Objective To explore the regulatory effects of cytokines interleukin(IL)-1β,IL-6,tumor necrosis factor alpha(TNF-ɑ),gamma interferon(IFN-γ),granulocyte colony-stimulating factor(G-CSF),and granulocyte-macrophage colony-stimulating factor(GM-CSF)on spontaneous pyroptosis in neutrophils.Methods Neutrophils isolated from mouse bone marrow by density-gradient centrifugation were cultured in vitro for 20 h with or without 10,50 or 100 ng/mL IL-1β,IL-6,IFN-γ,G-CSF or GM-CSF,or for 12 h with or without 1,10 or 50 ng/mL TNF-α.After incubation,cells were stained with annexin Ⅴ(AV)/propidium iodide(PI),and the proportions and absolute number of neutrophils undergoing different forms of cell death were determined by fluorescence microscopy combined with manual counting.Pyroptotic neutrophils were identified by cell morphology in conjunction with AV/PI staining.Flow cytometry with counting beads was employed to measure the proportions and number of AV/PI-stained Ly6g⁺neutrophils in different forms of cell death.Western blotting was employed to assess the cleavage and activation levels of cysteinyl aspartate-specific proteinase-3(caspase-3)and gasdermin E(GSDME).Results Treatment with IL-1β or IL-6 had no significant effect on the proportion or number of neutrophils undergoing spontaneous pyroptosis.After 12 h of treatment with TNF-α at 1,10,and 50 ng/mL,the proportions of pyroptotic neutrophils were(14.79±0.45)%,(19.99±3.02)%,and(20.66±1.99)%,respectively,higher than that[(10.22±1.12)%]in the untreated control(P=0.024,P<0.001,and P<0.001,respectively).Treatment with 10,50,and 100 ng/mL IFN-γ for 20 h reduced the proportion of pyroptotic neutrophils from(17.43±1.88)%to 12.00%(all P<0.001).G-CSF at 10,50,and 100 ng/mL reduced the proportion of pyroptotic cells to around 6.00%and greatly inhibited the cleavage of both caspase-3 and GSDME.After 20 h of treatment with 10,50,and 100 ng/mL GM-CSF,the proportions of pyroptotic neutrophils decreased to(7.52±0.53)%,(5.27±2.30)%,and(0.64±1.11)%,respectively.Conclusions Neither IL-1β nor IL-6 affects GSDME-mediated spontaneous pyroptosis in neutrophils.TNF-ɑ induces spontaneous pyroptosis in neutrophils,whereas IFN-γ,G-CSF,and GM-CSF demonstrate inhibitory effects.
Pyroptosis/drug effects* ; Animals ; Neutrophils/cytology* ; Mice ; Cytokines/pharmacology* ; Interleukin-1beta/pharmacology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology* ; Cells, Cultured ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Interferon-gamma/pharmacology* ; Interleukin-6/pharmacology*

Neutrophils, as the most abundant immune cells in the human body, possess the inherent ability to rapidly migrate to sites of inflammation and infection. Novel drug delivery systems leveraging neutrophils capitalize on their natural targeting and phagocytic capabilities to achieve precise drug delivery. Efficient drug loading into neutrophils within neutrophil-based delivery systems can be achieved through physical adsorption, chemical conjugation, and phagocytosis. Design strategies emphasize carrier selection and targeting ligand design to enhance delivery precision. Compared to traditional drug delivery systems, neutrophil-based systems offer significant advantages, including excellent biocompatibility and strong tissue penetration. These properties can significantly improve drug bioavailability and reduce adverse reactions associated with non-target tissue accumulation. However, these systems also face several challenges that require resolution, such as difficulties in cell collection and preservation, the need for stability optimization, challenges in large-scale production, and a lengthy clinical translation cycle. In disease treatment applications, neutrophil-based drug delivery systems enable precise delivery of anti-cancer drugs to tumor sites, potentially disrupting immunosuppression of the tumor microenvironment and enhancing therapeutic efficacy. For brain diseases, their unique ability to cross the blood-brain barrier facilitates effective drug delivery. In chronic inflammatory diseases, neutrophil-based systems can precisely deliver anti-inflammatory agents to mitigate inflammation. Performance enhancements for neutrophil-based systems can be achieved by the development of novel nanomaterials and optimization of targeting ligand affinity, thereby improving the accuracy and efficiency of drug delivery. This review comprehensively explores the design strategies, advantages, challenges, and future directions of neutrophil-based drug delivery systems. It summarizes research progress in disease treatment applica-tions, aiming to offer key insights for the development of novel drug delivery systems and advance precision medicine and targeted therapy.
Humans ; Drug Delivery Systems/methods* ; Neutrophils ; Phagocytosis ; Drug Carriers ; Blood-Brain Barrier ; Neoplasms/drug therapy*

OBJECTIVE: To investigate the effects of thymoquinone on the proliferation of acute myeloid leukemia (AML) cells and its molecular mechanism, so as to provide theoretical basis for the basic research on the anti-leukemia of traditional Chinese medicine. METHODS: The HL-60 and THP-1 cells were treated with thymoquinone at different concentration gradients, cell proliferation was detected by CCK-8 method, morphological changes were detected by Wright-Giemsa method, apoptosis was detected by Annexin V/PI double staining flow cytometry, and apoptosis and signal pathway protein expression were detected by Western blot. Real-time quantitative fluorescence PCR and Western blot were used to detect the expression changes of high mobility family members of SRY-related proteins (SOX). RESULTS: Thymoquinone inhibited the malignant proliferation of HL-60 and THP-1 cells, up-regulated the expression of pro-apoptotic protein Bax, down-regulated the expression of anti-apoptotic protein Bcl-2 and Survivin, and hydrolyzed Caspase-3 to induce the apoptosis of HL-60 and THP-1 cells. Thymoquinone could also significantly down-regulate the phosphorylation of PI3K, Akt and mTOR, and inhibit the malignant biological characteristics of HL-60 and THP-1 cells by inhibiting the activation of PI3K/Akt/mTOR pathway. After thymoquinone intervention in HL-60 and THP-1 cells, the expression of SOX2 and SOX4 could be down-regulated significantly. At low concentration ( < 10 μmol/L), the expression of SOX12 was weakly affected by thymoquinone. With increasing concentration, the expression of SOX12 could be down-regulated, however, thymoquinone had no effect on SOX11 expression. CONCLUSION Thymoquinone can inhibit the proliferation of AML cells, and its mechanism may be related to inhibiting the activation of PI3K/Akt/mTOR signaling pathway, regulating the expression of apoptotic proteins and core members of SOX family.
Humans ; Benzoquinones/pharmacology* ; Cell Proliferation/drug effects* ; Leukemia, Myeloid, Acute/metabolism* ; Apoptosis/drug effects* ; HL-60 Cells ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Cell Line, Tumor ; Phosphatidylinositol 3-Kinases/metabolism* ; THP-1 Cells

OBJECTIVE: To investigate the metabolic characteristics of ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) in myeloid leukemia by in vitro culture of myeloid leukemia cells and construction of tumor-bearing nude mouse model. METHODS: U937, THP-1, HL60 and K562 cells were cultured in vitro. The cells in logarithmic growth phase (l×10 ⁵ cells/well) were added with ¹⁸F-FDG, and the uptake rate of ¹⁸F-FDG was measured at 15, 30, 60 and 120 min after addation, respectively. The four kinds of cells were inoculated subcutaneously into the hind limbs of nude mice to establish a tumor-bearing nude mouse model. When the tumor size was about 500 mm³, ¹⁸F-FDG was injected through the tail vein of the mice, and positron emission tomography/computed tomography was performed at 60 min after injection. The morphology of tumor-bearing cells was observed by hematoxylin-eosin (HE) staining in serial pathological sections. RESULTS: After co-incubation with ¹⁸F-FDG, the ¹⁸F-FDG uptake rates of U937 cells were significantly higher than THP-1, HL60 and K562 cells at 4 time points (all P <0.05), and THP-1 cells were higher than K562 cells (all P <0.05). The uptake rate of ¹⁸F-FDG by leukemia cells was rapid in the first 60 min, then tended to be stable. Pathological analysis showed that subcutaneous inoculation of U937, THP-1, HL60 and K562 cells could successfully establish tumor-bearing nude mouse models of myeloid leukemia. The ¹⁸F-FDG uptake value in U937 tumor-bearing nude mice was significantly higher than THP-1, HL60 and K562 tumor-bearing nude mice (all P <0.01). The ¹⁸F-FDG uptake values in THP-1 and HL60 tumor-bearing nude mice were significantly higher than that in K562 tumor-bearing nude mice (both P <0.01). CONCLUSION The tumor-bearing nude mouse model of myeloid leukemia can be successfully constructed by subcutaneous inoculation. The ¹⁸F-FDG uptake rate of acute myeloid leukemia (AML) cells is higher in cells cultured in vitro and tumor-bearing nude mouse model. ¹⁸F-FDG may have better clinical application value for AML.
Animals ; Fluorodeoxyglucose F18/metabolism* ; Mice, Nude ; Mice ; Humans ; Leukemia, Myeloid/diagnostic imaging* ; HL-60 Cells ; K562 Cells ; Cell Line, Tumor ; U937 Cells

OBJECTIVE: To explore the immunophenotypic characteristics of bone marrow granulocytes (G) and their clinical significance in patients with multiple myeloma (MM). METHODS: The granulocyte immunophenotypes of bone marrow in 70 MM patients (MM group) and 40 anemia patients (control group) were detected by flow cytometry, and its correlation with clinical characteristics was further analyzed. Univariate and multivariate regression analysis were used to screen factors that affected prognosis. RESULTS: The CD56⁺G%, CD13⁺G%, CD22⁺G% and CD117⁺G% in MM group were higher than those in the control group (all P <0.05). CD56⁺G% and CD117⁺G% in CR⁺VGPR group were significantly lower than those in PR+MR+PD group (both P <0.05). The CD10⁺G% in RISS Ⅲ stage and Ca²⁺ ≥2.65 mmol/L groups were increased (both P <0.05). The CD56⁺G% in elevated lactate dehydrogenase, β₂-microglobulin≥5.5 mg/L and hemoglobin <85 g/L groups were increased (all P <0.05), while the CD117⁺G% in high-risk cytogenetic positive group was decreased (P <0.05). The expression rate of CD molecules on granulocytes was divided into low (L) and high (H) groups according to the median value. The overall survival (OS) of the LCD56⁺G%, LCD13⁺G% and LCD22⁺G% groups was significantly prolonged (all P <0.05). CD13⁺G% and CD22⁺G% were independent risk factors for OS in MM patients (HR=0.443, 0.410, both P <0.05). CONCLUSION The CD56⁺G%, CD10⁺G% and CD117⁺G% are closely correlated with clinical features in MM patients, while CD13⁺G% and CD22⁺G% are closely correlated with prognosis. Detection of CD molecules expression on granulocytes may be used to evaluate prognosis and guide treatment.
Humans ; Multiple Myeloma/immunology* ; Granulocytes/immunology* ; Prognosis ; Immunophenotyping ; Male ; Bone Marrow ; Female ; Flow Cytometry ; Middle Aged ; Aged ; Clinical Relevance

OBJECTIVE: To explore the effect and mechanism of DNA methyltransferase 1 (DNMT1) knockout on the inhibition of acute myeloid leukemia (AML) by natural killer (NK) cells. METHODS: The peripheral blood NK cells of AML patients and controls were collected, and the mRNA and protein level of DNMT1 were measured by PCR and Western blot, respectively. The DNMT1 knockout mice were constructed to obtain NK^DNMT1-/- cells. The NK cells were stimulated with interleukin (IL)-12, IL-15, and IL-18 to construct memory NK cells, and then the interferon-γ (IFN-γ) levels were measured by ELISA. After co-culturing with memory NK cells and HL60 cells, the killing effect of NK^DNMT1-/- cells on HL60 cells was detected by LDH assay. Then, the HL60 cell apoptosis and NK cell NKG2D level were measured by flow cytometry. The perforin and granzyme B protein levels of NK cells were measured by Western blot. The AML model mice were constructed by injecting HL60 cells into the tail vein, meanwhile, memory NK cells were also injected, and then the mouse weights, CD33 positive rates, and survival time were detected. RESULTS: The mRNA and protein levels of DNMT1 in NK cells of AML patients were significantly higher than those in the control group (both P < 0.01), while the IFN-γ level induced by interleukin was significantly lower than that in the control group (P < 0.05). Compared with NK^DNMT1+/+ cells, the ability of NK^DNMT1-/- cells to secrete IFN-γ after interleukin stimulation was significantly increased (P < 0.05). The killing and apoptosis-inducing effects of NK^DNMT1-/- cells on HL60 cells were significantly stronger than those of NK^DNMT1+/+ cells (both P < 0.05). The NKG2D level and expression of perforin and granzyme B of NK^DNMT1-/- cells were significantly increased compared with NK^DNMT1+/+ cells (all P < 0.05). Compared with AML mice injected with NK^DNMT1+/+ cells, AML mice injected with NK^DNMT1-/- cells showed significantly increased body weight, decreased CD33 positive rate, and prolonged survival time (all P < 0.05). CONCLUSION Knocking out DNMT1 can enhance the inhibitory effect of NK cells on AML, which may be related to enhancing NK cell memory function.
Killer Cells, Natural/metabolism* ; Animals ; Leukemia, Myeloid, Acute ; Humans ; DNA (Cytosine-5-)-Methyltransferase 1 ; Mice ; Mice, Knockout ; HL-60 Cells ; Apoptosis ; Interferon-gamma/metabolism* ; Granzymes/metabolism* ; Perforin/metabolism* ; NK Cell Lectin-Like Receptor Subfamily K/metabolism*

OBJECTIVE: To screen factors affecting the prognosis of chronic myeloid leukemia (CML) patients, and construct a nomogram model for event-free survival (EFS). METHODS: To screen out meaningful variables by univariate and multivariate Cox regression analysis in CML patients, and construct a nomogram model using R software. The nomogram was validated using consistency index (C-index), receiver operating characteristic (ROC) curve, area under the ROC curve (AUC), calibration curve, decision curve analysis (DCA), and risk stratification analysis. RESULTS: This study analyzed data from 116 CML patients. Univariate and multivariate Cox regression analysis demonstrated that age, peripheral blood basophil percentage, BCR-ABL1 ^IS at 3 months, and red blood cell distribution width (RDW) were independent prognostic factors of EFS. Subsequently, a nomogram was constructed based on the above predictors. The C-index of the nomogram was 0.733(95%CI : 0.676-0.790). The AUC values for predicting 1-, 3-, and 5-year EFS rate were 0.765, 0.855, and 0.827, respectively. The results of the calibration curve and DCA curve showed that the predictive model had good consistency, as well as strong clinical utility. The patients were stratified into high-risk group and low-risk group based on the total score of the model, there was a significant difference in EFS between the two groups (P < 0.001). CONCLUSION Age, peripheral blood basophil percentage, BCR-ABL1 ^IS at 3 months, and RDW were associated with the prognosis of CML patients. The nomogram model constructed in this study can accurately predict the prognostic status of CML patients, but its widespread application still requires external and prospective validation.
Nomograms ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality* ; Proportional Hazards Models ; Erythrocyte Indices ; Risk Assessment/methods* ; Fusion Proteins, bcr-abl/genetics* ; Basophils ; Leukocyte Count ; Humans

OBJECTIVE: To investigate the mechanism of circular RNA RAD18 (CircRAD18 ) in regulating daunorubicin (DNR) resistance in acute myeloid leukemia (AML) cells through the miR-185-5p/hepatoma-derived growth factor ( HDGF) axis. METHODS: Real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CircRAD18 , miR-185-5p, and HDGF in human AML cell lines HL-60, U937, and human AML drug-resistant cell line KG1a. KG1a cells were cultured in vitro and randomly divided into control group, DNR group, DNR+negative control group, DNR+CircRAD18 knockdown group, and DNR+CircRAD18 knockdown+miR-185-5p inhibitor group. After transfection, real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CircRAD18 , miR-185-5p, and HDGF of cells, CCK-8 method and Ki-67 immunofluorescence staining were applied to detect cell proliferation, flow cytometry was applied to detect cell apoptosis, and immunoblotting was applied to detect the expression of cell proliferation, apoptosis and drug resistance related proteins in each group. The double luciferase reporter gene experiment was applied to detect the targeting regulation of CircRAD18 on miR-185-5p, and miR-185-5p on HDGF in KG1a cells. RESULTS: Compared with HL-60 and U937 cells, the expression of CircRAD18 , and HDGF mRNA and protein in KG1a cells increased (all P <0.05), while miR-185-5p decreased ( P <0.05). Compared with the control group, the CircRAD18 expression, HDGF mRNA and protein expression, cell viability, proliferation rate, and PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown group decreased (all P <0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression increased (all P <0.05). There were no obvious changes in all indicators of cells in the DNR group compared with control group ( P >0.05). Compared with the DNR group, the CircRAD18 expression, HDGF mRNA and protein expression, cell viability, proliferation rate, PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown group decreased (all P < 0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression increased (all P < 0.05). There were no obvious changes in all indicators of cells in the DNR+negative control group compared with DNR group (P >0.05). Compared with the DNR+CircRAD18 knockdown group, the HDGF mRNA and protein expression, cell viability, proliferation rate, PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown+miR-185-5p inhibitor group increased (all P < 0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression decreased (all P < 0.05). CircRAD18 was able to target and down-regulate the expression of miR-185-5p in KG1a cells, and miR-185-5p was able to target and down-regulate the HDGF expression. CONCLUSION Knocking down CircRAD18 can reduce HDGF expression by up-regulating miR-185-5p, thereby weakening DNR resistance in AML cells, inhibiting KG1a cell proliferation under DNR treatment, and promoting apoptosis.
Humans ; MicroRNAs/metabolism* ; Leukemia, Myeloid, Acute ; Daunorubicin/pharmacology* ; Drug Resistance, Neoplasm ; Apoptosis ; RNA, Circular ; Intercellular Signaling Peptides and Proteins/metabolism* ; Cell Proliferation ; HL-60 Cells ; Cell Line, Tumor

Lung cancer is the malignant tumor with the highest mortality rate worldwide. The tumor microenvironment (TME) is a key factor in the progression of lung cancer, composed of tumor cells, signaling molecules, fibroblasts, immune cells, etc. Among them, tumor associated neutrophil (TAN), as an important component of immune cells in the TME, plays multiple roles in tumor cell proliferation, invasion, angiogenesis, and metastasis due to its aberrant function, and is closely associated with poor prognosis. However, there are limited researches on the mechanism of TAN in lung cancer. This review aims to provide more scientific basis for studying the therapeutic targets of lung cancer and developing new drugs by elucidating the different subtypes of TAN and their mechanisms of action in the occurrence and development of lung cancer.
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Humans ; Neutrophils/pathology* ; Lung Neoplasms/pathology* ; Tumor Microenvironment ; Animals

Lung cancer is one of the most common and lethal malignancies in China. In the context of the tumor microenvironment, neutrophil extracellular traps (NETs) released by neutrophils exert a profound impact on the occurrence and progression of lung cancer. Although the exact mechanisms by which NETs promote tumor growth have not been fully elucidated, existing research has revealed their multiple roles in tumor growth, invasion, metastasis, and cancer-related thrombosis. This article will review the molecular biology mechanisms and research progress of NETs in lung cancer based on recent studies.
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Extracellular Traps/metabolism* ; Humans ; Lung Neoplasms/drug therapy* ; Neutrophils/immunology* ; Animals ; Tumor Microenvironment