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The Long-Acting Serine Protease Inhibitor mPEG-SPA-MDSPI16 Alleviates LPS-Induced Acute Lung Injury.International Journal of Molecular... Apr 2024Anti-inflammatory drugs have become the second-largest class of common drugs after anti-infective drugs in animal clinical care worldwide and are often combined with...
Anti-inflammatory drugs have become the second-largest class of common drugs after anti-infective drugs in animal clinical care worldwide and are often combined with other drugs to treat fever and viral diseases caused by various factors. In our previous study, a novel serine protease inhibitor-encoding gene () with improved anti-inflammatory activity was selected from a constructed suppressive subducted hybridization library of housefly larvae. This protein could easily induce an immune response in animals and had a short half-life, which limited its wide application in the clinic. Thus, in this study, mPEG-succinimidyl propionate (mPEG-SPA, Mw = 5 kDa) was used to molecularly modify the MDSPI16 protein, and the modified product mPEG-SPA-MDSPI16, which strongly inhibited elastase production, was purified. It had good stability and safety, low immunogenicity, and a long half-life, and the IC for elastase was 86 nM. mPEG-SPA-MDSPI16 effectively inhibited the expression of neutrophil elastase and decreased ROS levels. Moreover, mPEG-SPA-MDSPI16 exerted anti-inflammatory effects by inhibiting activation of the NF-κB signaling pathway and the MAPK signaling pathway in neutrophils. It also exerted therapeutic effects on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. In summary, mPEG-SPA-MDSPI16 is a novel anti-inflammatory protein modified with PEG that has the advantages of safety, nontoxicity, improved stability, and strong anti-inflammatory activity in vivo and in vitro and is expected to become an effective anti-inflammatory drug.
Topics: Animals; Acute Lung Injury; Lipopolysaccharides; Mice; Serine Proteinase Inhibitors; Anti-Inflammatory Agents; Polyethylene Glycols; NF-kappa B; Male; Leukocyte Elastase; Humans; Signal Transduction; Recombinant Fusion Proteins; Disease Models, Animal
PubMed: 38674153
DOI: 10.3390/ijms25084567 -
International Journal of Molecular... Apr 2024Neutrophil elastase (NE) is taken up by macrophages, retains intracellular protease activity, and induces a pro-inflammatory phenotype. However, the mechanism of...
Neutrophil elastase (NE) is taken up by macrophages, retains intracellular protease activity, and induces a pro-inflammatory phenotype. However, the mechanism of NE-induced pro-inflammatory polarization of macrophages is not well understood. We hypothesized that intracellular NE degrades histone deacetylases (HDAC) and Sirtuins, disrupting the balance of lysine acetylation and deacetylation and resulting in nuclear to cytoplasmic translocation of a major alarmin, High Mobility Group Box 1 (HMGB1), a pro-inflammatory response in macrophages. Human blood monocytes were obtained from healthy donors or from subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Monocytes were differentiated into blood monocyte derived macrophages (BMDMs) in vitro. Human BMDMs were exposed to NE or control vehicle, and the abundance of HDACs and Sirtuins was determined by Western blotting of total cell lysates or nuclear extracts or determined by ELISA. HDAC, Sirtuin, and Histone acetyltransferase (HAT) activities were measured. NE degraded most HDACs and Sirtuin (Sirt)1, resulting in decreased HDAC and sirtuin activities, with minimal change in HAT activity. We then evaluated whether the NE-induced loss of Sirt activity or loss of HDAC activities would alter the cellular localization of HMGB1. NE treatment or treatment with Trichostatin A (TSA), a global HDAC inhibitor, both increased HMGB1 translocation from the nucleus to the cytoplasm, consistent with HMGB1 activation. NE significantly degraded Class I and II HDAC family members and Sirt 1, which shifted BMDMs to a pro-inflammatory phenotype.
Topics: Humans; Acetylation; Cells, Cultured; Cystic Fibrosis; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; HMGB1 Protein; Hydroxamic Acids; Leukocyte Elastase; Macrophages; Monocytes; Proteolysis; Pulmonary Disease, Chronic Obstructive; Sirtuin 1
PubMed: 38673851
DOI: 10.3390/ijms25084265 -
Biomedicine & Pharmacotherapy =... May 2024Acute lung injury (ALI) is a common and critical respiratory disorder caused by various factors, with viral infection being the leading contributor....
Acute lung injury (ALI) is a common and critical respiratory disorder caused by various factors, with viral infection being the leading contributor. Dehydroandrographolide (DAP), a constituent of the Chinese herbal plant Andrographis paniculata, exhibits a range of activities including anti-inflammatory, in vitro antiviral and immune-enhancing effects. This study evaluated the anti-inflammatory effects and pharmacokinetics (PK) profile of DAP in ALI mice induced by intratracheal instillation of Poly(I:C) (PIC). The results showed that oral administration of DAP (10-40 mg/kg) effectively suppressed the increase in lung wet-dry weight ratio, total cells, total protein content, accumulation of immune cells, inflammatory cytokines and neutrophil elastase levels in bronchoalveolar lavage fluid of PIC-treated mice. DAP concentrations, determined by an LC-MS/MS method, in plasma after receiving DAP (20 mg/kg) were unchanged compared to those in normal mice. However, DAP concentrations and relative PK parameters in the lungs were significantly altered in PIC-treated mice, exhibiting a relatively higher maximum concentration, larger AUC, and longer elimination half-life than those in the lungs of normal mice. These results demonstrated that DAP could improve lung edema and inflammation in ALI mice, and suggested that lung injury might influence the PK properties of DAP, leading to increased lung distribution and residence. Our study provides evidence that DAP displays significant anti-inflammatory activity against viral lung injury and is more likely to distribute to damaged lung tissue.
Topics: Animals; Acute Lung Injury; Anti-Inflammatory Agents; Diterpenes; Male; Mice; Poly I-C; Bronchoalveolar Lavage Fluid; Andrographis; Cytokines; Lung; Leukocyte Elastase
PubMed: 38552441
DOI: 10.1016/j.biopha.2024.116456 -
Anaerobe Mar 2024Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we...
OBJECTIVES
Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers.
METHODS
We stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology.
RESULTS
CD66b neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10 staining of neutrophils started to recover within 3-7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes.
CONCLUSIONS
Neutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.
PubMed: 38514010
DOI: 10.1016/j.anaerobe.2024.102840 -
Molecules (Basel, Switzerland) Mar 2024Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil...
Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with k/K value over 2,000,000 [Ms]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.
Topics: Humans; Leukocyte Elastase; Cathepsin G; Myeloblastin; Serine Proteases; Serine Proteinase Inhibitors
PubMed: 38474630
DOI: 10.3390/molecules29051120 -
Physiological Research Mar 2024The objective of this study was to evaluate whether RSV inhibits neutrophil extracellular traps (NETs) that induce joint hyperalgesia in C57BL/6 mice after...
The objective of this study was to evaluate whether RSV inhibits neutrophil extracellular traps (NETs) that induce joint hyperalgesia in C57BL/6 mice after adjuvant-induced arthritis. A subplantar injection of Freund's complete adjuvant was administered to C57BL/6 mice on day 0 for immunization in the AIA model. Resveratrol (RSV, 25 mg/kg) was administered intraperitoneally once daily starting on day 22 and continuing for two weeks. The effects of mechanical hyperalgesia and edema formation have been assessed in addition to histopathological scoring. Mice were sacrificed on day 35 to determine cytokine levels and PADI4 and COX-2 expression levels. ELISA was used to quantify neutrophil extracellular traps (NETs) along with neutrophil elastase-DNA and myeloperoxidase-DNA complexes in neutrophils. An immunohistochemical stain was performed on knee joints to determine the presence of nuclear factor kappa B p65 (NF-kappaB p65). AIA mice were found to have higher levels of NET in joints and their joint cells demonstrated an increased expression of the PADI4 gene. Treatment with RSV in AIA mice (25 mg/kg, i.p.) significantly (P<0.05) inhibited joint hyperalgesia, resulting in a significant increase in mechanical threshold, a decrease in articular edema, a decrease in the production of inflammatory cytokines, increased COX-2 expression, and a decrease in the immunostaining of NF-kappaB. Furthermore, treatment with RSV significantly reduced the amount of neutrophil elastase (NE)-DNA and MPO-DNA complexes, which were used as indicators of NET formation (P<0.05). This study indicates that RSV reduces NET production and hyperalgesia by reducing inflammation mediated by PADI4 and COX-2. According to these data, NETs contribute to joint pain and resveratrol can be used to treat pain in RA through this pathway.
Topics: Mice; Animals; Extracellular Traps; Resveratrol; Leukocyte Elastase; Toll-Like Receptor 4; Hyperalgesia; NF-kappa B; Cyclooxygenase 2; Mice, Inbred C57BL; Inflammation; Arthritis, Rheumatoid; Neutrophils; Cytokines; DNA; Edema
PubMed: 38466008
DOI: 10.33549/physiolres.935172 -
The European Respiratory Journal Mar 2024https://bit.ly/3SS1tKk
https://bit.ly/3SS1tKk
Topics: Humans; Leukocyte Elastase; Cystic Fibrosis; Proteolysis; Aminopeptidases; Neutrophils
PubMed: 38387968
DOI: 10.1183/13993003.01512-2023 -
European Journal of Pharmaceutical... Apr 2024Neutrophil elastase has been identified as a potential therapeutic target for acute lung injury or acute respiratory distress syndrome, and Sivelestat is a selective,... (Randomized Controlled Trial)
Randomized Controlled Trial
Safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation study in Chinese healthy subjects.
BACKGROUND AND OBJECTIVE
Neutrophil elastase has been identified as a potential therapeutic target for acute lung injury or acute respiratory distress syndrome, and Sivelestat is a selective, reversible and competitive neutrophil elastase inhibitor. This study was designed to investigate the safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat in healthy Chinese subjects.
METHODS
A randomized, double-blind, placebo-controlled single- and multiple-dose escalation clinical trial was carried out. Briefly, healthy volunteers in twelve cohorts with 8 per cohort received 1.0-20.2 mg/kg/h Sivelestat or placebo in an intravenous infusion manner for two hours, and healthy volunteers in four cohorts received two hours intravenous infusion of 2.0-5.0 mg/kg/h Sivelestat or placebo with an interval of twelve hours for seven times. The safety and tolerability were evaluated and serial blood samples were collected for pharmacokinetics and neutrophil elastase inhibitory effects analysis at the specified time-point.
RESULTS
A total of 128 subjects were enrolled and all participants completed the study except one. Sivelestat exhibited satisfactory safety and tolerability up to 20.2 mg/kg/h in single-dose cohorts and 5.0 mg/kg/h in multiple-dose cohorts. Even so, more attention should be paid to the safety risks when using high doses. The C and AUC of Sivelestat increased in a dose dependent manner, and T was similar for different dose cohorts. In multiple-dose cohorts, the plasma concentrations reached steady state 48 h after first administration and the accumulation of C and AUC was not obvious. Furthermore, the C of 5.0 mg/kg/h dose cohort could meet the needs of clinical treatment. For some reason, the pharmacodynamics data revealed that the inhibitory effect of Sivelestat on neutrophil elastase content in healthy subjects was inconclusive.
CONCLUSION
Sivelestat was safe and well tolerated with appropriate pharmacokinetic parameters, which provided support for more diverse dosing regimen in clinical application.
CLINICAL TRIAL REGISTRATION
www.chinadrugtrials.org.cn identifier is CTR20210072.
Topics: Humans; Healthy Volunteers; Leukocyte Elastase; Area Under Curve; Double-Blind Method; China; Dose-Response Relationship, Drug; Glycine; Sulfonamides
PubMed: 38336251
DOI: 10.1016/j.ejps.2024.106723 -
MBio Feb 2024Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic (UPEC) causes most of these...
Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes, and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial, and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a preclinical model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC-infected SLPI-deficient () mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type () controls. Combined with bulk bladder RNA sequencing, our data indicate that mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with a history of recent or recurrent UTI, suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI promotes clearance of UPEC in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.IMPORTANCEAnnually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.
Topics: Adolescent; Adult; Animals; Female; Humans; Mice; Middle Aged; Young Adult; Anti-Infective Agents; Cystitis; Escherichia coli Infections; Secretory Leukocyte Peptidase Inhibitor; Urinary Tract Infections; Uropathogenic Escherichia coli
PubMed: 38270443
DOI: 10.1128/mbio.02554-23 -
Medicina (Kaunas, Lithuania) Jan 2024: The role and the levels of ghrelin in diabetes-induced retinal damage have not yet been explored. The present study aimed to measure the serum levels of total ghrelin...
: The role and the levels of ghrelin in diabetes-induced retinal damage have not yet been explored. The present study aimed to measure the serum levels of total ghrelin (TG), and its acylated (AG) and des-acylated (DAG) forms in patients with the two stages of diabetic retinopathy (DR), non-proliferative (NPDR) and proliferative (PDR). Moreover, the correlation between serum ghrelin and neutrophil elastase (NE) levels was investigated. : The serum markers were determined via enzyme-linked immunosorbent assays in 12 non-diabetic subjects (CTRL), 15 diabetic patients without DR (Diabetic), 15 patients with NPDR, and 15 patients with PDR. : TG and AG serum levels were significantly decreased in Diabetic (respectively, < 0.05 and < 0.01 vs. CTRL), NPDR ( < 0.01 vs. Diabetic), and in PDR patients ( < 0.01 vs. NPDR). AG serum levels were inversely associated with DR abnormalities (microhemorrhages, microaneurysms, and exudates) progression (r = -0.83, < 0.01), serum neutrophil percentage (r = -0.74, < 0.01), and serum NE levels (r = -0.73, < 0.01). The latter were significantly increased in the Diabetic ( < 0.05 vs. CTRL), NPDR ( < 0.01 vs. Diabetic), and PDR ( < 0.01 vs. PDR) groups. : The two DR stages were characterized by decreased AG and increased NE levels. In particular, serum AG levels were lower in PDR compared to NPDR patients, and serum NE levels were higher in the PDR vs. the NPDR group. Together with the greater presence of retinal abnormalities, this could underline a distinctive role of AG in PDR compared to NPDR.
Topics: Humans; Leukocyte Elastase; Diabetic Retinopathy; Ghrelin; Enzyme-Linked Immunosorbent Assay; Exudates and Transudates; Diabetes Mellitus
PubMed: 38256379
DOI: 10.3390/medicina60010118