Sytox Green Staining Research Articles

Resolution of acute inflammation induced by monosodium urate crystals (MSU) through neutrophil extracellular trap-MSU aggregate-mediated negative signaling

BackgroundPolymorphonuclear neutrophils (PMN) activation by monosodium urate crystals (MSU) is crucial to acute gouty arthritis and subsequent spontaneous remission within 7–10 days. Activated PMNs release neutrophil extracellular traps (NETs) that entrap MSU crystals, forming NET-MSU aggregates. Whether NET-MSU aggregates contribute to the resolution of acute inflammation remains to be elucidated. This study uses a cell-based approach to unveil their molecular bases.MethodsAll-trans retinoic acid-differentiated HL-60 cells (dHL-60) served as surrogate PMNs. NET release from MSU-activated dHL-60 was confirmed by detecting DNA, neutrophil elastase, and citrullinated histone 3, forming large NET-MSU aggregates. NET area was measured with Fiji software after SYTOX Green staining. Released pro-inflammatory cytokines IL-8 and TNF-α, and the anti-inflammatory cytokine IL-1RA in culture supernatants were quantified to calculate the estimate inflammation score (EIS). Cellular redox state was determined by a FRET-based sensor. Expression of intracellular positive (ERK1/2) and negative (SHP-1 and SHIP-1) cytokine signaling regulators was detected by western blot. qPCR detected mRNA expressions of CISH and SOCS1–SOCS7. Flow cytometry measured neutrophil N1 (CD54) and N2 (CD182) surface markers after staining with fluorescent-conjugated antibodies.ResultsIncubating dHL-60 with MSU for 4 h maximized NET-MSU aggregate formation and acute inflammation with an EIS of 11.6. Prolonging the incubation of dHL-60 + MSU to 22 h gradually raised the EIS to 19.40 without increasing NET area, due to reduced cellular redox capacity. Adding both new dHL-60 and new MSU crystals to the culture, mimicking the clinical scenario, increased NET area but conversely suppressed EIS to 1.53, indicating acute inflammation resolution. The resolution of acute inflammation following prolonged incubation was attributed to decreases in P-ERK and increases in P-SHP-1, SOCS2, SOCS3, and CISH gene expressions, which may suppress pro-inflammatory and enhance anti-inflammatory cytokine production. Moreover, the large NET-MSU aggregates facilitated N1 to N2 polarization, crucial for accelerating inflammation resolution.ConclusionWe explored the potential molecular basis for the spontaneous resolution of MSU induced acute inflammation using a cell-based model in that huge NET-MSU aggregates frustrate the transformation of newly entering PMNs to the N2 phenotype, enhancing the production of the anti-inflammatory cytokine IL-1RA.

The Antifungal Potential of Ozonated Extra-Virgin Olive Oil Against Candida albicans: Mechanisms and Efficacy.

The growing emergence of resistance mechanisms and side effects associated with antifungal agents highlight the need for alternative therapies. This study aims to investigate the antifungal potential of ozonated extra-virgin olive oil (EOO) against Candida albicans, with the goal of developing eco-friendly and highly effective treatments based on natural products. Antifungal activity was evaluated via cell viability and biofilm formation assays using Crystal Violet and Sytox green staining. The results showed that EOO reduced C. albicans viability in a dose-dependent manner, achieving over 90% cell death at a 3% (v/v) concentration. Transmission Electron Microscopy (TEM) revealed cell wall structural damage, and ROS levels increased by approximately 60% compared to untreated controls within 10 min of treatment. Additionally, the expression of autophagy-related genes atg-7 and atg-13was upregulated by 2- and 3.5-fold, respectively, after 15 min, suggesting a stress-induced cell death response. EOO also significantly inhibited hyphal formation and biofilm development, thus reducing C. albicans pathogenicity while preserving cell biocompatibility. EOO antifungal activity was also observed in the case of Candida glabrata. In conclusion, ozonated olive oil demonstrates potent antifungal activity against C. albicans by reducing cell viability, inhibiting hyphal and biofilm formation, and triggering oxidative stress and autophagy pathways. These findings position EOO as a promising alternative therapy for fungal infections.

Antimicrobial and Antioxidant Hydrogels Based on the Water‐Insoluble β‐Glucan From Poria cocos Loaded With Total Flavonoids of Ampelopsis grossedentata

AbstractThis study aims to develop and assess an antimicrobial hydrogel that provides a sanitizer effect while avoiding skin oxidative damage. The hydrogel is composed of Poria cocos polysaccharide and loaded with total flavonoids extracted from Ampelopsis grossedentata (TF@PCPA‐G). The antibacterial activity of TF@PCPA‐G was assessed through minimum inhibitory concentration (MIC), zone of inhibition, and SYTOX green staining measurements. The findings indicate that TF@PCPA‐G exhibited remarkable antibacterial potency. Additionally, the antioxidant properties of the hydrogel were examined using DPPH, ABTS, and hydroxyl radical scavenging assay models, revealing that TF@PCPA‐G possessed a persistent ability to neutralize free radicals. Subsequently, the antioxidant properties of TF@PCPA‐G were further investigated using cellular and animal models of oxidative damage. As anticipated, TF@PCPA‐G effectively alleviated oxidative stress in cells and animal skin. In conclusion, TF@PCPA‐G has been demonstrated to eliminate bacteria effectively and prevent skin damage.

GGPPS Negatively Regulates the Formation of Neutrophil Extracellular Traps in Lipopolysaccharide-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are life-threatening diseases. Neutrophil extracellular traps (NETs) play a key role in lung damage. Geranylgeranyl diphosphate synthase (GGPPS) is associated with the development of inflammatory diseases. We aimed to explore the role of GGPPS in NETs formation in ARDS/ALI. First, lung pathological changes in lipopolysaccharide (LPS)-induced ALI mice after myeloid-specific GGPPS deletion were evaluated. The level of NETs formation was analyzed by immunofluorescence, PicoGreen assay and Western blotting. Next, we determined the role of GGPPS in NETs formation and underlying mechanisms using immunofluorescence, flow cytometry, DCFH-DA, and SYTOX GREEN staining in vitro. Finally, the correlation between GGPPS expression incirculating neutrophils and dsDNA levels in plasma was evaluated. Myeloid-specific GGPPS deletion mice showed increased NETs deposition in lung tissue and aggravated histopathological damage of lung tissue. In vitro, GGPPS deficiency in neutrophils resulted in increased NETs formation by Phorbol-12-myristate-13-acetate (PMA), which was reversed by Geranylgeranyl diphosphate (GGPP). In addition, inhibitors blocking protein kinase C (PKC) and NADPH-oxidase (NOX) decreased NETs formation induced by GGPPS deletion. Importantly, GGPPS expression in circulating neutrophils was decreased in ARDS patients compared with the healthy control, and the level of dsDNA in plasma of ARDS patients was negatively correlated with the GGPPS expression. Taken together, GGPPS deficiency in neutrophils aggravates LPS-induced lung injury by promoting NETs formation via PKC/NOX signaling. Thus, neutrophil GGPPS could be a key therapeutic target for ARDS.

Sanxiapeptin is an ideal preservative with a dual effect of controlling green mold and inducing systemic defense in postharvest citrus

Green mold is a common postharvest disease infected by Penicillium digitatum that causes citrus fruit decay, and severely affects fruit storage quality. This work aimed to investigate the antifungal activity of Sanxiapeptin against P. digitatum, and elucidate the possible mechanisms involved. Sanxiapeptin was capable of inhibiting spore germination, germ tube length and mycelial growth. The SYTOX green staining assay revealed that Sanxiapeptin targeted the fungal membrane, and changed the membrane permeability, leading to the leakage of cell constituents. Meanwhile, Sanxiapeptin could influence the cell wall permeability and integrity by increasing the activities of chitinase and glucanase, resulting in abnormal chitin consumption and the decrease of glucan. Intriguingly, Sanxiapeptin could effectively control postharvest decay in citrus fruits, and activate the host resistance responses by regulating the phenylpropanoid pathway. In conclusion, Sanxiapeptin exhibits multiphasic antifungal mechanisms of action to control green mold in citrus fruits, shows great potential as novel food preservatives.

The role of PPP1R3G in promoting necroptosis in ischemia reperfusion induced acute kidney injury

Recent discoveries have revealed necroptosis as a major contributor to the pathogenesis of acute kidney injury (AKI). Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) has been linked to the essentiality of RIPK1-dependent apoptosis and type I necroptosis. Nonetheless, the involvement of PPP1R3G in the regulation of necroptosis during ischemia reperfusion-induced acute kidney injury (IR-AKI) has yet to be elucidated. Here, we investigated the role of PPP1R3G in the regulation of necroptosis in IR using an in vitro cellular I/R model and in vivo IRI-AKI mouse model. Primary proximal tubular cells (PTCs) from C57BL/6 and Ppp1r3g−/− mice were cultured and cell I/R was induced through hypoxia/reoxygenation. Cell viability and necroptosis were analyzed using CellTiter-Glo assay and Annexin V-Cy3/ SYTOX green staining, respectively. The expression levels of necroptosis, oxidative stress, and inflammation factors were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. In the in vivo IRI-AKI model, bilateral renal pedicles clamping was conducted in both C57BL/6 and Ppp1r3g−/− mice. The kidney injury and function were evaluated by plasma creatine, BUN, and KIM-1 levels and GFR values, respectively. Kidney morphological evidence of injury was assessed by PAS staining and cell death by immunofluorescent staining. We found that knockout of PPP1R3G in the primary tubular cells significantly preserved cell viability and attenuated tubular cell death. This protective effect was associated with the reduced expression and phosphorylation of necroptosis factors (RIP1, RIP3, and MLKL), and inflammatory factors (IL-6, IL-10 and TNF-α) in I/R injury cells. Deletion of PPP1R3G attenuated the I/R induced kidney injury indicated by 54% decrease in the plasma creatine (2.28±0.12mg/dL vs. 1.09±0.07mg/dL, p<0.0001, n=7) and 45% higher GFR values than WT mice at day 3 after reperfusion. Histology showed less diffuse renal tubular necrosis and casts comprising necrotic cells and debris in Ppp1r3g−/− mice, as well as 60% less cell death compared to WT mice. Moreover, the application of chemical compounds to prevent inhibitory phosphorylation of RIPK1 significantly reinstated cell death in Ppp1r3g−/− cells. In conclusion, our findings demonstrate, for the first time, that Ppp1r3g-mediated necroptosis significantly promotes IRI-AKI. Therefore, targeting Ppp1r3g to reduce necroptosis in the kidney holds promising potential for significant clinical benefits. National Institutes of Health Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differentiation and quantification of extracellular polymeric substances from microalgae and bacteria in the mixed culture

Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

Imbalance of programmed cell death patterns mediated by dendritic cell subsets in systemic lupus erythematosus and lupus nephritis.

Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns. Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 μmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 μmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group. Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group. This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.

Computational analysis of curcumin-mediated alleviation of inflammation in periodontitis patients with experimental validation in mice.

Using network pharmacology and experimental validation to explore the therapeutic efficacy and mechanism of curcumin (Cur) in periodontitis treatment. Network pharmacology was utilized to predict target gene interactions of Cur-Periodontitis. Molecular docking was used to investigate the binding affinity of Cur for the predicted targets. A mouse model with ligature-induced periodontitis (LIP) was used to verify the therapeutic effect of Cur. Microcomputed tomography (micro-CT) was used to evaluate alveolar bone resorption, while western blotting, haematoxylin-eosin staining and immunohistochemistry were used to analyse the change in immunopathology. SYTOX Green staining was used to assess the in vitro effect of Cur in a mouse bone marrow-isolated neutrophil model exposed to lipopolysaccharide. Network pharmacology identified 114 potential target genes. Enrichment analysis showed that Cur can modulate the production of neutrophil extracellular traps (NETs). Molecular docking experiments suggested that Cur effectively binds to neutrophil elastase (ELANE), peptidylarginine deiminase 4 (PAD4) and cathepsin G, three enzymes involved in NETs. In LIP mice, Cur alleviated alveolar bone resorption and reduced the expression of ELANE and PAD4 in a time-dependent but dose-independent manner. Cur can directly inhibit NET formation in the cell model. Our research suggested that Cur may alleviate experimental periodontitis by inhibiting NET formation.

(113) A Novel 3D Cellular Model for Peyronie's Disease Treatment Screening: PD.SPHERE

Abstract Introduction Peyronie's disease (PD) is a common issue that affects approximately 10% of males. Currently, researchers primarily study the pathogenesis and potential treatments for PD using monolayer fibroblast cells and a limited number of animal models. However, these models do not accurately represent the complex cellular structure and interactions involved in PD pathogenesis. We have successfully developed a three-dimensional (3D) cellular model, called PD.SPHERE, using a ground-breaking 'micro-gravity' cell culture method. This model is cultivated from extracted human PD tissues and serves as a valuable tool for in vitro treatment screening for PD. However, we have yet to use this model to evaluate the effectiveness of available and potential treatments. Objective Our objective is to determine the efficacy of the novel 3D cellular PD model, PD.SPHERE, as a treatment screening tool. Methods To achieve this, we cultured fibroblasts isolated from human PD tissues using the 'micro-gravity' approach. In this method, cells were seeded in a non-adhesive 96-well plate, placed on an orbital shaker, and incubated at 37°C with 5% CO2 and 95% relative humidity. We confirmed the proper morphology and cellular composition through microscopic assays. This 3D model was then used to assess the effects of collagenase clostridium histolyticum (CCH) and the actinidin enzyme, obtained from kiwi fruits. The PD.SPHERE was treated with actinidin at 10 mg/ml for 48 hours. We subsequently quantified collagen content using a soluble collagen quantification kit and observed viability, morphology, and changes in cellular compositions of the spheroids through immunofluorescent techniques and confocal microscopy. Results Preliminary results from our model have indicated significant changes in cellular morphology. Treatment with actinidin led to a significant decrease in spheroid size and density compared to the control group (P&amp;lt;0.01). The ratio of vimentin and actin filaments to nuclei staining was slightly but significantly reduced (P&amp;lt;0.05) in the spheroids treated with actinidin at 10 mg/ml. The cellular viability of the treated group showed a slight decrease, as revealed by Sytox Green staining. The assessment of collagen contents in the treated spheroids is ongoing. Conclusions Preliminary results suggest that our PD.SPHERE model can be effectively used to assess morphological and cellular component changes in PD tissues after treatment. Actinidin appears to compromise the plasma membrane of fibroblasts in PD.SPHERE. Our model holds promise for personalized treatment planning and potential for developing high-throughput treatment screening platforms. It also demonstrates consistency and reproducibility while closely mimicking the morphology of a PD plaque. Further research is required to collect signature gene expression data, physiological and immunological information, such as collagen production, extracellular matrix organization, and cellular expression of tight junctions, chemokines, and cytokines. These additional measures will help verify our model's pathophysiological, immunological, and treatment response aspects. Disclosure No.

LC-AMP-F1 Derived from the Venom of the Wolf Spider Lycosa coelestis, Exhibits Antimicrobial and Antibiofilm Activities.

In recent years, there has been a growing interest in antimicrobial peptides as innovative antimicrobial agents for combating drug-resistant bacterial infections, particularly in the fields of biofilm control and eradication. In the present study, a novel cationic antimicrobial peptide, named LC-AMP-F1, was derived from the cDNA library of the Lycosa coelestis venom gland. The sequence, physicochemical properties and secondary structure of LC-AMP-F1 were predicted and studied. LC-AMP-F1 was tested for stability, cytotoxicity, drug resistance, antibacterial activity, and antibiofilm activity in vitro compared with melittin, a well-studied antimicrobial peptide. The findings indicated that LC-AMP-F1 exhibited inhibitory effects on the growth of various bacteria, including five strains of multidrug-resistant bacteria commonly found in clinical settings. Additionally, LC-AMP-F1 demonstrated effective inhibition of biofilm formation and disruption of mature biofilms. Furthermore, LC-AMP-F1 exhibited favorable stability, minimal hemolytic activity, and low toxicity towards different types of eukaryotic cells. Also, it was found that the combination of LC-AMP-F1 with conventional antibiotics exhibited either synergistic or additive therapeutic benefits. Concerning the antibacterial mechanism, scanning electron microscopy and SYTOX Green staining results showed that LC-AMP-F1 increased cell membrane permeability and swiftly disrupted bacterial cell membranes to exert its antibacterial effects. In summary, the findings and studies facilitated the development and clinical application of novel antimicrobial agents.

Neutrophil extracellular trap-related mechanisms in acne vulgaris inspire a novel treatment strategy with adipose-derived stem cells

Acne vulgaris is a type of chronic skin disorder caused by Propionibacterium acnes (P. acnes). Neutrophil extrinsic traps (NETs) play key role in many types of inflammatory skin diseases. Adipose-derived stem cells (ADSCs) was reported modulate immune responses and neutrophil activity. Here, we explored the potential role of ADSCs and the potential mechanism associated with neutrophil extracellular traps (NETs) in relieving acne vulgaris. In the P. acnes-infected ear skin model, histological staining was used to evaluate the inflammatory infiltration and NET formation in control, P. acnes, and P. acnes + ADSCs groups. Besides, western blot was used to detect the expression levels of cit-H3, MPO, and Nrf2 in ear tissue. In vitro, the immunofluorescence staining of MPO and cit-H3, and SYTOX green staining were performed to measure the NET formation. CCK-8 assay, EdU staining, and wound healing assay were used to detect the proliferation and migration abilities of keratinocytes. ELISA assay was utilized to detect the secretion of inflammatory cytokines. In P. acnes-infected ear skin, ADSC treatment significantly attenuated inflammation and NET formation via activating Nrf2 signaling pathway. In vitro, the conditioned medium of ADSCs reduced the formation of P. acne-induced NETs. Besides, ADSCs could inhibit that the NETs efficiently promoted the proliferation, migration, and inflammatory cytokine secretion of keratinocytes. Our study suggested that ADSCs could attenuate P. acne-related inflammation by inhibiting NET formation. This study provides a novel therapeutic perspective of ADSCs in combating acne vulgaris.

Aspergillus Enhances Eosinophil and Neutrophil Extracellular DNA Trap Formation in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is characterized by inflammatory cell infiltration in the sinonasal mucosa. Eosinophil and neutrophil extracellular traps (EETs and NETs, respectively) are prominently found in CRS. This study aimed to investigate the effect of airborne fungi, Alternaria alternata and Aspergillus fumigatus, on EET and NET formation. Nasal epithelial cells, eosinophils, and neutrophils were isolated from eosinophilic CRS (ECRS), non-ECRS (NECRS), and healthy control. We determined eosinophil and neutrophil transepithelial migration after fungal treatment. We then determined the release of EETs and NETs by fungi using Sytox Green staining and determined the role of reactive oxygen species (ROS) using ROS inhibitors. We identified more abundant EETs and NETs in ECRS than in NECRS. A. alternata and A. fumigatus enhanced eosinophil and neutrophil transepithelial migration. A. fumigatus strongly induced EET and NET formation in CRS and, simultaneously, suppressed fungal metabolic activity. EET formation in CRS is associated with nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and NET formation with NADPH-oxidase and mitochondrial ROS. A. fumigatus, but not A. alternata, induced EET and NET formation, and peripheral blood eosinophils and neutrophils exhibited different immune responses against A. fumigatus following the inflammatory status of the host. Aspergillus-fumigatus-induced EET and NET formation plays a crucial role in CRS pathogenesis.

Neutrophil extracellular traps in upper respiratory tract secretions: insights into infectious and allergic rhinitis.

Neutrophil extracellular traps (NETs) are structures released by neutrophils in response to various infections. NETs have a biocidal role and have been demonstrated to be effective against bacteria, fungi, viruses, and parasites. Depending on the situation, NETs can protect the host from pathogen invasion or contribute to the development of autoimmune diseases such as cystic fibrosis and rheumatoid arthritis. In this study, we aimed to investigate the occurrence of NET as one of the components in upper respiratory tract secretions in infectious and allergic diseases. Nasal mucus was collected from donors diagnosed with infectious rhinitis or allergic rhinitis. The extracellular DNA content was determined using SytoxGreen staining, and the total protein pool was determined using the microBCA method. Micrococcal nuclease was used to digest the samples and ELISA was employed to identify the NET proteins. The enzymatic activity of elastase was determined. Our findings showed that nasal mucus collected from patients with infectious rhinosinusitis contained extracellular DNA that could come from a variety of sources, responsible for increasing the density and viscosity of secretions, as well as NETs proteins. The identified enzymatic activity of NET elastase indicates the possible irritation of nasal tissues. However, the DNA content was not identified in the samples from allergic patients. In addition, we have shown in preliminary studies that therapy using N-acetylcysteine can liquefy nasal secretions. The study suggests that the composition of nasal mucus varies according to the cause of mucosal irritation. The presence of DNA and NET proteins can have severe consequences for the therapeutic process prolonging treatment. The low viscosity of nasal mucus in allergic patients facilitates mucosal flushing and the removal of allergens. Understanding the occurrence and role of NETs in various respiratory diseases is critical for developing effective treatment strategies that consider the complex interaction between the immune system and pathogens. The results of this study suggest that NETs may be present in upper respiratory tract secretions with an infectious background, supporting basic defense mechanisms using eosinophils and EETs. Further research is needed to explore the potential of NETs as a therapeutic target in respiratory diseases.

The Role and Mechanism of Ferroptosis Mediated by METTL3-m6A Modification in Regulating Radioresistance of Esophageal Cancer

Radioresistance remains the major cause of recurrence within two years after radical radiotherapy in approximately 50% of patients with esophageal cancer (ESCC). Recently, it has been reported that enhancing ferroptosis can reverse tumor radioresistance. However, the underlying mechanisms remain elusive. The aim of this study was to elucidate the role and mechanism of ferroptosis mediated by METTL3 N6-methyladenosine (m6A) modification in regulating radioresistance of Esophageal Cancer. We hypothesize that inhibition of ferroptosis confers radioresistance in Esophageal Cancer by METTL3-induced m6A modification which increase the expression of m6A in the 3'UTR region of SOCS6 and inhibits its expression. To explore the relationship between radioresistance and ferroptosis. Firstly, ferroptosis was analyzed by 4-HNE staining in ESCC specimens; Furthermore, cell death was detected by propidium iodide (PI) or SYTOX Green staining combined with microscopy or flow cytometry in ESCC radioresistance cells and parental cells, and Iron Assay Kit,C11-BODIPY were used to examine whether ferroptosis were inhibited in radioresistance cells. Subsequently, MeRIP-seq and MeRIP-PCR were performed for discussing the relationship between ferroptosis and m6A. Finally, Mettl3 depleted cells were generated with CRISPR/Cas9-mediated knockout system. Then, m6A methylation level, sensitivity to radiation and ferroptosis of Mettl3 depleted cells by EpiQuik m6A RNA methylation quantification kit, colony formation, C11-BODIPY and Western Blot. Firstly, we found that the level of lipid peroxide 4-HNE in ESCC samples that recurred after radical radiotherapy was significantly lower than that in radiotherapy (40Gy), and the ferroptosis of radioresistance cells was inhibited. MeRIP-seq found that the level of m6A in radioresistance cells increased significantly, and then the KEGG pathway analysis of MeRIP-seq results showed that hypermethylation of m6A was closely related to ferroptosis signal pathway. and we also found that the level of m6A was significantly decreased by adding agonist (Erastin), whereas the modification level was significantly increased by adding inhibitor Fer-1. Meanwhile, we found that the level of m6A was significantly decreased in radioresistance cells silencing METTL3, while the ferroptosis was activated, which caused the radiosensitivity of ESCC. Lastly, MeRIP-PCR data showed that METTLT3 induced a significant increase in m6A level in 3' UTR region of SOCS6 mRNA and inhibited its expression, which eventually led to radioresistance of ESCC. m6A-regulated ferroptosis inhibition confers radioresistance of ESCC. METTL3 up-regulated m6A in SOCS6 3' UTR and inhibited the expression of SOCS6, which caused the inhibition of ferroptosis.

Quizartinib inhibits necroptosis by targeting receptor-interacting serine/threonine protein kinase 1.

Systemic inflammatory response syndrome (SIRS), at least in part driven by necroptosis, is characterized by life-threatening multiple organ failure. Blocking the progression of SIRS and consequent multiple organ dysfunction is challenging. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important cell death and inflammatory mediator, making it a potential treatment target in several diseases. Here, using a drug repurposing approach, we show that inhibiting RIPK1 is also an effective treatment for SIRS. We performed cell-based high-throughput drug screening of an US Food and Drug Administration (FDA)-approved drug library that contains 1953 drugs to identify effective inhibitors of necroptotic cell death by SYTOX green staining. Dose-response validation of the top candidate, quizartinib, was conducted in two cell lines of HT-22 and MEFs. The effect of quizartinib on necroptosis-related proteins was evaluated using western blotting, immunoprecipitation, and an in vitro RIPK1 kinase assay. The in vivo effects of quizartinib were assessed in a murine tumor necrosis factor α (TNFα)-induced SIRS model. High-throughput screening identified quizartinib as the top "hit" in the compound library that rescued cells from necroptosis in vitro. Quizartinib inhibited necroptosis by directly inhibiting RIPK1 kinase activity and blocking downstream complex IIb formation. Furthermore, quizartinib protected mice against TNFα-induced SIRS. Quizartinib, as an FDA-approved drug with proven safety and efficacy, was repurposed for targeted inhibition of RIPK1. This work provides essential preclinical data for transferring quizartinib to the treatment of RIPK1-dependent necroptosis-induced inflammatory diseases, including SIRS.

Macrophage extracellular traps promote tumor-like biologic behaviors of fibroblast-like synoviocytes through cGAS-mediated PI3K/Akt signaling pathway in patients with rheumatoid arthritis.

Rheumatoid arthritis is an autoimmune disease characterized by synovium hyperplasia and bone destruction. Macrophage extracellular traps are released from macrophages under various stimuli and may generate stable autoantigen-DNA complexes, as well as aggravate autoantibody generation and autoimmune responses. We aimed to investigate the role of macrophage extracellular traps on the biologic behaviors of rheumatoid arthritis fibroblast-like synoviocytes. Synovial tissues and fibroblast-like synoviocytes were obtained from patients with rheumatoid arthritis. Extracellular traps in synovium and synovial fluids were detected by immunofluorescence, immunohistochemistry, and SYTOX Green staining. Cell viability, migration, invasion, and cytokine expression of rheumatoid arthritis fibroblast-like synoviocytes were assessed by CCK-8, wound-healing assay, Transwell assays, and quantitative real-time polymerase chain reaction, respectively. RNA sequencing analysis was performed to explore the underlying mechanism, and Western blot was used to validate the active signaling pathways. We found that extracellular trap formation was abundant in rheumatoid arthritis and positively correlated to anti-CCP. Rheumatoid arthritis fibroblast-like synoviocytes stimulated with purified macrophage extracellular traps demonstrated the obvious promotion in tumor-like biologic behaviors. The DNA sensor cGAS in rheumatoid arthritis fibroblast-like synoviocytes was activated after macrophage extracellular trap stimuli. RNA sequencing revealed that differential genes were significantly enriched in the PI3K/Akt signaling pathway, and cGAS inhibitor RU.521 effectively reversed the promotion of tumor-like biologic behaviors in macrophage extracellular trap-treated rheumatoid arthritis fibroblast-like synoviocytes and downregulated the PI3K/Akt activation. In summary, our study demonstrates that macrophage extracellular traps promote the pathogenically biological behaviors of rheumatoid arthritis fibroblast-like synoviocytes through cGAS-mediated activation of the PI3K/Akt signaling pathway. These findings provide a novel insight into the pathogenesis of rheumatoid arthritis and the mechanisms of macrophages in modulating rheumatoid arthritis fibroblast-like synoviocyte tumor-like behaviors.

Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo.

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Network pharmacological analysis of corosolic acid reveals P4HA2 inhibits hepatocellular carcinoma progression

BackgroundCorosolic acid is a pentacyclic triterpene acid with hypoglycemic, anti-inflammatory, and anti-cancer effects. However, its potential targets in hepatocellular carcinoma (HCC) are unknown, hindering clinical utilization.MethodsDifferentially expressed proteins of the Bel-7404 cell line were identified with tandem mass tag analysis and differentially expressed genes (DEGs) of an HCC TCGA dataset using bioinformatics. Gene functions and pathways were inferred using the DAVID database. Online databases were used to establish P4HA2 expression in HCC (GEPIA2) and its relationship with patient survival (UALCAN and The Human Protein Atlas), the association between P4HA2 expression and immune cell infiltration (TIMER2), and DNA methylation of the P4HA2 gene (MethSurv). Cell proliferation, cell cycle, and cell death were assessed with PI and SYTOX-Green staining, CCK-8, and colony formation assays. Protein expression levels were detected by Western blotting.ResultsA total of 44 differentially expressed proteins and 4498 DEGs were identified. Four genes whose proteins were also found in the differential protein profile but with opposing expressions were selected as candidate targets. The candidate gene prolyl 4-hydroxylase subunit alpha 2 (P4HA2) was recognized as the only potential target due to its high expression in public datasets, association with poor patient survival, and relation to immune cell infiltration in HCC tissues. Moreover, the DNA methylation status in 4 CpG islands of the P4HA2 gene correlated with a poor prognosis. Furthermore, corosolic acid treatment inhibited the proliferation of HCC cell lines Bel-7404 and HepG2 in a dose-dependent manner, caused G2/M phase cell cycle arrest, and promoted cell death. In addition, the treatment reduced P4HA2 protein levels.ConclusionOur results indicate that P4HA2 is a potential target of corosolic acid. Thus, they contribute to understanding molecular changes in HCC after corosolic acid treatment and facilitate finding new treatment regimens.

Ozone-oxidized black carbon particles change macrophage fate: Crosstalk between necroptosis and macrophage extracellular traps

The impacts of environmental PM 2.5 on public health have become a major concern all over the world. Many studies have shown that PM 2.5 still poses a threat to public health even at very low levels. Physical or chemical reactions occur between primary particles and other components in the environment, which changes the properties of primary particles. Such newly formed particles with changed properties are called secondary particles. Ozone-oxidized black carbon (oBC) is a key part of PM 2.5 and a representative secondary particle. Macrophages extracellular traps (METs) is a means for macrophages to capture and destroy invading pathogens, thereby exercising innate immunity. Necroptosis is a kind of programmed cell death, which is accompanied by the destruction of membrane integrity, thus inducing inflammatory reaction. However, there is no research on the crosstalk mechanism between necroptosis and MET after oBC exposure. In our study, AO/EB staining, SYTOX Green staining, fluorescent probe, qPCR, Western blot, and immunofluorescence were applied. This experiment found that under normal physiological conditions, when macrophages receive external stimuli (such as pathogens; in our experiment: phorbol 12-myristate 13-acetate (PMA)), they will form METs, capture and kill pathogens, thus exerting innate immune function. However, exposure to oBC can cause necroptosis in macrophages, accompanied by increased levels of reactive oxygen species (ROS) and cytosolic calcium ions, as well as the expression disorder of inflammatory factors and chemokines, and prevent the formation of METs, lose the function of capturing and killing pathogens, and weaken the innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibited the formation of METs. This study was the first to explore the crosstalk mechanism between necroptosis and METs after oBC exposure.