[Sivelestat sodium inhibits neutrophil elastase to regulate intrahepatic biliary mucin 5AC expression].

OBJECTIVE: To explore whether sivelestat sodium could reduce the expression of mucin 5AC (MUC5AC) in intrahepatic bile duct epithelial cells by inhibiting neutrophil elastase (NE) and thus provide new potential therapeutic ideas for the treatment of intrahepatic bile duct stone (IBDS). METHODS: (1) Bioinformatics analysis: differential gene analysis was performed on gallbladder stone cholecystitis sequencing data based on the gene expression omnibus (GEO) to screen for significantly different genes related to neutrophils and mucins. The search tool for the retrieval of interacting genes database (STRING) was used for protein interaction analysis to predict whether there was an interaction between NE and MUC5AC genes. (2) Animal experiment: a total of 18 male SD rats were divided into the sham-operated group, cholangitis model group and sivelestat sodium treatment group according to the random number table method, with 6 rats in each group. The cholangitis rat model was established by a one-time injection of 1.25 mg/kg lipopolysaccharide (LPS) into the right anterior lobe of the liver of rats in combination with the pre-experiment; the liver of the sham-operated group was injected with an equal volume of saline. After the modelling, 100 mg/kg of sivelestat sodium was injected into the tail vein of the cevalexin treatment group once a day for 5 days, and an equal volume of saline was injected into the tail vein of the sham-operated group and the cholangitis model group. Two weeks later, the rats were euthanized and their liver and bile duct tissues were taken. The pathological changes in the liver and bile duct tissues were observed under the light microscope. Immunohistochemical staining was used to detect the expressions of NE and MUC5AC in liver and bile duct tissues. The protein expressions of NE, MUC5AC and Toll-like receptor 4 (TLR4) were detected by Western blotting. (3) Cell experiment: primary human intrahepatic biliary epithelial cell line (HiBEpiC) was divided into blank control group, NE group (10 nmol/L NE), NE+sivelestat sodium low dose group (10 nmol/L NE+1×10-8 g/L sivelestat sodium 1 mL), NE+sivelestat sodium medium dose group (10 nmol/L NE+1×10-7 g/L sivelestat sodium 1 mL), NE+sivelestat sodium high dose group (10 nmol/L NE+1×10-6 g/L sivelestat sodium 1 mL). Cells were collected after 48 hours of culture, and EdU was performed to detect the proliferative activity of cells; enzyme linked immunosorbent assay (ELISA) and Western blotting were performed to detect the expression of MUC5AC in cells. RESULTS: (1) Bioinformatics analysis: the NE gene (ELANE) had a reciprocal relationship with MUC5AC. (2) Animal experiment: light microscopy showed that hepatocyte edema, hepatocyte diffuse point and focal necrosis, confluent area fibrous tissue and intrahepatic bile ducts hyperplasia and inflammatory cell infiltration in the cholangitis model group; hepatic lobule structure of sivelestat sodium treatment group was clear, and the degree of peripheral inflammatory cell infiltration was reduced compared with the cholangitis model group. Immunohistochemical staining showed that the expressions of NE and MUC5AC were increased in the cholangitis model group compared with the sham-operated group, and the expressions of NE and MUC5AC were decreased in the sivelestat sodium group compared with the cholangitis model group [NE (A value): 5.23±2.02 vs. 116.67±23.06, MUC5AC (A value): 5.40±3.09 vs. 23.81±7.09, both P < 0.05]. Western blotting showed that the protein expressions of NE, MUC5AC, and TLR4 in the hepatic biliary tissues of the cholangitis model group were significantly higher than those of the sham-operated group; and the protein expressions of NE, MUC5AC, and TLR4 in the liver biliary tissues of the sivelestat sodium treatment group were significantly higher than those of the sham-operated group (NE/ß-actin: 0.38±0.04 vs. 0.70±0.10, MUC5AC/ß-actin: 0.37±0.03 vs. 0.61±0.05, TLR4/ß-actin: 0.39±0.10 vs. 0.93±0.15, all P < 0.05). (3) Cell experiment: fluorescence microscopy showed that the proliferation of HiBEpiC cells in each group was good, and there was no significant difference in the proportion of positive cells. ELISA and Western blotting showed that the expressions of MUC5AC in cells of the NE group were significantly higher than those of the blank control group. The expressions of MUC5AC in the NE+different dose of sivelestat sodium group were significantly lower than those in the NE group, and showed a decreasing trend with the increase of sevastatin sodium concentration, especially in the highest dose group [MUC5AC (µg/L): 3.46±0.20 vs. 6.33±0.52, MUC5AC/ß-actin: 0.45±0.07 vs. 1.75±0.10, both P < 0.05]. CONCLUSIONS: LPS can upregulate the expression of NE and MUC5AC in rats with cholangitis, while sodium sivelestat can reduce the expression of MUC5AC in in intrahepatic biliary epithelial cells by inhibiting NE, providing a new direction for the treatment of IBDS.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.

Effect of Neurotropic and Immunotropic Drugs on Leukocyte Elastase Activity In Vitro.

Leukocyte elastase is a marker of inflammation. Previously, a relationship was found between the severity of mental disorders in patients and elastase-like activity of blood plasma. The effect of various neurotropic drugs on leukocyte elastase activity was analyzed in an in vitro experiment. We revealed an inhibitory effect of the benzodiazepine tranquilizers diazepam and bromodihydrochlorophenylbenzodiazepine and immunomodulators aminodihydrophthalazinedione and diclofenac on the plasma elastase-like activity of healthy donors and pure human neutrophil elastase. The antipsychotics chlorpromazine and alimemazine, as well as the nootropic vinpocetine increased elastase-like activity in a dose-dependent manner. The activating effect of chlorpromazine and vinpocetine, but not alimemazine, was reproduced in neutrophil elastase. We hypothesized that these drugs can affect the development of inflammatory reactions in the complex therapy of mental disorders.

A Pilot Study to Investigate Peripheral Low-Level Chronic LPS Injection as a Model of Neutrophil Activation in the Periphery and Brain in Mice.

Lipopolysaccharide-induced (LPS) inflammation is used as model to understand the role of inflammation in brain diseases. However, no studies have assessed the ability of peripheral low-level chronic LPS to induce neutrophil activation in the periphery and brain. Subclinical levels of LPS were injected intraperitoneally into mice to investigate its impacts on neutrophil frequency and activation. Neutrophil activation, as measured by CD11b expression, was higher in LPS-injected mice compared to saline-injected mice after 4 weeks but not 8 weeks of injections. Neutrophil frequency and activation increased in the periphery 4-12 h and 4-8 h after the fourth and final injection, respectively. Increased levels of G-CSF, TNFa, IL-6, and CXCL2 were observed in the plasma along with increased neutrophil elastase, a marker of neutrophil extracellular traps, peaking 4 h following the final injection. Neutrophil activation was increased in the brain of LPS-injected mice when compared to saline-injected mice 4-8 h after the final injection. These results indicate that subclinical levels of peripheral LPS induces neutrophil activation in the periphery and brain. This model of chronic low-level systemic inflammation could be used to understand how neutrophils may act as mediators of the periphery-brain axis of inflammation with age and/or in mouse models of neurodegenerative or neuroinflammatory disease.

Neutrophil-Derived Peptidyl Arginine Deiminase Activity Contributes to Pulmonary Emphysema by Enhancing Elastin Degradation.

In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and in vitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Endotypes of Exacerbation in Bronchiectasis: An Observational Cohort Study.

Rationale: Bronchiectasis is characterized by acute exacerbations, but the biological mechanisms underlying these events are poorly characterized. Objectives: To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. Methods: A total of 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation before receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral, or both. Sputum inflammatory assessments included label-free liquid chromatography-tandem mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16 s rRNA sequencing was used to characterize the microbiome. Measurements and Main Results: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacterium was identified in 103 samples (86%), and a high bacterial load (total bacterial load > 107 copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients, with rhinovirus being the most common virus (31%). PCR testing was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, IL-1ß, and CXCL8. These markers were particularly associated with bacterial and bacterial plus viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral, and eosinophilic events in both hypothesis-led and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating four subtypes of exacerbation. Conclusions: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses, and inflammatory dysregulation.

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.

Killer Function of Circulating Neutrophils in Relation to Cytokines in Uterine Myoma and Endometrial Cancer.

The study presents the killer functions of circulating neutrophils: myeloperoxidase activity, the ability to generate ROS, phagocytic activity, receptor status, NETosis, as well as the level of cytokines IL-2, IL-4, IL-6, IL-17A, and IL-18, granulocyte CSF, monocyte chemotactic protein 1, and neutrophil elastase in the serum of patients with uterine myoma and endometrial cancer (FIGO stages I-III). The phagocytic ability of neutrophils in uterine myoma was influenced by serum levels of granulocyte CSF and IL-2 in 54% of the total variance. The degranulation ability of neutrophils in endometrial cancer was determined by circulating IL-18 in 50% of the total variance. In uterine myoma, 66% of the total variance in neutrophil myeloperoxidase activity was explained by a model dependent on blood levels of IL-17A, IL-6, and IL-4. The risk of endometrial cancer increases when elevated levels of monocyte chemotactic protein 1 in circulating neutrophils are associated with reduced ability to capture particles via extracellular traps (96% probability).

Unveiling the metabolites underlying the skin anti-ageing properties of Cytinus hypocistis (L.) L. through a biochemometric approach.

BACKGROUND: The genus Cytinus, recognised as one of the most enigmatic in the plant kingdom, has garnered attention for its bioactive potential, particularly its skin anti-ageing properties. Despite this recognition, much remains to be accomplished regarding deciphering and isolating its most active compounds. HYPOTHESIS: This study aimed to identify the compounds responsible for C. hypocistis skin anti-ageing potential. METHODS: Using multivariate analysis, a biochemometric approach was applied to identify the discriminant metabolites by integrating extracts' chemical profile (Liquid Chromatography-High-Resolution Mass Spectrometry, LCHRMS) and bioactive properties. The identified bioactive metabolite was structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR). RESULTS: Among the studied bioactivities, the anti-elastase results exhibited a significant variation among the samples from different years. After the biochemometric analysis, the compound 2,3:4,6-bis(hexahydroxydiphenoyl)glucose, with a molecular mass of 784.075 Da, was structurally elucidated as the discriminant feature responsible for the outstanding human neutrophil elastase inhibition. Remarkably, the subfraction containing this compound exhibited a tenfold improvement in neutrophil elastase inhibition efficacy compared to the crude extract; its effectiveness fell within the same range as SPCK, a potent irreversible neutrophil elastase inhibitor. Moreover, this subfraction displayed no cytotoxicity or phototoxicity and excellent efficacy for the tested anti-ageing properties. CONCLUSIONS: Hydrolysable tannins were confirmed as the metabolites behind C. hypocistis skin anti-ageing properties, effectively mitigating critical molecular mechanisms that influence the phenotypically distinct ageing clinical manifestations. Pedunculagin was particularly effective in inhibiting neutrophil elastase, considered one of the most destructive enzymes in skin ageing.

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Blockade of neutrophil extracellular trap components ameliorates cholestatic liver disease in Mdr2 (Abcb4) knockout mice.

Primary sclerosing cholangitis (PSC) is an (auto)immune-mediated cholestatic liver disease with a yet unclear etiology. Increasing evidence points to an involvement of neutrophils in chronic liver inflammation and cirrhosis but also liver repair. Here, we investigate the role of the neutrophil extracellular trap (NET) component myeloperoxidase (MPO) and the therapeutic potential of DNase I and of neutrophil elastase (NE) inhibitor GW311616A on disease outcome in the multidrug resistance 2 knockout (Mdr2-/-) mouse, a PSC animal model. Initially, we observed the recruitment of MPO expressing cells and the formation of NETs in liver biopsies of PSC patients and in Mdr2-/- livers. Furthermore, sera of Mdr2-/- mice contained perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA)-like reactivity similar to PSC patient sera. Also, hepatic NE activity was significantly higher in Mdr2-/- mice than in wild type littermates. Flow cytometry analyses revealed that during disease development a highly active neutrophil subpopulation established specifically in the liver of Mdr2-/- mice. However, absence of their MPO activity, as in MPO-deficient Mdr2-/- mice, showed no effect on hepatobiliary disease severity. In contrast, clearance of extracellular DNA by DNase I reduced the frequency of liver-resident neutrophils, plasmacytoid dendritic cells (pDCs) and CD103+ conventional DCs and decreased cholangiocyte injury. Combination of DNase I with a pDC-depleting antibody was additionally hepatocyte-protective. Most importantly, GW311616A, an orally bioavailable inhibitor of human NE, attenuated hepatobiliary injury in a TNFα-dependent manner and damped hyperproliferation of biliary epithelial cells. Further, hepatic immigration and activity of CD11b+ DCs as well as the secretion of IFNγ by hepatic CD4 and CD8 T cells were reduced. Our findings delineate neutrophils as important participants in the immune cell crosstalk that drives cholestatic liver disease and identify NET components as potential therapeutic targets.

Neutrophil Elastase Degrades Histone Deacetylases and Sirtuin 1 in Primary Human Monocyte Derived Macrophages.

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.

The Long-Acting Serine Protease Inhibitor mPEG-SPA-MDSPI16 Alleviates LPS-Induced Acute Lung Injury.

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 (MDSPI16) 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 IC50 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.

Resveratrol Attenuates Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via TLR-4 Mediated Inflammation in C57BL/6 Mice.

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.

[The clinical characteristics of neutrophil extracellular trap in patients with severe cerebral venous sinus thrombosis and its prognostic value in acute and subacute patients].

Objective: To explore the clinical characteristics of neutrophil extracellular trap (NET) in patients with severe cerebral venous sinus thrombosis (CVST) and to study their prognostic value in the acute and subacute phases. Methods: This study is a retrospective case series analysis. Clinical and pathological data of 52 patients with severe cerebral venous sinus thrombosis who underwent endovascular treatment in the Department of Neurosurgery, Tianjin Huanhu Hospital from June 2019 to June 2022 were retrospectively analyzed. There were 20 males and 32 females, with an age of (40.1±13.6) years(range:18 to 66 years). Forty-five healthy physical examinees were included in the control group. High-resolution MRI was used to stage the thrombus, with 11 cases in the acute group, 28 cases in the subacute group, and 13 cases in the chronic group. Thrombus specimens were obtained through endovascular treatment, and the fluorescence intensity of NET in peripheral blood at different time points was analyzed by immunofluorescence contrast,including the double-stranded DNA structure and adhesion protein components (citrolinated histone H3 (CitH3), myeloperoxidase-DNA complex(MPO-DNA), neutrophil elastase (NE)). The NET markers were determined by ELISA. Spearman rank correlation analysis was used to analyze the correlation between the NET markers in peripheral blood of patients with severe cerebral venous sinus thrombosis in the acute and subacute phases and the volume of venous sinus thrombus, the degree of venous sinus recanalization after treatment, and the discharge modified Rankin scale(mRS)score. The accuracy of NET markers in predicting the prognosis of patients with severe cerebral venous sinus thrombosis was analyzed by drawing receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Results: The results of immunofluorescence staining and ELISA showed that no NET structure was formed in the peripheral blood of the control group, while CitH3, MPO-DNA and NE levels in the peripheral blood of CVST patients were increased, among which the acute stage group was the highest, followed by the subacute group, and the chronic group was the lowest. Spearman correlation analysis showed that CitH3, MPO-DNA and NE levels in peripheral blood of patients in acute group and subacute group were positively correlated with thrombus volume and mRS score at discharge (P<0.05). The levels of CitH3 and MPO-DNA in peripheral blood of patients with complete venous sinus recanalization were lower than those of patients with partial venous sinus recanalization (P<0.01). ROC curve analysis results showed that MPO-DNA and NE had no predictive ability for the prognosis of CVST patients (P values were 0.614 and 0.324, respectively), and the AUC of CitH3 was 0.800 (95%CI: 0.638~0.962, P=0.032), the best cut-off value was 13.5 µg/L, the sensitivity was 100%, and the specificity was 58.8%. Conclusions: A large number of NET are formed in patients with severe cerebral venous sinus thrombosis in acute stage. Patients with severe cerebral venous sinus thrombosis in acute stage and subacute stage with high peripheral blood NET content has a low rate of complete sinus revascularization and poor neurological function recovery after treatment.

Anti-inflammatory effect and pharmacokinetics of dehydroandrographolide, an active component of Andrographis paniculata, on Poly(I:C)-induced acute lung injury.

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.

Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase.

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 kinact/KI value over 2,000,000 [M-1s-1]. 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.

SPLUNC1 as a biomarker of pulmonary exacerbations in children with cystic fibrosis.

BACKGROUND: Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is an innate defence protein that acts as an anti-microbial agent and regulates airway surface liquid volume through inhibition of the epithelial sodium channel (ENaC). SPLUNC1 levels were found to be reduced in airway secretions of adults with cystic fibrosis (CF). The potential of SPLUNC1 as a biomarker in children with CF is unknown. METHODS: We quantified SPLUNC1, interleukin-8 (IL-8) and neutrophil elastase (NE) in sputum of CF children treated with either intravenous antibiotics or oral antibiotics for a pulmonary exacerbation (PEx)s, and in participants of a prospective cohort of CF children with preserved lung function on spirometry, followed over a period of two years. RESULTS: Sputum SPLUNC1 levels were significantly lower before compared to after intravenous and oral antibiotic therapy for PEx. In the longitudinal cohort, SPLUNC1 levels were found to be decreased at PEx visits compared to both previous and subsequent stable visits. Higher SPLUNC1 levels at stable visits were associated with longer PEx-free time (hazard ratio 0.85, p = 0.04). SPLUNC1 at PEx visits did not correlate with IL-8 or NE levels in sputum or forced expiratory volume in one second (FEV1) but did correlate with the lung clearance index (LCI) (r=-0.53, p < 0.001). CONCLUSION: SPLUNC1 demonstrates promising clinometric properties as a biomarker of PEx in children with CF.

Key inflammatory markers in bronchoalveolar lavage predict bronchiectasis progression in young children with CF.

INTRODUCTION: Inflammation appears early in cystic fibrosis (CF) pathogenesis, with specific elevated inflammatory markers in bronchoalveolar lavage fluid (BALF) correlating with structural lung disease. Our aim was to identify markers of airway inflammation able to predict bronchiectasis progression over two years with high sensitivity and specificity. METHODS: Children with CF with two chest computed tomography (CT) scans and bronchoscopies at a two-year interval were included (n= 10 at 1 and 3 years and n= 27 at 3 and 5 years). Chest CTs were scored for increase in bronchiectasis (Δ%Bx), using the PRAGMA-CF score. BALF collected with the first CT scan were analyzed for neutrophil% (n= 36), myeloperoxidase (MPO) (n= 25), neutrophil elastase (NE) (n= 26), and with a protein array for inflammatory and fibrotic markers (n= 26). RESULTS: MPO, neutrophil%, and inducible T-cell costimulator ligand (ICOSLG), but not clinical characteristics, correlated significantly with Δ%Bx. Evaluation of neutrophil%, NE, MPO, interleukin-8 (IL-8), ICOSLG, and hepatocyte growth factor (HGF), for predicting an increase of > 0.5% of Δ%Bx in two years, showed that IL-8 had the best sensitivity (82%) and specificity (73%). Neutrophil%, ICOSLG and HGF had sensitivities of 85, 82, and 82% and specificities of 59, 67 and 60%, respectively. The odds ratio for risk of >0.5% Δ%Bx was higher for IL-8 (12.4) than for neutrophil%, ICOSLG, and HGF (5.9, 5.3, and 6.7, respectively). Sensitivity and specificity were lower for NE and MPO). CONCLUSIONS: High levels of IL-8, neutrophil%, ICOSGL and HGF in BALF may be good predictors for progression of bronchiectasis in young children with CF.