Liriope platyphylla seeds (LLPS) using network pharmacology, molecular docking, and in vitro experiments. Here GC–MS analysis tentatively identified forty-three lipophilic compounds in LLPS. LLPS exhibited powerful antioxidant activity, according to the results from chemical-based antioxidant assays on DPPH, ABTS+, superoxide anion, hydrogen peroxide, nitric oxide, and hydroxyl radicals scavenging, lipid peroxidation, reducing antioxidant powers, and total antioxidant capacity. Additionally, LLPS enhanced cellular antioxidant capacity by inhibiting reactive oxygen species formation and elevating antioxidant enzyme levels, including catalase and heme oxygenase-1. Moreover, LLPS attenuated inflammatory response by reducing nitric oxide secretion and downregulating the expression of inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-1β in lipopolysaccharide-treated macrophages. Network pharmacology and molecular docking analyses showed that key compounds in LPPS, particularly phytosterols and fatty acid esters, exerted antioxidant and anti-inflammatory properties through regulating NFKB1, PTGS1, PTGS2, TLR4, PRKCA, PRKCD, KEAP1, NFE2L2, and NR1l2. Overall, these data suggest that LLPS may be a potential antioxidant and anti-inflammatory agent for developing functional foods.Antioxidant and anti-inflammatory mechanisms counteract the pathogenesis of chronic diseases, such as diabetes, aging, and cancer. Therefore, enhancing antioxidant and anti-inflammatory functions may help manage these pathological conditions. This study aimed to assess the antioxidant and anti-inflammatory potentials of lipophilic fraction of  Systemic, or chronic, inflammation is closely associated with the pathologies of a wide variety of diseases, such as inflammatory bowel disease, psoriasis, atherosclerosis, and cancer . During ROS and RNS, including both free radical and non-free radical species such as superoxide anion radical, hydrogen peroxide, hydroxyl radical, and nitric oxide, are the products of normal cellular metabolisms. ROS/RNS overproduction overwhelms antioxidant defense systems, leading to oxidative/nitrosative stresses that damage cellular structures and signaling pathways and ultimately trigger pathogenesis and the aging process ,5. ThereLiriope platyphylla is an herbaceous perennial seed-producing plant belonging to the Liliaceae family and is widely distributed in mountainous areas of temperate regions of East Asia. It has been used as food and traditional medicine for cough, asthma, lung inflammation, and neurodegenerative diseases in Korea, China, and Japan -associated protein 1 (KEAP1) is a negative regulator of nuclear factor erythroid 2-related factor 2 , which is a master transcription factor mediating redox homeostasis. Under physiological condition, NFE2L2 is sequestered in the cytosol by KEAP1. However, in response to electrophiles and oxidative stress, NFE2L2-KEAP1 complex is dissociated, leading to NFE2L2 liberation and subsequent nuclear translocation to drive the transcription of a number of antioxidant genes, namely, heme oxygenase 1 (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) .S-transferase  × 100Lipid peroxidation was determined using the TBA method . The samThe FRAP was estimated using a slightly modified procedure as outlined in a previous study . The samThe PFRAP was estimated using a previously outlined method with minor modifications . The samThe TAC was determined following the phosphomolybdenum method . The sam2.Murine RAW 264.7 monocyte–macrophage cells, purchased from the American Type Culture Collection , were nourished in Dulbecco’s modified Eagle’s medium added with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified incubator with 5% CORAW 264.7 cells were seeded in a 96-well plate and incubated for 24 h. After pretreatment with LLPS (10 or 100 µg/mL) for 1 h, the cells were challenged with LPS (1 µg/mL) for an additional 6 h. Afterward, the cells were incubated with 20 µM of DCFH-DA in the dark at 37 °C for 1 h. After washing off the probe, the production of intracellular ROS was measured at the wavelengths of 282/20 nm excitation and 528/20 nm emission with a microplate reader (BioTek).2.The NO level in culture media was determined by the Griess reagent. After treatment, the culture medium was incubated with an equal amount of the Griess reagent for 10 min, and absorbance was read at 546 nm using a microplate reader (BioTek). The NO concentration was quantified based on a standard curve of NaNOTreated cells were homogenized in RIPA buffer  and incubated on ice for 1 h. After centrifugation, total protein content was quantified using a BCA protein assay kit (Thermo Scientific). Equal amounts of protein were separated on SDS–PAGE gels and transferred onto PVDF. The membranes were blocked with 5% milk in TBST , followed by incubation with specific primary antibodies at 4 °C for 24 h. After washing, the membranes were hybridized with appropriate secondary antibodies at 4 °C for 3 h. Eventually, blots were visualized using a wsE-7120 Ez WestLumi plus reagent .https://pubchem.ncbi.nlm.nih.gov/, accessed on 1 June 2023). The potential targets of bioactive compounds were predicted using SwissTargetPrediction  [https://prediction.charite.de/, accessed on 1 June 2023) [https://www.uniprot.org/, accessed on 1 June 2023) for standardizing gene names. The GeneCards database , Online Mendelian Inheritance in Man (OMIM) database , and Comparative Toxicogenomics database  were utilized to screen out target genes related to inflammation, anti-inflammation, oxidation, and antioxidant.The canonical SMILES structures of lipophilic compounds in LLPS were collected from the PubChem database (ne 2023)  and Supene 2023)  webtoolshttps://bioinformatics.psb.ugent.be/webtools/Venn/, accessed on 2 June 2023) to identify overlapping genes between LLPS compounds-, antioxidant-, and inflammation-target genes. These genes were then interconnected with the compounds to generate the compound–target network, which was visualized and analyzed using Cytoscape 3.9.1 software. Bioactive compounds and genes targets were represented by the nodes, while their interaction was explained by the edges. The 20 most important target nodes were then selected to analyze the protein–protein interaction network through the STRING database . The top 10 hub genes were then determined from the PPI network using the maximal clique centrality (MCC) method of CytoHubba in Cytoscape. The degree value was reflected by the color and the size of the node. The functional properties of LLPS were analyzed through the enrichment of the common genes using Database for Annotation, Visualization, and Integrated Discovery . The most important enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results were visualized through the SRPlot platform .A Venn diagram was generated using bioinformatic webtools . After removing water molecules, adding polar hydrogen, and calculating Kollman charges, simulation docking of bioactive compounds and target proteins was performed using AutoDock Vina 1.5.6 software. The best docking poses were visualized by Discovery Studio 2021 software.The 3D conformations of potential bioactive compounds were acquired from the PubChem database and charged with Gasteiger partial charges using OpenBabel 2.4.1. The structures of the target proteins were retrieved from the Protein Data Bank . Mean comparisons were executed using the one-way analysis of variance followed by Tukey’s post hoc test. The difference with a This study demonstrated that LLPS harbored a powerful antioxidant activity, as evident from the in vitro chemical- and cell-based assays. Also, LLPS exerted anti-inflammatory ability by reducing the levels of pro-inflammatory cytokines and mediators in LPS-stimulated RAW 264.7 cells. Network pharmacology and molecular docking analyses shed light on the underlying mechanisms of LLPS, revealing that bioactive compounds, primarily phytosterols and fatty acid esters, affected core targets, including NFKB1, PTGS1, PTGS2, TLR4, PRKCA, PRKCD, KEAP1, NFE2L2, and NR1l2. Most enriched pathways, such as the TLR, PI3k-Akt, MAPK, NF-κB, and HIF-1 signaling pathways, were associated with inflammation and oxidative stress, underscoring their pivotal roles in antioxidant and anti-inflammatory effects of LLPS. However, this study has limitations, notably the absence of molecular dynamics simulations and binding energy calculations, which could have provided a more detailed understanding of ligand–protein interactions and binding energetics. Further research should address these shortcomings and comprehensively investigate LLPS’s antioxidant and anti-inflammatory properties and its underlying mechanisms in specific models. In conclusion, these data suggest that LLPS may be a promising agent for developing nutraceuticals and functional foods with antioxidant and anti-inflammatory capacities.


To gain deeper insights into the microenvironment of breast cancer, we utilized GeoMx Digital Spatial Profiling (DSP) technology to analyze transcripts from 107 regions of interest in 65 untreated breast cancer tissue samples. Our study revealed spatial heterogeneity in the expression of marker genes in tumor cell enriched, immune cell enriched, and normal epithelial areas. We evaluated a total of 55 prognostic markers in tumor cell enriched regions and 15 in immune cell enriched regions, identifying that tumor cell enriched regions had higher levels of follicular helper T cells, resting dendritic cells, and plasma cells than immune cell enriched regions, while the levels of resting CD4 memory in T cells and regulatory (Treg) T cells were lower. Additionally, we analyzed the heterogeneity of HLA gene families, immunological checkpoints, and metabolic genes in these areas. Through univariate Cox analysis, we identified 5 prognosis-related metabolic genes. Furthermore, we conducted immunostaining experiments, including EMILIN2, SURF4, and LYPLA1, to verify our findings. Our investigation into the spatial heterogeneity of the breast cancer tumor environment has led to the discovery of specific diagnostic and prognostic markers in breast cancer.The online version contains supplementary material available at 10.1186/s12967-023-04395-x. Breast cancer is the most prevalent and lethal malignant tumor in women, representing a significant global threat to their life and health. Traditional clinical and pathological staging of breast cancer, along with molecular biological characteristics, offers clinical criteria for prognostic evaluation of breast cancer patients . NeverthThe tumor microenvironment (TME) is a complex network system composed of various cells and cytokines that exhibit high heterogeneity. This heterogeneity contributes to different treatment responses and clinical outcomes in cancer patients , posing There is substantial evidence showing a noteworthy association between breast cancer progression and prognosis with metabolic alterations . One exaThe heterogeneity of tumors plays a significant role in disease progression. Identification of the molecular drivers of heterogeneity, particularly in the TME will guide future diagnostic and therapeutic strategies. In this study, we employed GeoMx Digital Spatial Profiling (DSP) technology to conduct spatial transcriptomic sequencing on different regions of clinical breast cancer tissue. Our goal was to investigate the spatial expression patterns of genes and explore the interactions between tumor cells and immune cells in the TME. The objective was to reveal the spatial heterogeneity of gene expression patterns and their clinical significance in the TME, and to provide new strategies for prognostic evaluation and immune therapy of breast cancer.2 tissue cores from 125 patients. We acknowledged that the GeoMx Digital Spatial Profiler has spatial limitations in selecting regions of interest (ROI) within a certain range and may not cover the entire tissue chip. Additionally, we experienced some technical challenges during the experimental process, resulting in the loss of a few cores. As a result, we were able to obtain 65 relatively intact cores for further analysis. Subsequently, 65 cores comprising 107 regions of interest  were subjected to DSP RNA assays.Paraffin-embedded tumor tissue samples were obtained from 125 breast cancer patients who underwent initial surgical operation without prior treatment at the Houjie Hospital of Dongguan, Affiliated Houjie Hospital of Guangdong Medical University. The Ethics and Scientific Committee of the Houjie Hospital of Dongguan, Affiliated Houjie Hospital of Guangdong Medical University approved this study (2021002). A tissue microarray (TMA) was constructed using a TMArrayer (Pathology Devices) to obtain 1.5 mmFor the immunohistochemistry analysis, 125 instances of breast cancer tissue were included in the study. However, 26 cores were excluded from EMILIN2 analysis, 23 cores were excluded from LYPLA1 analysis, and 28 cores were excluded from SURF4 analysis due to loss of tissue integrity during staining.The NanoString GeoMx DSP RNA assays were conducted at CapitalBio Technology  according to the recommended procedure as previously described . To distThe Mann-Whitney U test was employed to compare two groups. Genes were considered significant if they had a fold change of at least 1.5 and a p-value less than 0.05. To conduct gene ontology (GO) enrichment and KEGG enrichment analysis of differentially expressed genes (DEGs), EnrichProfiler R-packages were used, with Benjamini-Hochberg multiple testing correction.http://www.string-db.org/), with network type (complete STRING network), meaning of network edges (confidence), and minimum interaction score parameters being set. The protein interaction data was input into the Cytoscape software using the STRING database, and the core genes in the protein-protein interaction (PPI) network were selected using the cytoHubba plugin.The WGCNA and limma software were employed to conduct a weighted correlation network analysis (WGCNA) on differentially expressed genes (DEGs) from 65 PanCK-expressing and 36 CD45-expressing areas. A Pearson correlation coefficient matrix was constructed from the gene expression matrix, and an adjacency matrix was generated using the optimal power value of the Pearson correlation coefficient matrix. To perform hierarchical clustering by module, a topology overlap matrix (TOM) was constructed, and the clustered modules were automatically separated and merged using the cutree Dynamic function. Both turquoise and blue module genes were subjected to functional enrichment analysis, including gene ontology (GO) and KEGG, using R packages such as clusterProfiler, enrichplot, org.Hs.eg.db, and ggplot2. Genes from the turquoise module and the blue module were separately uploaded to the STRING database . Differences in immune cell levels among the three regions were assessed using the Kruskal-Wallis test and the reshape2 and ggpubr packages. A gene set related to immunological function was identified based on previous studies . Single-The levels of HLA gene expression in the three areas were assessed using the Kruskal-Wallis test with the aid of the limma, reshape2, ggplot2, and ggpubr packages. The divergent expression of immune checkpoint-related genes across the three areas was investigated through the Wilcoxon test using the limma, ggplot2, and ggpubr packages. A correlation analysis was conducted between immune checkpoint gene expression and immune cell levels in PanCK and CD45-expressing areas using the Spearman correlation coefficient and the limma, reshape2, tidyverse, and ggplot2 packages. The findings were presented in a visual format.http://www.gsea-msigdb.org/gsea/downloads.jsp) provided the “c2.cp.kegg.v7.5.1.symbols.gmt” file, which was used to perform GSVA on the ROIs using the GSEABase, GSVA, limma, and pheatmap packages. Only 20 pathways were selected, and the parameters were set to P < 0.05. A Venn diagram was created by intersecting the 944 metabolic genes with 3515 differential genes (PanCK- versus CD45-expressing areas) obtained from the Bioinformatics website (http://bioinformatics.psb.ugent.be/webtools/Venn/), resulting in 182 genes. Univariate Cox analysis was conducted on the 182 metabolic genes in the 65 PanCK-expressing ROIs to identify genes associated with prognosis, followed by survival analysis and ROC curve plotting using the Survivor, Survminer, and timeROC packages in R.Based on previous literature, we identified 944 genes involved in metabolic processes . The GenThe TMA sections were incubated overnight with anti-human EMILIN2 , SURF4 , or LYPLA1  antibodies, followed by incubation with a secondary antibody and then subjected to the liquid DAB substrate-chromogen system. The experiments were conducted in accordance with the clinical pathological protocols of the Affiliated Houjie Hospital of Guangdong Medical University.Fluorescent anti-PanCK and anti-CD45 antibodies were employed to identify the 107 regions of interest (ROIs) in 65 patients with breast cancer. The pathologic morphology of PanCK-expressing ROIs allowed us to differentiate between normal epithelial cells and tumor cells. Consequently, 65 ROIs represented tumor cells (PanCK-expressing), 36 ROIs represented immune cells (CD45-expressing), and 6 ROIs represented normal epithelial cells (NC), as shown in Fig. ALCAM, ARL6IP1, CCNG2, CCT2, CFB, COBL, COX7C, CTNND1, DCTN1, DEPTOR, GUCD1, MRPL19, MRPL49, NDUFB1, NFIC, NUMA1, OAT, PKP3, PPT1, PRKACA, RPS13, SIPA1L3, SREBF1, TNFRSF25, TNRC18, UGDH, and YIPF3, while APOBR, C12orf40, CD226, CD248, CD93, CDH5, COL6A1, COL6A2, EMILIN2, FCHO1, FOXP3, HIC1, ISM2, ITPRIPL1, LAMA4, MFAP4, MMP1, MMP2, MSR1, PDE4D, PIK3CG, PRKCQ, PRRX1, PTPN7, RNASE6, SLC8A1, TGFBI, and TPSAB1 acted as risk factors. Higher expression of the protective factors was associated with better prognosis for breast cancer patients, whereas higher expression of risk factors was associated with poorer prognosis , while the blue module was significantly positively associated with CD45 expression (r = 0.34 and P = 7e − 04) Fig. A. Based The relative levels of 22 immune cells in 107 ROIs are shown in Fig. The expression of immune cells and their functions varied among different regions Fig. : Fig. 2.TNFRSF8, CD27, CD28, CD40, CD44, CD48, CD86, HAVCR2, ICOS, IDO1, IDO2, KIR3DL1, LAG3, LAIR1, TIGIT, TNFRSF25, BTLA, BTNL2, CD160, CD200, CD200R1, CD244, CD274, CD40LG, CD70, CD80, CTLA4, ICOSLG, PDCD1, PDCD1LG2, TMIGD2, TNFRSF14, TNFRSF4, TNFRSF9, TNFSF14, TNFSF15, TNFSF18, TNFSF4, and TNFSF9, compared to PanCK-expressing areas .The expression of metabolic genes varied between PanCK-expressing and CD45-expressing regions. In PanCK-expressing regions, metabolic genes were primarily involved in citrate cycle TCA cycle, glycosphingolipid biosynthesis LATCO and NEOLATCO series, N glycan biosynthesis, glycosaminoglycan biosynthesis keratan sulfate, RNA polymerase, and pyrimidine metabolism. In contrast, in CD45-expressing regions, metabolic genes were predominantly involved in the calcium signaling pathway, VEGF signaling pathway, FC epsilon RI signaling pathway, vascular smooth muscle contraction, GNRH signaling pathway, retinol metabolism, arachidonic acid metabolism, and linoleic acid metabolism Fig. A. The inect Fig. C. Higherval Fig. D. FigureEstrogens signaling pathways play a critical role in the development, progression, and survival of breast cancer. In our study, we categorized the estrogen receptor (ER) expression levels in breast cancer patient tissues and identified differentially expressed genes between the negative and positive groups. In ER-positive tissues, the differentially expressed genes were primarily associated with porphyrin and chlorophyll metabolism, fructose and mannose metabolism, lysosome, and glycosphingolipid biosynthesis globo series. Conversely, in ER-negative tissues, the genes were predominantly associated with primary immunodeficiency, alzheimer’s disease, and pathways in cancer  and CD45-expressing (immune cells) regions, the study identified distinct sets of genes associated with breast cancer prognosis. These findings shed light on the role of specific genes and immune cell populations in tumor development and patient outcomes.Using DSP technology, we examined 15 prognosis markers in immune cell-enriched regions and 55 prognostic gene markers in tumor cell-enriched regions. The sets of genes identified through WGCNA analysis and univariate Cox regression analysis are particularly noteworthy. The turquoise module genes were positively correlated with PanCK expression. These genes, including rognosis . High exrognosis , 17.The analysis of various tumors through histopathology has confirmed that the composition of infiltrating immune cells within the tumor is distinct from that in the surrounding non-tumor area. The interaction between tumor cells and adjacent stromal cells is an important driving force in promoting tumor progression . The stuThe development and progression of tumors are closely linked to metabolism, and metabolic genes may play a significant role in the prognosis of cancer patients . This stIn conclusion, this study enhances our understanding of breast cancer biology, focusing on gene expression, immune infiltration, and metabolic processes. The identified gene sets and immune-related factors have potential as prognostic biomarkers and therapeutic targets for breast cancer patients. These insights into immune modulation and metabolic dysregulation offer novel treatment opportunities that can complement traditional approaches. The findings underscore the importance of personalized treatment approaches that consider the unique molecular and immune characteristics of individual tumors. Overall, this research opens up new avenues for clinical interventions and may improve patient outcomes in breast cancer management.Additional file 1: Figure S1. Survival analysis of prognostic genes.A Survival analysis of genes in the turquoise module in tumor cell enriched regions. B Survival analysis of genes in the blue module in immune cell enriched regions. Figure S2. Differential expression of immune checkpoint-related genes among the three regions. Data shows the expression of immune checkpoint-related genes in tumor cell (PanCK), immune cell (CD45), and normal epithelial cells enriched regions (NC). Figure S3. Analysis of ER-related genes. A GSVA enrichment analysis of PanCK-expressing and CD45-expressing regions. B There were 944 metabolic genes, 3515 differentially expressed genes (PanCK-expressing regions vs. CD45-expressing regions), and 182 intersecting genes. C Univariate Cox analysis was performed on 134 intersecting genes in PanCK-expressing regions.D Survival analysis was conducted on prognostic metabolic genes in PanCK-expressing regions. E ROC curves were used to predict the survival rates of breast cancer patients. For the Kaplan Meier curves, the p values were calculated using the log-rank test. Table S1. Characteristics of breast cancer patients (n=125).


Diabetic cardiomyopathy (DCM) is a serious complication and death cause of diabetes mellitus (DM). Recent cardiology studies suggest that spermidine (SPD) has cardioprotective effects. Here, we verified the hypothesis of SPD’s protective effects on DCM. Therefore, db/db mice and primary neonatal mouse cardiomyocytes were used to observe the effects of SPD. Immunoblotting showed that ornithine decarboxylase (ODC) and SPD/spermine N1-acetyltransferase (SSAT) were downregulated and upregulated in the myocardium of db/db mice, respectively. We found that diabetic mice showed cardiac dysfunction in 12 weeks. Conversely, exogenous SPD could improve cardiac functions and reduce the deposition of collagens, myocardial damage, reactive oxygen species (ROS) levels, and endoplasmic reticulum stress (ERS) in diabetic mouse hearts. Our results also demonstrated that cardiomyocytes displayed ferroptosis and then activated Pannexin-1 expression, which resulted in the increase of the extracellular adenosine triphosphate (ATP). Subsequently, increased ATP as a paracrine molecule combined to purinergic receptor P2X7 to activate ERK1/2 signaling pathway in cardiomyocytes and activated NCOA4-mediated ferroptinophagy to promote lipid peroxidation and ferroptosis. Interestingly, SPD could reverse these molecular processes. Our findings indicate an important new mechanism for DCM and suggest that SPD has potential applicability to protect against deterioration of cardiac function with DCM. Diabetes mellitus (DM) is a metabolic disease, divided into type 1 and type 2. The former is usually induced by congenital pancreatic islet dysfunction, and the latter with the higher incidence is mostly caused by insulin resistance . DCM is Polyamines (PAs) are small branched chain cationic molecule derived from a linear amino acid, including spermine (SP), spermidine (SPD), and putrescine (PU), and are widely distributed in all types of mammalian cells . IntraceOur team, and others, recently corroborated that SPD and SP levels were decreased in diabetic rats , 16, andn ═ 8 per group): (1) Wild-type (WT) group: eight-week-old male C57BL/6 mice were injected with stroke-physiological saline solution buffer (SPSS); (2) WT+SPD group: C57BL/6 mice intraperitoneal injection of SPD  dissolved in SPSS (10 mg/kg); (3) Type 2 diabetes (T2D) group: db/db mice; (4) T2D+SPD group: db/db mice intraperitoneal injection of SPD (10 mg/kg in SPSS), the dosing interval of animals in each group was every other day. All mice in the four groups were sacrificed in week 12 and determined relevant experimental indexes according to the protocols.Homozygous eight-week-old male db/db mice (22 ± 0.5 g) on a C57BL/6 background were provided by the Animal Research Institute of Mudanjiang Medical University (MMU), and the study was approved by the MMU Medical Science Ethics Committee. All mice were maintained on a 12-h light/dark cycle and fed with a standard chow and clean water ad libitum. The mice were randomly divided into four groups  containing 10% fetal bovine serum and 1% penicillin and streptomycin, and then abandoned DMEM, added 7 ml type II collagenase . Then put it on a shaker, 37 ∘C, 160 r/min, 10 min, and collected the supernatant DMEM in a 50 ml centrifuge tube (store at 4 ∘C). The abovementioned process was repeated 3–5 times until all hearts were completely digested. The cells in the 50 ml centrifuge tube were collected by centrifugation at 4 ∘C, 800 r/min, 5 min. Subsequently, added the medium and mixed well, inoculated in Petri dishes, and 2 h of incubation, the unattached cells were cardiomyocytes. The cardiomyocytes were planted in another Petri dish and maintained at 37 ∘C in a 5% CO2 humidified incubator. The media of cells was changed three times per week. Cardiomyocytes were treated with high glucose , SPD (10 µM), Erastin , P2X7 specific inhibitor , LY3214996 , according to the experiment protocol.Primary neonatal mouse cardiomyocytes were isolated from the hearts of 1–3-day old C57BL/6 mice. In brief, in a sterile operating table, the hearts were quickly removed, immediately placed in pre-chilled D-hanks solution. All hearts in the dish were washed and transferred to a 15 ml centrifuge tube, added 3 ml D-Hanks and 5 ml 0.25% trypsin, sealed, and placed on a shaker at 4 Blood glucose was measured from the mouse tail vein blood by a blood glucose meter . In week 12, blood samples taken from the medial canthal vein were centrifuged and serum was used to detect relevant indicators. Serum insulin levels were determined by ELISA kit . Serum levels of triacylglycerol (TG) and total cholesterol (TC) were analyzed using a standard biochemistry panel . Lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), and cardiac troponin-I (cTnI) in the blood serum were measured by using commercially available kits . All kits were operated according to the manufacturer’s instructions.Mice were intraperitoneally injected with D-glucose (2 g/kg mass). Tail vein blood was collected, and blood glucose was detected using a glucometer .Heart tissue and cardiomyocytes were homogenized with phosphate-buffered saline (PBS) and the supernatant was collected after centrifugation. The iron levels of tissue and cells were detected by the Iron Assay Kit  according to the manufacturer’s instructions. As described method in the previous protocol , glutath After anesthesia, the heart was quickly excised and washed with pre-cooled PBS buffer. The cardiac tissue was fixed in 10% buffered paraformaldehyde, embedded in paraffin, sliced at 4 mm, and used for morphological observation. Hematoxylin and eosin staining (HE), Masson trichrome staining, and Sirius red staining were conducted according to the dyeing protocol, tissue-stained sections were observed using computer-assisted color image analysis system .A Vivid 7 Dimension echocardiography machine was used to assess cardiac function and dimensions . All the mice were anesthetized with 2% isoflurane to perform echocardiography. Left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular internal dimension systole (LVIDs), and left ventricular internal dimension diastole (LVIDd) were measured.∘C, fixed in 1% osmium tetroxide for 2 h. Subsequently, heart tissue was dehydrated using a graded ethanol series, embedded in epoxy resin, and observed using a H-7650 transmission electron microscope .The ultrastructural analysis was performed as previously described . BrieflyHeart samples were lysed, and trypsin digested according to LC-MS/MS analysis procedure. Mass spectral data were retrieved using Proteome Discoverer (v2.4.1.15). The searching database is Mus_musculus_10090_SP_20201214.fasta (17063 sequences). A heat map was drawn by using the “gplots” R-package. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, enrichment of gene ontology (GO) biological process, cellular component, and molecular function terms were analyzed by using DAVIDs Functional Annotation Chart tool (Version 6.8) , 24.∘C in the dark. The images were then taken with a laser scanning confocal microscope  at an excitation 488 nm wavelength and emission 530 nm wavelengths and analyzed with Image J 1.46 software. Red fluorescence was JC1 aggregate, green fluorescence was JC1 monomer in the mitochondria.Δ ψm was determined using a JC1 Mitochondrial Membrane Potential Assay Kit . In brief, after treatment with HG and SPD, cardiomyocytes were incubated with 10 µM JC1 for 30 min at 37 ∘C for 30 min, followed by two rinses with cold PBS. Photos were captured immediately, and the fluorescence density was measured using Image J.The level of reactive oxygen species (ROS) was assessed through the use of dihydroethidium (DHE)  staining on freshly frozen tissue sections. A solution of 5 µM DHE dissolved in DMSO was added to the sections of mouse heart tissue after being diluted in PBS. The sections were then incubated in darkness at a temperature of 37 ∘C condition for 20 min. The cells were washed three times with serum-free cell culture medium to fully remove DCFH-DA, and then used laser scanning confocal microscope  at an excitation 488 nm wavelength and emission 525 nm wavelength to take photograph and analyzed with Image J 1.46 software. Lipid ROS was detected by using BODIPY 581/591 C11 fluorescent probe  as the previous protocol [Oxidative stress was detected using an ROS kit . Briefly, diluted DCFH-DA with serum-free medium at 1:1000 to make the final concentration 10 µM, removed the cardiomyocytes culture medium and added an appropriate volume of diluted DCFH-DA, incubated in 37 ∘C. After rinsing, the slices were incubated with corresponding fluorescent-conjugated secondary antibody , stained nuclei with DAPI, and observed using laser scanning confocal microscope .Frozen sections of heart tissue were washed, fixed, and permeabilized. Subsequently, the slices were immunolabelled with specific primary antibodies (Pannexin-1 at 1:50 ratio) overnight at 4 The concentration of adenosine triphosphate (ATP) in cardiomyocytes lysate was measured using ATP Assay Kit , and intracellular ATP was measured according to the instructions provided by the manufacturer.To detect the concentration of extracellular ATP, the culture medium was processed by a special method . BrieflyThe cardiomyocytes were seeded in a 35 mm dish treated with Pannexin-1 short interfering RNAs , following the experimental protocol. In brief, cells were transfected by siRNA using PolyJetTM (Signagen), Con-siRNA, and Pannexin-1–siRNA and the transfection reagent was incubated for 12 h with serum-free medium to inhibit the relevant protein expression according to the manufacturer’s instructions and replaced with normal medium containing 10% FBS to continue to incubate for 12 h, and then the next experiment could be processed.∘C, the above process repeats four times. Solubilized proteins were centrifugated at 13,500 rpm for 25 min, the supernatant was then collected. The protein concentration of each sample was quantified using the BCA Protein Assay kit . Protein lysates of each group were separated by electrophoresis with SDS-PAGE and electro-transferred onto a PVDF membrane (Millipore). Non-specific proteins on membranes were blocked with 5% non-fat dried milk for 2 h at room temperature, the membranes were incubated overnight with the following primary antibodies : P2X7, ACSL-4, GPX-4, FTH-1, HO-1, NCOA-4, SSAT ; Prdx-1, TfR-1, p-p38, t-p38, p-JNK, t-JNK, GRP94, GRP78, ATF-4, Chop ; and ODC, ERK1/2, p-ERK1/2, Pannexin-1, β-actin and β-tubulin . And then the membranes were incubated with anti-mouse/anti-rabbit IgG antibody  at a 1:10000 dilution for 1 h at room temperature. The specific complex was determined by an enhanced chemiluminescent (ECL) kit  and a multiplex fluorescent imaging system .The mouse heart tissue and cardiomyocytes were homogenized in 0.5 ml of RIPA  buffer using small tubes and vibrated for every 10 min for 5 s at 4  To determine mRNA expression of Pannexin-1, GRP94, GRP78, ATF-4, and Chop, the total RNA was extracted from myocardial tissue and cardiac fibroblasts using the Qiagen RNeasy mini kit. One microgram of total RNA was used to synthesize cDNA, a portion of which  was used in a PCR with two appropriate primers . The mRNThe study was approved by the Mudanjiang Medical University (MMU) Medical Science Ethics Committee (20220516-17).P-value < 0.05 was considered statistically significant.All experiments were replicated at least three times independently. All data were expressed as mean value ± standard error of mean (SEM). Statistical analysis was performed by two-tailed Student t-test or one-way ANOVA, followed by the Bonferroni multiple comparison test using GraphPad Prism 9.0. To better observe the effects of SPD, we selected 8-week-old wild-type mice and db/db mice as our research objects, and intraperitoneally injected with SPD every other day for 12 weeks, respectively. Glucose levels, glucose intolerance, insulin levels, triglyceride (TG), and TC levels were examined on week 12, which recapitulated the hallmark features of type 2 diabetes. The results showed that compared with the WT group, the blood glucose levels and glucose intolerance at each time point were higher, the insulin levels were significantly increased, and TG and TC were also increased in the T2D group and T2D+SPD group, Interestingly, compared with T2D group, there were no differences of the above indexes in the T2D+SPD group, also SPD had no effects in the WT group –1F. It fTo determine whether SPD can improve heart function in db/db mice, echocardiography was performed. Compared with the T2D group, we observed that EF and FS were increased, LVIDs and LVIDd were decreased in the T2D+SPD group, and WT mice treated with SPD had no obvious changes of cardiac functions –2E.To verify that DCM can cause serious cardiac injury, the hearts of mice in each group were excised in week 12, HE staining indicated that the cardiac myocytes were disordered and hypertrophic, TEM results revealed myocardial myofilament lysis and mitochondrial edema, and SPD could mitigate these pathological changes in db/db mice . Masson We also detected the serum myocardial injury markers  in each group. The results showed that the contents of serum LDH, CK-MB, and cTnI in the T2D group were significantly higher than those in the WT group. Compared with the T2D group, the serum contents of above enzymes in T2D+SPD group decreased significantly. These indicated SPD has protective and therapeutic effects on myocardial injury caused by high glucose –2K. MoreP-value < 0.05, 169 proteins were identified as differentially expressed (DE) between the two groups (115 upregulated proteins and 54 downregulated proteins)  analysis of cardiac tissues in WT and db/db mice group was performed. Using the criteria fold change > 1.5 and roteins) . The majroteins)  and 3C. roteins) . DE protroteins) , we founWe detected the levels of pivotal PA metabolic enzymes in vivo and in vitro, the results showed that the cardiac expression of ODC was downregulated but SSAT was upregulated in db/db mice, and the expression trend is consistent with cell model results  and 4B. Excessive ROS deposition can activate ferroptosis-related pathways of cells by a series of cascade reactions , 28. In Immunofluorescence results showed that compared with the WT group, the Pannexin-1 fluorescence intensity significantly increased in the T2D group, and significantly decreased in the T2D+SPD group compared to the T2D group , which wTo further study the mechanism of SPD alleviating DCM, we used P2X7 specific inhibitor (A-438079) and ERK1/2 signaling pathway blocker (LY3214996) to treat with cardiomyocytes separately. The results showed that HG could increase p-ERK1/2 expression and had no effect on t-ERK1/2 expression, indicating that it can activate phosphorylated ERK1/2 to activate the pathway. Conversely, A-438079 and SPD could significantly reduce its expression . FurtherIn the present study, we selected db/db mouse, a model of type 2 diabetes as the research object, whose genetic background is C57BL/6 mouse diabetes gene knocked out and even insulin intervention fails to control hyperglycemia in db/db mouse. Therefore, db/db homozygous mice are widely used in the study of endocrine defects, neurological diseases, and cardiac diseases induced by abnormal glucose and lipid metabolism , 31. At At 12 weeks, echocardiography displayed that EF and FS were decreased, LVIDs and LVIDd were increased in the T2D group, indicating heart systolic dysfunction. Further myocardial injury marker enzyme test confirmed that LDH, CK-MB, and cTnI levels were increased in the blood serum, and SPD could attenuate the above indicators of heart damage. In the T2D group and T2D+SPD group, db/db mice showed a significant increase in body weight, however, treatment with SPD could decrease the HW/TL ratio and we suspected it was due to remodeling of the heart and increasing the myocardial extracellular matrix. This speculation is supported by cardiac morphology. In the T2D group, Masson and Sirius red staining showed large amounts of collagen deposition in the interstitial areas, TEM results presented obvious myofilament dissolution and mitochondrial edema, HE staining displayed db/db mouse heart eventuated degeneration and necrosis of cardiomyocytes, and SPD could inhibit these changes effectively. These results demonstrated that SPD could alleviate high glucose-induced myocardial damages.To further explore the mechanism of DCM in db/db mice, we used proteomics to analyze the differential expression of proteins in cardiac tissue between C57BL/6 and db/db mouse, and enrich in KEGG pathways. The results showed that DCM was closely related to ferroptosis pathway, MAPK signaling pathway, and gap junction, which indicated the next research direction of our project.ACSL-4 and GPX-4 are the central regulators of ferroptosis, and they are often used as a marker of ferroptosis , 33. In Oxidative stress causes disruptions in signaling pathways and oxidizes cellular components. As a result, the affected cells may become dysfunctional or undergo cell death through necrosis or apoptosis. Mitochondrial overproduction of ROS is the primary cause of damage, triggering increased polyol pathway flux, increased formation of advanced glycation end products (AGEs), and activation of the receptor for AGEs (RAGE) in DCM . OxidatiPannexin-1 belongs to the ATP-releasing pathway family and its function is the regulation of ATP flow out of the cell . By immuOur proteomic analyses have confirmed that MAPK signaling pathway is involved in the pathological process of DCM, and previous studies have shown that ferroptosis could activate the MAPK pathway through ERK1/2 , 42, we Taken as a whole, based on the aforementioned experimental results and present literature, it is hypothesized that in db/db mouse, persistent hyperglycemic stimulation leads to cardiomyocyte damages (ROS and ERS) and activation of ferroptosis pathway, and then upregulates the Pannexin-1 to open ATP outflow channel, resulting in an increase of extracellular ATP. Subsequently, increased ATP as a paracrine molecule combines to P2X7 receptors to activate ERK1/2 signaling pathway which regulate NCOA-4-mediated ferroptinophagy and antioxidant gene HO-1 expression. Conversely, SPD can reverse the above molecular regulation process see . Hopeful


Glucolipid metabolism plays an important role in the occurrence and development of diabetes mellitus. However, there is limited research on the characteristics of glucolipid metabolism and complications in different subgroups of newly diagnosed diabetes. This study aimed to investigate the characteristics of glucolipid metabolism and complications in novel cluster-based diabetes subgroups and explore the contributions of different glucolipid metabolism indicators to the occurrence of complications and pancreatic function.1C), homeostasis model assessment-2 beta-cell function (HOMA2-β), and homeostasis model assessment-2 insulin resistance (HOMA2-IR) were used as clustering variables. The participants were divided into 4 groups by k-means cluster analysis. The characteristics of glucolipid indicators and complications in each subgroup were analyzed. Regression analyses were used to evaluate the impact of glucolipid metabolism indicators on complications and pancreatic function.This retrospective study included 547 newly diagnosed type 2 diabetes patients. Age, body mass index (BMI), glycated hemoglobin , and 2-h postprandial plasma glucose (2hPG) were higher in the severe insulin-resistant diabetes (SIRD) and severe insulin-deficient diabetes (SIDD) groups. Fasting insulin (FINS), fasting C-peptide (FCP), 2-h postprandial insulin (2hINS), 2-h postprandial C-peptide (2hCP), and the monocyte-to-high-density lipoprotein cholesterol ratio (MHR) were higher in mild obesity-related diabetes (MOD) and SIRD. 2hCP, FCP, and FINS were positively correlated with HOMA2-β, while FPG, TyG, HbA1C, and TG were negatively correlated with HOMA2-β. FINS, FPG, FCP, and HbA1C were positively correlated with HOMA2-IR, while high-density lipoprotein (HDL) was negatively correlated with HOMA2-IR. FINS (odds ratio (OR),1.043;95% confidence interval (CI) 1.006 ~ 1.081), FCP , and TyG  contributed to increase the risk of nonalcoholic fatty liver disease (NAFLD); 2hINS  contributed to increase the risk of atherosclerotic cardiovascular disease (ASCVD); FCP  significantly increased the risk of chronic kidney disease (CKD).Total cholesterol (TC), triglycerides (TG), triglyceride glucose index (TyG), HbA1C, HDL and TG influenced the function of insulin. FINS, TyG, 2hINS, and FCP were associated with ASCVD, NAFLD, and CKD in newly diagnosed T2DM patients.There were differences in the characteristics of glucolipid metabolism as well as complications among different subgroups of newly diagnosed type 2 diabetes. 2hCP, FCP, FINS, FPG, TyG, HbA Diabetes mellitus has become the third largest noncommunicable chronic disease after cardiovascular diseases and cancer, severely impacting people's quality of life. In 2021, approximately 537 million adults worldwide had diabetes, and this number is projected to rise to 643 million by 2030 and 784 million by 2045 [1C), homeostasis model assessment-2 of insulin resistance (HOMA2-IR), homeostasis model assessment-2 of beta-cell function (HOMA2-β), and glutamic acid decarboxylase antibody (GADA), they identified five subgroups: severe autoimmune diabetes (SAID), severe insulin-deficient diabetes (SIDD), severe insulin-resistant diabetes (SIRD), mild obesity-related diabetes (MOD), and mild age-related diabetes (MARD) [The Ahlqvist team first applied cluster analysis to newly diagnosed diabetes patients. By using variables such as age at diagnosis, body mass index (BMI), glycated hemoglobin (HbAs (MARD) . The SAIs (MARD) –15. Howes (MARD) , 16–18.Trajectory analysis indicates that changes in metabolism and inflammation-related biomarkers begin more than 10 years before the onset of T2DM –21. NotaHowever, there is currently limited research on the characteristics of glucolipid metabolism in each subgroup and their relationship with complications. The contribution of various metabolism indicators to complications and pancreatic function is still unclear. This study explored the differences in glucolipid metabolism indicators among the subgroups and analyzed the occurrence of chronic complications. Additionally, the contributions of various glucolipid metabolism indicators to pancreatic function and complications were also discussed separately.Cross-sectional descriptive research was conducted on this issue. According to the diagnostic criteria of the World Health Organization , a total1C, total cholesterol (TC), triglycerides (TG), triglyceride glucose index (TyG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and monocyte/high-density lipoprotein ratio (MHR). The OGTT test , which involved measuring a glucose load of 75 g, was used to measure blood sugar levels and indicators related to pancreatic function, such as FPG (fasting plasma glucose), 2hPG , FCP (fasting C-peptide), 2hCP , FINS (fasting insulin level), and 2hINS . Apart from that, the other indicators were obtained from the venous blood collected in the antecubital fossa after the patient fasted for more than 8 h overnight, and the serum was separated at room temperature. All indicators were measured by a Hitachi 7600-DDP fully automatic biochemical analyzer  in our hospital's laboratory. Insulin determination was performed using electrochemiluminescence immunoassay , and HbA1C determination was performed using high-performance liquid chromatography . The HOMA-related indices were calculated by the Oxford University HOMA2 calculator [The data collection for the present study included demographic characteristics, glycemic and lipid metabolism indicators, and the occurrence of complications. Demographic characteristics included age, gender, height, weight, BMI, systolic blood pressure, diastolic blood pressure, history of hypertension, smoking history, and alcohol consumption history. Glycemic and lipid metabolism indicators were measured by using standard methods upon the patient's admission to the hospital, including fasting plasma glucose (FPG), fasting insulin (FINS), fasting C-peptide (FCP), 2-h postprandial plasma glucose (2hPG), 2-h postprandial insulin (2hINS), 2-h postprandial C-peptide (2hCP), HbAlculator . The trilculator . The datComplications were defined based on the ICD-10 codes  E11.4; CKD E11.2; diabetic retinopathy (DR) E11.5; peripheral vascular disease (PVD) I73; ASCVD ; NAFLD K76.0 or on the original diagnosis text of the physicians.1C, BMI, HOMA2-β, and HOMA2-IR were used as cluster variables for cluster analysis. The variables were standardized before clustering. Patients with extreme outliers (mean > 5 standard deviations) were excluded. K-means clustering was performed with a k value of 4 in SPSS 25.0.Based on the study of Ahlqvist et al. , age, HbAnalyses were conducted by SPSS 25.0 software . Non-normally distributed data were transformed using logarithmic conversion before clustering. Missing values were less than 5% and were imputed using the median or mode. Continuous variables that followed a normal distribution were presented as mean ± standard deviation, while non-normally distributed continuous variables were presented as median and interquartile range. All categorical variables were represented as numbers (proportions). Group comparisons of continuous variables were performed using one-way ANOVA or the Mann–Whitney U test. When comparing pairwise between groups, the least significant difference method was utilized. Group comparisons of categorical variables were conducted using the chi-square test. For conducting multiple comparisons of proportions among multiple groups, the Bonferroni correction was applied to adjust the significance level. Spearman correlation analysis was used to investigate the correlation between pancreatic function and glycemic and lipid metabolism indicators. Binary stepwise logistic regression analysis and multiple stepwise linear regression analysis were utilized to analyze the factors that influence the independent variables. The forward selection method was employed for regression analysis, with the first group being used as the reference category.1C, HOMA2-β, and HOMA2-IR, which were used as clustering variables. The four groups were SIDD, SIRD, MOD, and MARD. In the clustering analysis, the MARD group had the highest proportion of individuals, with 168 people (30.71%), followed by 141 people (25.78%) in the SIDD group and 136 people (24.86%) in the MOD group. The SIRD group had the fewest individuals, with 102 people (18.65%).A total of 547 patients with newly diagnosed T2DM were ultimately included in this study. As shown in Fig. P < 0.05). When conducting pairwise comparisons between groups, the MOD group had the highest number of people with a history of hypertension, while the SIRD group had the lowest number of people with a history of hypertension. The MOD group had the highest diastolic blood pressure, while the SIDD group had the lowest diastolic blood pressure. The MOD group had the highest weight and BMI, while the SIDD group had the lowest weight and BMI (P < 0.05).Table 1C, FPG and 2hPG in SIRD and SIDD groups were significantly higher than those in MOD and MARD groups. In addition, FINS, FCP, 2hINS, 2hCP, and MHR were higher in MOD and SIRD, especially in the MOD group. In the SIDD group, FINS, FCP, 2hINS, 2hCP, and MHR were the lowest, while LDL was relatively higher. In the SIRD group, HDL was the lowest while TG/HDL was the highest. As shown in Fig. P < 0.05). There was no statistically significant difference in the incidence rates of DPN, DR, and PVD among the groups (P > 0.05).Table 1C, and TG were negatively correlated with HOMA2-β. FINS, FPG, FCP, and HbA1C were positively correlated with HOMA2-IR, while HDL was negatively correlated with HOMA2-IR , FCP  and TyG  were associated with increased risk of NAFLD; 2hINS  was associated with increased risk of ASCVD; FCP  was associated with increased risk of CKD. When FINS, FCP, and TyG increased by one unit each, the risk of developing NAFLD increased by 1.043, 2.881, and 1.649 times respectively. When 2hINS increased by one unit, the risk of developing ASCVD increased by 1.015 times. When FCP increased by one unit, the risk of developing CKD increased by 1.297 times.Both the univariate and multivariate logistic analyses of NAFLD, ASCVD, and CKD were summarized in Table This cross-sectional research revealed the different glucolipid metabolism characteristics among subgroups of newly diagnosed T2DM patients. These characteristics were associated with varying incidence rates of complications in different subgroups. Different glucolipid metabolism indicators also contributed differently to pancreatic function and complications.1C, FPG, and 2hPG compared to the MARD group and the MOD group, indicating poorer glycemic control. The SIDD group had the lowest HOMA2-β index, as well as lower levels of insulin and C-peptide, suggesting impaired pancreatic beta-cell function. On the other hand, the MOD group had the highest HOMA2-β index and higher levels of insulin and C-peptide, indicating better pancreatic beta-cell function. MHR is a novel marker of inflammation that combines both inflammatory and anti-inflammatory factors. It reflects the inflammatory status and severity of the body better than a single inflammatory marker. It was demonstrated to be the highest in the SIRD group in our study. In terms of lipid metabolism, the SIRD group had the lowest HDL levels and the highest levels of TG/HDL, TC, TG, and TyG, indicating a more dyslipidemia profile. Previous studies have shown that TG/HDL can serve as a marker of insulin resistance in prediabetes [It has been confirmed that triglycerides, inflammatory processes, and insulin resistance are significantly related in different populations , 32. As diabetes . It has diabetes , 38.Due to poor compliance and inadequate control of blood glucose and other metabolic targets, there is a higher risk of complications in individuals with diabetes . It remaThere is a positive correlation between serum inflammatory biomarkers, lipid metabolism, and the occurrence and progression of complications in diabetic patients –47. When1C, and TG were correlated with HOMA2-β, while FINS, FPG, FCP, HbA1C and HDL were correlated with HOMA2-IR. Regarding the contributions of glucolipid metabolism markers to complications, FINS, TyG, 2hINS, and FCP showed differential superiority over other indicators. Cluster classification has promoted the progress of precision medicine, but clustering methods are often complex, requiring tedious calculation formulas and clustering software to categorize patients. This process needs further improvement to better guide clinical practice. Therefore, a classification pattern centered around pancreatic function may be more beneficial for the advancement of clinical work. Our study not only clarified the characteristics of cluster classification and the relative contributions of glycemic and lipid metabolism markers but also established a foundation for further improvements in classification methods.Of note, only a few studies have investigated the relative contributions of glucolipid metabolism markers to different complications and pancreatic function. The application of the TyG index, MHR, and TG/HDL in cluster analysis for newly diagnosed diabetes is also limited in relevant research. We found that 2hCP, FCP, FINS, FPG, TyG, HbAOur advantage lies in incorporating novel indicators that reflect pancreatic function and inflammatory factors and exploring the contributions of glucolipid metabolism markers to pancreatic function and complications. However, this is a single-center retrospective clinical study, future prospective studies and larger multicenter clinical studies should be conducted to clarify its causal relationship. In addition, due to limitations in conditions, indicators such as GADA, waist circumference, and visceral fat area were missing. Future studies should include the aforementioned indicators to further enhance the research.1C, HDL, and TG were correlated with the function of insulin. FINS, TyG, 2hINS, and FCP were associated with ASCVD, NAFLD, and CKD in newly diagnosed T2DM patients.Insulin resistance was closely associated with inflammation and lipid metabolism disorders. 2hCP, FCP, FINS, FPG, TyG, HbA


This report elucidates a unique case of a 39-year-old female with immune thrombocytopenic purpura (ITP) who developed a rare and severe complication: diffuse alveolar hemorrhage (DAH). Despite initial treatments for ITP, the patient experienced fluctuating platelet (PLT) counts and shortness of breath, which were later identified as symptoms of DAH. An urgent splenectomy improved the patient's platelet counts and overall condition. This case underscores the imperative to recognize DAH as a possible ITP complication, requiring clinicians' vigilance for prompt diagnosis and intervention. The intricate nature of ITP in adults necessitates individualized, patient-centered treatment approaches to enhance outcomes. This report provides invaluable insights into the clinical understanding and management of ITP and its complications through detailed analysis and documentation of the patient's treatment journey. Immune thrombocytopenic purpura (ITP), previously known as idiopathic thrombocytopenic purpura, is commonly diagnosed when platelet (PLT) counts fall below 100,000/uL. However, clinical symptoms usually manifest at even lower platelet counts. This disorder is characterized by the immune-mediated destruction of platelets, primarily through the reticuloendothelial system, and can manifest in various forms depending on its duration and the patient's age .In the United States, the annual incidence rate of ITP is 3.3 per 100,000, while in Europe, it varies between 1 and 4 per 100,000 depending on the country. Preliminary studies in the Middle East and North Africa (MENA) region aim to characterize the epidemiology of ITP, with findings showing a female predominance and a mean age of 28.29 (±17.34) years in the Africa, Middle East, and Asia (AMEA) regions [Over the past decade, our understanding of ITP's pathophysiology has evolved. We now know that it involves not only antibody-mediated platelet destruction but also impaired platelet production due to megakaryocyte destruction, as well as processes not evidently antibody-mediated . A subseStratified classifications have been developed to tailor treatment regimens based on age and presentation, helping to avoid unnecessary interventions given the self-resolving nature of ITP in children and the more complex manifestations in adults .ITP's clinical presentations are diverse, ranging from asymptomatic findings to severe outcomes such as genitourinary, gastrointestinal, and intracranial bleeding. In females, heavy menstrual bleeding can also be a symptom . FatigueDiffuse alveolar hemorrhage (DAH), identified through widespread radiographic alveolar infiltrates, hemoptysis, anemia, and respiratory failure, is rarely associated with ITP . Only tw9/L. Despite treatment with steroids, intravenous immunoglobulin (IVIG), azathioprine, eltrombopag, and four doses of infliximab , her platelet count would transiently increase before plummeting again. A bone marrow biopsy in Yemen further confirmed the ITP diagnosis.A 39-year-old female was diagnosed with idiopathic thrombocytopenia (ITP) in May 2023 in Yemen, presenting with symptoms of epistaxis, heavy menstrual bleeding, and skin bleeding spots. The diagnosis was incidentally discovered through a complete blood count (CBC) performed before labor, indicating a platelet count of 48,000 × 10During a recent admission to our department, the patient reported experiencing shortness of breath for three days with a gradual onset, progressively worsening and exacerbated by exertion. This symptom was partially relieved by rest, increased at night, and was described as feeling like choking. The shortness of breath was unrelated to body position and did not awaken her from sleep. She reported no chest pain, palpitations, cough, sore throat, or nasal congestion. There was no audible wheezing or lower limb swelling.9/L, hemoglobin (HB) of 12.5 g/dL, white blood cell (WBC) count of 7.1 × 109/L, C-reactive protein (CRP) of 23 mg/L, D-dimer of 0.4 mg/L, and random blood sugar (RBS) of 577 mg/dL  of 126/83 mmHg, heart rate (HR) of 100 beats/minute, respiratory rate (RR) of 21 breaths/minute, and O2 saturation of 89% on room air. Laboratory results highlighted a critical platelet count of <5,000 × 10A normal chest X-ray was noted Figure . Her med9/L, HB of 10.7 g/dL, triglycerides (TG) of 284 mg/dL, high-density lipoprotein (HDL) of 17 mg/dL, low-density lipoprotein (LDL) of 120 mg/dL, and hemoglobin A1C (HbA1C) of 10%.On the second day of her hospital stay, a high-resolution chest computed tomography (CT) revealed diffuse alveolar hemorrhage Figure , necessi9/L, HB of 9.9 g/dL, WBC of 6.6 × 109/L, and CRP of 20 mg/L, with creatinine (CR), sodium (Na), and potassium (K) all within normal ranges.On day 3, the patient received an additional eight units of platelets with other management unchanged. The patient was clinically stable, requiring O2 therapy at 3 L/minute via nasal cannula. Laboratory results were as follows: PLT of 6,000 × 109/L, HB of 9.1 g/dL, and WBC of 4.4 × 109/L, with both creatinine (CR) and potassium (K) within normal parameters.On day 4, the patient exhibited signs of oxygen desaturation, necessitating increased oxygen support via a non-rebreather mask at 15 L/minute. Her laboratory results indicated PLT of 39,000 × 109/L, HB of 8.5 g/dL, and WBC of 4.3 × 109/L, with creatinine (CR) and potassium (K) remaining normal.By day 5, she continued to need a non-rebreather face mask. A chest X-ray showed bilateral perihilar infiltration with a "flask-shaped" heart Figure . An echo9/L, HB of 9.1 g/dL, and WBC of 3.1 × 109/L, with potassium (K) remaining normal.On day 6, the patient still required a non-rebreather mask. Pulsed steroid therapy was discontinued, and she was started on methylprednisolone 40 mg IV every 12 hours. Additionally, she was transitioned to basal-bolus insulin with insulin glargine 25 units subcutaneously every 24 hours and insulin aspart at 20/15/20 units subcutaneously. Laboratory results showed PLT of 290,000 × 109/L, HB of 9.4 g/dL, and WBC of 3.9 × 109/L, with potassium (K) remaining normal.On day 7, her oxygen needs decreased, and she was maintained on a face mask with oxygen at 5 L/minute. Tranexamic acid was discontinued, and her insulin regimen was modified to insulin glargine 30 units subcutaneously every 24 hours and insulin aspart at 30/30/30 units subcutaneously. Laboratory results displayed PLT of 393,000 × 109/L, HB of 9.8 g/dL, and WBC of 3.2 × 109/L. Discharge medications prescribed were esomeprazole 40 mg once daily, norethisterone acetate 10 mg once daily, nystatin three times daily, insulin aspart at 30/30/30 units subcutaneously, insulin glargine 35 units subcutaneously once daily, dapagliflozin/metformin 5/1000 mg orally every 12 hours, and glyburide 5 mg orally once daily. For a comprehensive understanding, Table By day 8, the day of her discharge, she appeared well and was set to be released with home oxygen therapy and scheduled for subsequent clinic follow-ups. Laboratory results at discharge showed PLT of 440,000 × 109/L. Preoperative preparations included 10 units of platelets, IV tranexamic acid, and IV fluids. The surgical procedure proceeded without complications. In the immediate postoperative period, her platelet count surged from 14,000 × 109/L on the day of surgery to 168,000 × 109/L by postoperative day 3, and further to 198,000 × 109/L by postoperative day 4, without any signs of fever. The pathology report of the splenectomy corroborated the clinical diagnosis of ITP, noting red pulp expansion and hyperplasia.However, the patient was readmitted four days after discharge for an urgent splenectomy due to persistent thrombocytopenia with a platelet drop to 14,000 × 10Before discharge, the patient was educated and strongly advised on crucial preventive measures to mitigate her lifelong elevated risk of infections. She was counseled to receive essential vaccinations, including the 13-valent and 23-valent pneumococcal vaccines, quadrivalent and recombinant meningococcal B vaccines, the Hib vaccine, and the annual influenza vaccine to safeguard against overwhelming post-splenectomy infections. Always carrying a supply of high-dose antibiotics for self-administration in emergencies, recognizing signs of severe infections, and notifying healthcare providers about her splenectomy were emphasized. Prior to traveling, especially to malaria-endemic regions, seeking medical advice is imperative. Participation in a clinical registry for asplenic patients was strongly endorsed for continued education, management, and support. Adherence to these recommendations and consistent follow-up appointments are vital for her long-term health and well-being.ITP manifests as an autoimmune disorder, with autoantibodies precipitating the early breakdown of platelets, leading to diminished platelet counts. The spleen and the mononuclear phagocyte system play pivotal roles in this process . IndividIn the case presented here, the patient diagnosed with ITP developed diffuse alveolar hemorrhage (DAH), a condition rarely associated with ITP. DAH results from damage to the alveolar microcirculation, often due to immune-related processes. It presents with symptoms such as dyspnea, hemoptysis, anemia, and radiological signs of diffuse alveolar filling. The DAH in our patient likely arose in connection with acute ITP, as suggested by the absence of other autoantibodies and the patient's immediate response to high-dose IVIG and corticosteroid therapy ,13.Various disorders, including different vasculitides and collagen vascular diseases, may also lead to DAH. Therefore, careful clinical evaluation is imperative when patients with ITP present with respiratory symptoms suggestive of DAH. DAH associated with ITP is treated with high-dose IVIG therapy, corticosteroids, and occasionally platelet transfusions. IVIG therapy has proven to be an effective first-line emergency treatment ,14.Additionally, the subject of this case displayed a form of ITP that was unresponsive to corticosteroids, necessitating alternative therapeutic interventions. Adults enduring corticosteroid-resistant or corticosteroid-dependent ITP for over three months might benefit from thrombopoietin receptor agonists (TPO-RAs), such as romiplostim or eltrombopag, depending on their preference for specific administration methods. Studies comparing these agents have found them to be similarly effective in sustaining response, preventing bleeding episodes, and reducing corticosteroid use with minimal variation -17.For patients whose ITP does not respond to corticosteroids and persists for over three months, TPO-RA is often preferred over splenectomy. Splenectomy is typically delayed for at least a year due to the possibility of spontaneous remission. If ITP continues for more than 12 months, both splenectomy and TPO-RA are considered viable options, with the patient's preferences, age, overall health, and desire for either a durable response or long-term treatment influencing the decision-making process -28.With splenectomy's decline as a favored second-line treatment due to postoperative complications, especially in older patients, alternatives such as TPO-RA are gaining prominence. TPO-RAs are reserved for patients unresponsive to other medical treatments, in accordance with international guidelines. The decision between TPO-RA and splenectomy should be individualized, taking into account various factors such as age, comorbidities, lifestyle, treatment compliance, and patient preferences ,30.In the presented case, the patient underwent elective splenectomy after initial treatments for refractory ITP proved partially successful. The procedure led to a significant improvement in platelet counts, aligning with pathology findings indicative of ITP. The clinical decisions made during the treatment course reflected the complex interplay of the patient's symptoms, ITP and DAH progression, and responses to different interventions, highlighting the necessity for individualized treatment approaches in managing ITP and its complications.ITP is a complex disorder exhibiting a range of clinical presentations. Its rare association with DAH poses significant challenges for clinicians, underscoring the need for increased awareness and careful clinical management. The case presented here underscores the importance of individualized treatment, demonstrating the effectiveness of various therapeutic approaches for refractory ITP. The choice between medical management and surgical interventions, such as splenectomy, should be based on a comprehensive clinical evaluation, taking into account the patient's overall presentation, disease progression, and response to treatment.


Neutrophils are crucial innate immune cells and comprise 50–70% of the white blood cell population under homeostatic conditions. Upon infection and in cancer, blood neutrophil numbers significantly increase because of the secretion of various chemo- and cytokines by, e.g., leukocytes, pericytes, fibroblasts and endothelial cells present in the inflamed tissue or in the tumor microenvironment (TME). The function of neutrophils in cancer has recently gained considerable attention, as they can exert both pro- and anti-tumorigenic functions, dependent on the cytokine milieu present in the TME. Here, we review the effect of cytokines on neutrophil development, tissue homing, function and plasticity in cancer and autoimmune diseases as well as under physiological conditions in the bone marrow, bloodstream and various organs like the spleen, kidney, liver, lung and lymph nodes. In addition, we address several promising therapeutic options, such as cytokine therapy, immunocytokines and immunotherapy, which aim to exploit the anti-tumorigenic potential of neutrophils in cancer treatment or block excessive neutrophil-mediated inflammation in autoimmune diseases. Neutrophils are the body’s first line of defense against pathogens, e.g., bacteria and fungi, and comprise 50–70% of the white blood cell population in human circulation. They are essential immune cells, and patients that lack (mature) neutrophils often succumb to severe opportunistic bacterial infections . NeutropNeutrophils have a short half-life in blood, ranging from 13 to 19 hours under homeostatic conditions ,5,6. ForCsf3 or its receptor (Csf3r) are severely neutropenic, indicating that this signaling pathway is essential for normal neutrophil production and release [CSF3R gene are found in humans and result in severe congenital neutropenia, characterized by low absolute neutrophil counts (<0.5 × 109/L) in the circulation [Neutrophils are derived from long-term hematopoietic stem cells (LT-HSCs) in the bone marrow and, via several stem and (multipotent) progenitor cell stages, differentiate into granulocyte–monocyte progenitors (GMPs) ,12. Thes release ,16. In aculation . Activatculation .In addition to CSF3, colony-stimulating factor 2 (CSF2), also known as granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6) are described as being involved in the production of neutrophils, especially during inflammatory responses in a process called emergency granulopoiesis . Zilionis et al. described the identification of six different circulating neutrophil subsets (termed N1 to N6) based on single-cell RNA sequencing blood from six treatment-naïve patients with non-small-cell lung cancer . WhetherIn addition to the subdivision of circulating neutrophils based on their transcriptomes, Zhu and colleagues used a CyTOF panel containing 40 of the most commonly used surface markers of neutrophil maturation, activation and function to investigate neutrophil subsets in blood from 21 treatment-naïve patients with melanoma . In addiAs first responders upon microbial infection or injury, neutrophils rapidly migrate into affected tissues to regulate pathogen dissemination and contribute to the resolution of inflammation. Neutrophil extravasation and emigration to infected sites are complex multi-step cascades, highly dependent on the interplay between neutrophils and various cell types, such as endothelial cells, perivascular cells and stromal cells  29,30,3,330,31. Research on neutrophil recruitment into the lungs, spleen, lymph nodes, kidneys and other organs has highlighted the existence of different neutrophil subpopulations with distinct functions. Interestingly, the presence of neutrophils in these organs occurs not only upon infection or inflammation but also under homeostatic conditions. Marginated pools of neutrophils that adhere to the endothelium and serve as a reservoir upon stimulation have been identified in the liver, spleen and lungs . The biodistribution of neutrophils is dependent on their maturation and activation state and is, among other things, affected by microvascular blood flow and factors such as exercise, drugs and infection ,39,40,41int immature and Ly6Ghigh mature) in the red pulp (RP) of the spleen both under steady state and upon S. pneumoniae infection. Upon infection, the mature neutrophils played an immune surveillance role and facilitated bacterial clearance along with RP macrophages, whereas the immature neutrophils served as a reservoir in case of emergency [Puga et al. were the first to identify two neutrophil subpopulations residing in the marginal zone (MZ) of the spleen in mice, which showed a different marker profile compared with circulating neutrophils. They demonstrated that IL-10 secreted by sinusoidal endothelial cells upon microbial TLR signaling would reprogram neutrophils residing in the spleen into B helper neutrophils. This novel neutrophil subset would then activate MZ B cells by secreting B cell-activating factor (BAFF), a proliferation-inducing ligand (APRIL) and IL-21, thus inducing Ig class switching, somatic hypermutation and antibody production . The eximergency .E. coli, neutrophil recruitment was dependent on TNF-α, CXCL1 and CXCL2 produced by tissue-resident macrophages and was directly associated with kidney injury and poor disease outcomes [Single-cell sequencing studies have provided new insights into the presence and heterogeneity of neutrophils in kidneys in both healthy and pathological conditions ,47,48. Poutcomes . This isoutcomes ,52,53.The dual role of neutrophils has also been investigated in liver tissues. Although the routine patrolling of neutrophils in liver sinusoids fortifies the liver upon infection, increased hepatic infiltration is also a key feature of most liver pathologies. Neutrophils are able to contribute to liver regeneration following hepatectomy by promoting the Kupffer cell/tissue-resident macrophage-dependent secretion of IL-6 and TNF-α. However, their excessive secretion of ROS and cytokines such as IL-1β, TNF-α, transforming growth factor beta (TGF-β) and IL-17, as well as their degranulation and NET formation, can aggravate the liver upon injury, ischemia-reperfusion, cirrhosis, fibrosis and cancer ,55.+ monocytes recruited in cystic fibrosis airways showed a skewed pro-inflammatory profile with an increased expression of cytokines, e.g., Cxcl1, Cxcl2 and Csf3, known to drive neutrophil chemotaxis and differentiation [Owing to the COVID-19 pandemic, the role of neutrophils in the lung has gained increasing attention and heightened research conducted in this area. Excessive neutrophil infiltration in the lung is considered a hallmark of acute respiratory distress disease (ARDS), observed in 29–42% of COVID patients, often resulting in fatality ,57. Undentiation . Additiontiation . Followintiation ,63,64. Intiation . Furtherntiation ,67,68.P. aeruginosa, S. aureus and Salmonella enterica or the injection of Bacillus Calmette–Guérin (BCG), neutrophils have been shown to localize in different zones of the lymph nodes and be in close proximity to T and B cells, as well as innate-like lymphocytes such as γδ T cells, natural killer (NK) cells and natural killer T (NKT) cells [L. major infection. Conversely, they can also negatively impact the immune response by facilitating the removal of subcapsular sinus macrophages during parasite infection [S. aureus and Leishmania major) in human and mouse neutrophils, respectively [+ and CD8+ T cells [In recent years, a lot of attention has been drawn to the recruitment and role of neutrophils in the lymph nodes. Neutrophils routinely patrol the lymph nodes during steady-state conditions, and upon infection, they recruit additional neutrophils by releasing LTB4 . As reveT) cells ,76,77,78nfection . Their anfection ,78,79,80ectively ,82. Infeectively ,84. The  T cells ,86,87. A T cells , it has  T cells ,88,89. low/CXCR1high) and tissue-resident neutrophils (ICAM1high/CXCR1high), are able to migrate from the peripheral organs back to the circulation [Neutrophil clearance at inflammatory sites is essential to maintain homeostasis. The established theory in which activated neutrophils undergo apoptosis/necrosis and subsequent phagocytosis via macrophages after executing their effector functions has been modified over the last two decades. Several groups utilizing in vivo advanced imaging technologies have demonstrated that activated neutrophils show high expressions of ICAM1 and low expressions of CXCR1, a unique phenotype compared with circulatory , caused by the enhanced de novo generation of neutrophils as a result of increased myeloid progenitor cell proliferation ,98. This tissues ,104. IL-nfection . In addiin vitro .In contrast to their protective function against pathogens, increasing evidence links an abundance of pro-inflammatory neutrophils to the pathogenesis of several autoimmune diseases like multiple sclerosis (MS); systemic lupus erythematosus (SLE); rheumatoid arthritis (RA); type I diabetes; and inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis ,105. In + T cell recruitment and activation, e.g., CCL3, CXCL9 and CXCL10, as well as pro-inflammatory cytokines like IL-12, TNF-α and CSF2 (+ regulatory T cells (Tregs) by secreting high levels of CCL17 , respectively . Pro- anand CSF2  [119,120of CCL17  [121,122A transition from N1 to N2 TANs can occur and is described as being regulated by TGF-β, while CSF3 and IL-6 have also been linked to inducing a pro-tumorigenic neutrophil phenotype  119,123123. In cOn top of the pathogen/tumor cell killing effect of granules described above, several granule contents are associated with pro-tumorigenic effects. The serine proteases present in the azurophilic granules, such as neutrophil elastase and cathepsin G , are deshN1-5), in contrast to seven distinct mouse neutrophil populations  [MMP8, MMP9, ARG1, S100A8 and S100A9 could be detected in both mN1 and hN1. Based on SPRING/nearest-neighbor analyses of the neutrophil subsets, the majority of other subpopulations were found to originate from these mN1 and hN1 neutrophils. Among the other subsets were the tumor-specific mN5 and hN5 neutrophils, which showed the mRNA expression of cytokines, including CCL3, CSF1, IRAK2 and MIF. The mN2 and hN2 neutrophils formed a rare subpopulation, which was characterized by the expression of type I interferon response genes . In addition, mouse neutrophil populations 4–6 (mN4-6) were highly tumor-enriched and showed the expression of Siglecf, previously linked to several pro-tumorigenic functions, including angiogenesis; extracellular matrix remodeling; the suppression of T cell responses; and tumor cell proliferation and growth [Recent single-cell RNA-sequencing studies have added more complexity to TANs by identifying additional sub-groups of neutrophils in the tumor tissues of treatment-naïve patients with non-small-cell lung cancer and tumor-bearing mice . Five huand N1b) ,126. Cand growth .PMN-MDSCs are immature suppressive neutrophils that expand under pathological conditions, e.g., inflammation and cancer, and share similarities with neutrophils, including their origin . Whether+Ly6G+Ly6Clow cells, while in humans, they are defined as CD14−CD11b+CD15+(CD66b+) cells, similar to neutrophils [In mice, PMN-MDSCs are defined as CD11btrophils ,143. Humtrophils . Additiotrophils . Additiotrophils . Moreovetrophils . They altrophils . The suptrophils ,150. Nottrophils . Converstrophils . Additiotrophils . It is ntrophils .Various cellular components within the TME, such as cancer cells, stromal cells (including cancer-associated fibroblasts/CAFs) and immune cells secrete a wide array of cytokines and chemokines. These cytokines and chemokines have the ability to diffuse through the surrounding tissues, serving as potential signals to circulating or tissue-patrolling neutrophils or more immature neutrophils in the bone marrow , ultimately attracting them to the tumor microenvironment.CXCR2, IL17 and IL17R increased in Cl66, a doxorubicin- and paclitaxel-resistant murine breast cancer cell line. In addition, it was reported that breast cancer cells secrete IL-1β, resulting in IL-17 production via γδ T cells [The upregulation of chemokines within the TME represents the initial step in neutrophil recruitment, which is mainly regulated by chemokines like CXCL1, CXCL2, CXCL8 and their corresponding receptors, CXCR1 and CXCR2 ,157,158. T cells . Increas T cells . Further T cells .Additionally, CCL20 produced by breast cancer cells has been reported to modulate PMN-MDSC to promote cancer cell stemness through the CXCL2-CXCR2 pathway . MoreoveSeveral studies have indicated the modulatory roles of cytokines in the phenotypes of neutrophils and PMN-MDSCs. For instance, type I interferons like IFN-β have been shown to increase the tumor cytotoxicity of neutrophils; increase NET, ICAM1 and TNF-α expression; and polarize TANs toward an anti-tumor N1 phenotype in vivo . Cheng e+ MDSC colonization to the lung was promoted in a lung metastasis model of breast cancer via the tumor secretion of CXCL17 and CSF3 [Chemokines play a pivotal role in recruiting neutrophils and PMN-MDSC, not only into primary tumor sites but also pre-metastatic niches and metastatic sites. In a mouse model of breast cancer, tumor-associated mesenchymal stromal cells released CXCL1, CXCL2 and CXCL5, leading to increased neutrophil recruitment at primary tumor sites . Furtherand CSF3 . PMN-MDSand CSF3 .Tumor cells, CAFs and immune cells have been reported to induce the formation of NETs via neutrophils through various mechanisms. In the TME, cytokines such as CXCL8, IL-1β and TNF-α have been demonstrated to stimulate the production of ROS, leading to the release of NETs ,175,176.With increasing knowledge about neutrophil plasticity and function, there is growing interest in exploring new therapeutic interventions to harness neutrophils’ innate capabilities to target and eliminate pathogens and cancer cells. Such potential strategies could target neutrophil recruitment and polarity; modulate neutrophil activation; or reduce excessive inflammation. As the field of immunotherapy is continuously evolving, several innovative therapeutic approaches have been developed or are being developed and could be used to leverage the anti-tumorigenic potential of neutrophils and block excessive neutrophil-mediated inflammation in autoimmune diseases.In conditions of exacerbated cytokine production, e.g., inflammatory and autoimmune disease, the inhibition of cytokine functions caused by monoclonal antibodies or receptor blockers has been successfully used in the clinic. For example, patients with rheumatoid arthritis and Crohn’s disease are effectively treated with various TNF-blocking monoclonal antibodies, while a human IL-12/IL-23 monoclonal antibody is used to treat psoriasis patients, both resulting in reduced neutrophil infiltration into affected tissues ,180. CytGenetically fusing a cytokine to another protein can help reshape the cytokine’s biodistribution profile, overcome poor pharmacokinetic properties and help promote tumor localization. This application is especially interesting in cancer, where a cytokine can be fused to a therapeutic antibody, specifically recognizing a tumor-associated antigen. These fusion constructs, called immunocytokines, hold promise as potential treatments and are currently undergoing evaluation in clinical trials . For exaIn addition to the cytokine part of immunocytokines, the antibody itself can also affect neutrophils by initiating neutrophil-mediated tumor cell killing via ADCC. All antibodies used for immunotherapy purposes are of the IgG isotype, which can bind various Fc gamma receptors on immune cells. Human neutrophils express the activating Fc-gamma receptors FcγRI (CD64), FcγRIIa (CD32a) and FcγRIIIa (CD16a) ,190. In In contrast to IgG antibodies, IgA antibodies strictly bind the activating FcαR (CD89), making them very efficient in activating neutrophils and inducing ADCC . HoweverThe identification of different neutrophil subsets and the dual role of neutrophils in cancer have shifted the field of neutrophil biology tremendously. Gaining a comprehensive understanding of neutrophil functions in tissues in various tissue states , as well as their plasticity and their role in the TME, is essential for harnessing their anti-tumorigenic potential effectively. The identification of cytokines that polarize neutrophils toward an anti-tumorigenic phenotype, e.g., IFN-β or IL-12, as well as cytokines that promote a pro-tumorigenic phenotype, e.g., TGF-β, has been essential for understanding the effect of the TME on neutrophil plasticity and opened up possibilities for cytokine therapy. The field of bioengineering has made remarkable advancements, leading to the study and in vivo or clinical trial testing of synthekines, immunocytokines and IgA antibodies as potential anti-cancer therapies. To facilitate the transition from pro-tumorigenic neutrophils to anti-tumorigenic neutrophils that effectively combat cancer cells, immunocytokines containing IFN-β or IL-12, known to induce the transition to N1 neutrophils , should