natural IgM and IL-10.In sepsis,B-1a cell numbers are decreased in the peritoneal cavity as they robustly migrate to the spleen.Within the spleen,migrating B-1a cells differentiate into plasma cells,leading to alterations in their original phenotype and functionality.We discovered a key player,sialic acid-binding immunoglobulin-like lectin-G(Siglec-G),which is expressed predominantly on B-1a cells and negatively regulates B-1a cell migration to maintain homeostasis.Siglec-G interacts with CXCR4/CXCL12 to modulate B-1a cell migration.Neutrophils aid B-1a cell migration via neutrophil elastase(NE)-mediated Siglec-G cleavage.Human studies revealed increased NE expression in septic patients.We identified an NE cleavage sequence in silico,leading to the discovery of a decoy peptide that protects Siglec-G,preserves peritoneal B-1a cells,reduces inflammation,and enhances sepsis survival.The role of Siglec-G in inhibiting B-1a cell migration to maintain their inherent phenotype and function is compromised by NE in sepsis,offering valuable insights into B-1a cell homeostasis.Employing a small decoy peptide to prevent NE-mediated Siglec-G cleavage has emerged as a promising strategy to sustain peritoneal B-1a cell homeostasis,alleviate inflammation,and ultimately improve outcomes in sepsis patients.

Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients

Objectives:Elevated circulating DNA(cirDNA)concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release.We carried out the first prospective,multicenter study of the dynamics of cirDNA and neutrophil extracellular trap(NETs)markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients.Methods:We examined the plasma levels of two NETs protein markers[myeloperoxidase(MPO)and neutrophil elastase(NE)],as well as levels of cirDNA of nuclear(cir-nDNA)and mitochondrial(cir-mtDNA)origin in 29 colon,prostate,and breast cancer patients and in 114 healthy individuals(HI).Results:The synergistic analytical information provided by these markers revealed that:(i)NETs formation contributes to post-surgery conditions;(i)post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer[r=0.60(P<0.001)and r=0.53(P<0.01),respectivelyl,but not in prostate and breast cancer;(i)each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics,but overall the pre-and post-surgery median values of cir-nDNA,NE,and MPO were significantly higher in cancer patients than in HI.Conclusion:Taken as a whole,our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period,depending on surgical procedure or cancer type.By contrast,cir-mtDNA is poorly associated with NETs formation in the studied perioperative period,which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

α_1-ANTITRYPSIN ATTENUATES ENDOTOXIN-INDUCED ACUTE LUNG INJURY IN RABBITS

Objective To investigate whether pretreatment with α 1-AT can attenuate acute lung injury (ALI) in rabbits induced with endotoxin. Methods Thirty-two New Zealand rabbits were randomly assigned to four groups(n=8):1.Infusion of endotoxin(Lipopolysaccharide,LPS 500μg/kg)without α 1-AT (group LPS).2.Infusion α 1-AT 120mg/kg at 15min before challenge with LPS(group LAV).3.Infusion of α 1-AT 120mg/kg(group AAT).4 Infusion of saline 4ml/kg as control (group NS).Arterial blood gases,peripheral leukocyte counts and airway pressure were recorded every 1h.Physiologic intrapulmonary shunting (Qs/Qt) was measured every 4h.After 8h the bloods were collected for measurement of plasma concentration and activity of α 1-AT.Then bronchoalveolar lavage fluid (BALF)was collected for measurement of concentrations of total protein (TP),interleukin-8(IL-8),tumor necrosis factor(TNF-α),the activities of elastase-like and α 1-AT,total phospholipids(TPL) and disaturated phosphatidylcholine (DSPC).In addition,the wet-to-dry lung weight ratio(W/D) was measured. Results After infusion of endotoxin,it was observed that PaO 2,peripheral luekocyte counts,total respiratory compliance progressively decreased and P peak and Qs/Qt increased comparing with the baseline values.In contrast to group NS,the increased plasma concentration but reduced activity of α 1-AT was found in group LPS.In the BALF,the activity of α 1-AT,TPL,DSPC/TPL were lower,but the concentrations of albumin,IL-8,TNF-α,and the activity of NE were higher.The ratio of W/D also increased.The pretreatment of α 1-AT attenuated the deterioration of oxygenation,the reduction of compliance and the deterioration of other physiological,biochemical parameters mentioned above. Conclusion Pretreatment with α 1-AT could attenuate endotoxin-induced lung injury in rabbits.Those beneficial effects of α 1-AT might be due in part to the inhibitory effect on neutrophil elastase.

Atramacronoids A-C,three eudesmanolide sesquiterpene-phenol hybrids with an unprecedented C-C linkage from the rhizomes of Atractylodes macrocephala

Three eudesmanolide sesquiterpene-phenol hybrids,atramacronoids A-C(1-3),featuring an unusual6/6/5/5/6 skeleton furnished by forming an unexpected C-8-C-16 linkage,were obtained from the rhizomes of Atractylodes macrocephala.Their structures and absolute configurations were elucidated by spectroscopic data analysis,chemical calculations,combined with X-ray diffractions.The plausible biosynthetic pathways for compounds 1-3 are proposed.Surprisingly,compound 1 exhibited cytotoxicity against SGC-7901 cells by inducing cells apoptosis,which might relate to the promotion of synthesis of neutrophil elastase.

Effect of Tanreqing Injection(痰热清注射液)on Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease with Chinese Medicine Syndrome of Retention of Phlegm and Heat in Fei(肺)

Objective：To explore the effect of Tanreqing Injection（痰热清注射液,TRQI） on the treatment of acute exacerbation of chronic obstructive pulmonary disease（AECOPD） with Chinese medicine syndrome of retention of phlegm and heat in Fei（痰热阻肺证,RPHF）.Methods：In a prospective randomized controlled clinical trial,90 patients with AECOPD of RPHF syndrome were randomly assigned to 3 groups,TRQI and controls A and B,each with 30 cases.The TRQI group was administered with the intravenous injections of 20 mL TRQI once a day and conventional Western medicine treatment.Control group A was administered with the intravenous injection of 15 mg ambroxol hydrochloride twice a day and conventional Western medicine treatment,and control group B was administered with conventional Western medicine treatment only.The treatments were administered for 10 days.Chinese medical symptoms and signs were scored,and plasma concentrations of interleukin（IL）-8 and neutrophil elastase（NE） were recorded.Results：（1） The Chinese medical symptoms （cough,sputum amount,expectoration,dyspnea and fever） and signs（tongue and pulse） improved significantly in the TRQI group（P〈0.05 or P〈0.01）,and improvements in cough,sputum amount and expectoration were better in the TRQI group than control group B（P〈0.05）;there was no significant difference between the TRQI group and control group A（P〉0.05）.The sign of tongue was also improved significantly in the TRQI group （P〈0.05）.（2） The overall effects in the TRQI group and control group A were significantly better than in control group B（P〈0.05）,with no significant differences between the TRQI group and control group A（P〉0.05）.There was no significant difference in the total effective rate among the three groups（P〉0.05）.（3） After treatment, the plasma concentrations of IL-8 and NE decreased in the TRQI group and control group A（P〈0.05）,and the concentration of IL-8 in control group B decreased（P〈0.05）.The difference in IL-8 was greater in the TRQI group than in control group A and B before and after treatment,and the change in NE was greater in control group A than in the TRQI group and control group B,but there was no statistical significance among the three groups with regards to the change in IL-8 or NE（P〉0.05）.Conclusion：TRQI could improved the Chinese medical signs and symptoms in the patients with AECOPD,possibly because of the decreasing plasma levels of IL-8 and NE which could improve response to airway inflammation and mucus hypersecretion.

Protective effects of α_1 -antitrypsin on acute lung injury in rabbits induced by endotoxin

Objective To investigate whether pretreatment with α1,-antitrypsin (AAT) can attenuate acute lung injury (ALI) in rabbits induced with endotoxin.Methods Thirty-two healthy adult New Zealand rabbits were anaesthetized, tracheotomized and mechanically ventilated. They were then randomly divided into four groups (n =8): (1) Infusion of Escherichia coli endotoxin [ Lipopolysaccharide (LPS) 500μg/kg ] without AAT (Group LPS). (2) Infusion of AAT 120 mg/kg at 15 minutes after LPS (Group LAV). (3) Infusion of AAT 120 mg/kg without endotoxin (Group AAT). (4) Infusion of saline 4 ml/kg as control (Group NS). Arterial blood gases, peripheral leukocyte counts and airway pressure were recorded every hour for eight hours. Physiologic intrapulmonary shunting (Qs/Qt) was measured every four hours. After eight hours, blood samples were collected for measurement of plasma concentration and activity of AAT. Then, the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected for measurement of concentrations of total protein (TP), interleukin-8 (IL-8), tumor necrosis factor (TNFa, the activities of NE and AAT, total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC). In addition, the wet-to-dry lung weight ratio (W/D) was measured.Results The infusion of endotoxin induced decreases in arterial oxygen pressure (PaO2), peripheral leukocyte counts, total respiratory compliance (TLC) and the increases in peak pressure (Ppeak), Qs/ Qt compared with the baseline values ( P < 0. 05). The increased plasma concentration but reduced activity of AAT was also found in contrast to that in Group NS (P<0. 05). In the BALF, the activity of AAT, TPL, DSPC/TPL were lower than those in Group NS (P<0. 05), but the concentrations of albumin, IL-8, TNFα, the activity of NE and the ratio of W/D were higher than those in Group NS (P <0. 05). The pretreatment of AAT attenuated the deterioration of oxygenation, the reduction of compliance and the deterioration of other physiological and biochemical parameters mentioned above.Conclusion Pretreatment with AAT could attenuate endotoxin-induced lung injury in rabbits. Those beneficial effects of AAT might be due, in part, to reduction in the levels of mediators that could activate neutrophils, in addition to the direct inhibitory effect on neutrophil elastase.