BackgroundEndometriosis (EM) is associated with immune dysregulation, while dysfunction of natural killer (NK) cells is regarded as a key mechanism underlying immune escape and the persistent growth of ectopic lesions.MethodThis study used single‐cell RNA sequencing (scRNA‐seq) on lesions from three patients with EM and on three normal endometrium samples and integrated these data with three bulk RNA‐seq datasets from GEO (GSE105765, GSE7305, and GSE6364). Seurat, Monocle, limma, least absolute shrinkage and selection operator (LASSO), and support vector machine recursive feature elimination (SVM‐RFE) were used for cell clustering, trajectory inference, differential expression analysis, and feature selection. Immune‐cell composition and pathway activity were evaluated with CIBERSORT and GSVA. Gene expression was validated by qPCR, and cell migration and invasiveness were assessed using wound healing and Transwell assays.ResultscRNA‐seq resolved 11 clusters assigned to eight major cell types. By integrating pseudotime features with bulk data, 20 differentially expressed genes (DEGs) were prioritized, and machine‐learning analyses identified three key genes: granulysin (GNLY), perforin 1 (PRF1), and ENTPD1. The three‐gene model showed good discrimination in the training set and two external validation cohorts (AUCs 0.84, 0.67, and 0.77, respectively). GNLY and PRF1 were predominantly expressed in NK cells and CD8+ T cells and correlated with activation signatures, whereas ENTPD1 was highly expressed in endometrial stromal cells and enhanced their migratory and invasive capacities. ENTPD1 may contribute to disease via adenosine signaling–mediated modulation of NK–cell function. In silico analyses also nominated candidate agents targeting this pathway, including resveratrol, ibuprofen, and danazol.ConclusionThis study highlights the central role of NK–cell dysfunction in EM pathogenesis and proposes GNLY, PRF1, and ENTPD1 as potential molecular diagnostic biomarkers. Notably, ENTPD1 appears to have dual functions, including immunomodulation and promotion of stromal cell migration, which promotes lesion formation. These findings provide a mechanistic rationale and actionable targets for earlier screening and targeted therapy in EM.

This study investigates the therapeutic effects of Zengwei Chengqi Decoction (ZW) on acute intestinal obstruction (AIO), focusing on its ability to alleviate inflammation and reduce associated tissue damage. Network pharmacology and experimental animal models were used to explore the mechanisms of action. Using network pharmacology, we identified 228 active compounds in ZW and 420 corresponding targets linked to inflammation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify the biological pathways influenced by ZW. In vivo, an SD rat model of AIO was induced, and the therapeutic effects of ZW were assessed by histological examination of tissue damage and serum cytokine levels. Treatment with ZW significantly mitigated tissue damage in AIO rats. Additionally, serum levels of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were substantially reduced in the ZW-treated group compared to the control group (p < 0.01). ZW shows strong potential for modulating inflammation in AIO, as evidenced by reduced cytokine levels and improved tissue integrity. These findings provide valuable insights into the mechanisms underlying its therapeutic effects, positioning ZW as a promising treatment for AIO.

Traumatic brain injury (TBI) is an important cause of disability and death worldwide. The development of neuroinflammation after TBI is related to the brain parenchyma. M1-type microglia play important roles in this process, but the specific mechanism through which regulating microglia M1 polarization is still not fully understood. This study aimed to investigate the role of ephrin receptor A4 (EPHA4) in the M1 polarization of microglia after TBI. A TBI rat model was established by the controlled cortical impact (CCI) method, and M1 polarization of GMI-R1 cells was induced by lipopolysaccharide (LPS) treatment. Target genes associated with the progression of TBI were screened by transcriptome sequencing; the expression of key genes and proteins was detected by real-time quantitative PCR (RT‒qPCR), Western blot, ELISA, and immunofluorescence, and the damage of the rat brain tissue and the blood-brain barrier (BBB) was evaluated by hematoxylin‒eosin (HE) staining and Evans blue staining. This study revealed that EPHA4 expression is upregulated in the brain tissue of TBI rats and that treatment with its inhibitor, KYL peptide, can improve the progression of TBI. KYL peptide intervention downregulated the levels of the proinflammatory cytokines and upregulated the levels of the anti-inflammatory cytokines and inhibited the M1 polarization of microglia. The levels of the endoplasmic reticulum stress (ERS)-related proteins and Ca2+ were upregulated in TBI rats and downregulated after KYL peptide treatment but subsequently increased after 123C4 treatment. Our results showed that EPHA4 promoted the M1 polarization of microglia by enhancing ERS. Notably, mitogen-activated protein kinase (MAPK) signaling was significantly enriched in TBI. In vitro studies revealed that LPS treatment promoted the activation of MAPK signaling in GMI-R1 cells. A mechanistic study revealed that EPHA4 may activate ERS by activating the MAPK signaling pathway and promote M1 polarization of microglia after TBI. Our study revealed that the EPHA4/MAPK axis may be a key regulatory factor in controlling microglial M1 polarization during brain injury. Blocking this signaling axis may represent a potential therapeutic approach for improving TBI.

Bone fracture healing is a multifaceted process that involves different stages and intercellular interactions. In this study, we aimed to investigate the effect of Taohong Siwu decoction (TSD) on bone fracture healing and the underlying mechanisms. First, a mouse model of femur fracture was constructed, and TSD intervention was administered for durations of 7, 14, and 21 days. Following this, immunofluorescence (IF) was employed to evaluate the expression of CD90 (a marker for mesenchymal stem cells [MSCs]), endomucin (Emcn), and CD31. We also treated MSCs with normal serum and 10% TSD-containing serum to investigate the effects of TSD. Molecular docking was applied to verify the binding of active compounds in TSD to pVon Hippel-Lindau (VHL). Additionally, MSCs were treated with paeoniflorin and 2-methoxyestradiol (2-ME2) to explore the effects of paeoniflorin. Subsequently, mouse aortic endothelial cells were extracted and identified. Furthermore, normally cultured MSCs were cocultured with endothelial cells. MSCs were exposed to control serum, 10% TSD-containing serum, and a combination of 10% TSD-containing serum with 2-ME2. Finally, we administered a combination of 2-ME2 over 21 days to evaluate its effects on the fractured mice. TSD significantly influenced H-type angiogenesis during the healing process of fractured mice. Compared to the sham group, the model group exhibited lower levels of Emcn, CD90, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF), while there was an increase in pVHL expression. After 7, 14, and 21 days of TSD intervention, the levels of Emcn, CD90, HIF-1α, VEGF, and pVHL gradually increased, whereas HIF-1α expression decreased. In vitro experiments revealed that TSD enhanced the proliferation and migration of MSCs while inhibiting the ubiquitination of pVHL/HIF-1α. Moreover, ferulic acid, amygdalin, hydroxysafflor yellow A, and paeoniflorin demonstrated a strong affinity for binding with pVHL. Notably, paeoniflorin promoted the proliferation and migration of MSCs through the pVHL/HIF-1α pathway to promote angiogenesis. Furthermore, TSD was found to enhance endothelial angiogenesis in MSCs. In summary, TSD affects H-type angiogenesis and MSCs homing during the healing process of fractured mice through the HIF-1α axis. TSD regulated MSC-mediated H-type angiogenesis to accelerate fracture healing through VHL/HIF-1α ubiquitination.

Psoriasis, a chronic inflammatory skin disease, arises from a dysregulated interaction between keratinocytes (KCs) and dendritic cells (DCs). We previously identified Rh family C glycoprotein (RHCG) as a key mediator of KC inflammation and DC activation. Here, we demonstrate that luteolin, a bioactive compound derived from the traditional Chinese formula cooling blood and detoxicating formula (CBDF), directly binds to RHCG, as confirmed by multiple computational methods and in vitro experiments. In vitro, luteolin suppressed RHCG expression in KCs, reducing CXCL14 secretion and subsequent DC activation. Spatial transcriptomics (STs) revealed that luteolin preferentially targets DC-enriched spatial domains and restores desmosomal protein expression (e.g., DSC2), which is dysregulated in psoriasis. In vivo, luteolin ameliorated psoriasis-like inflammation in imiquimod-induced mice, lowering Psoriasis Area and Severity Index (PASI) scores and normalizing pathological markers. Our findings indicate that luteolin disrupts KC-DC communication through multiple modes of action, thereby reversing tissue-level pathology and demonstrating its potential as a targeted therapy for psoriasis.

Helicobacter pylori (HP) is a major cause of gastritis, yet the epithelial mechanisms linking infection-induced stress to mast cell and Treg responses remain poorly defined. Three datasets (GSE5081, GSE27411, and GSE233973) were integrated and analyzed using weighted gene co-expression network analysis (WGCNA) and machine learning algorithms. Mast cell-related hub gene expressions were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), and inflammatory cytokines were quantified using the enzyme-linked immunosorbent assay (ELISA). Histopathological changes were evaluated using hematoxylin and eosin (HE), Giemsa, and Warthin-Starry silver staining. Cell apoptosis was assessed by flow cytometry, and mast cell and Treg cell activities were analyzed by Transwell assays, histamine detection, and immunohistochemistry (IHC). Fos proto-oncogene (FOS), ribonucleotide reductase regulatory subunit M2 (RRM2), and RAD51 recombinase (RAD51) were identified as mast cell-related hub genes, all of which were upregulated in HP-induced gastritis mice. In vitro, HP infection or CagA stimulation increased FOS expression in gastric epithelial cells. FOS knockdown in HP-infected mice alleviated gastric mucosal injury, reduced bacterial burden, and decreased pro-inflammatory cytokine levels. FOS silencing enhanced GES-1 cell viability and suppressed apoptosis. In HP-infected GES-1 cells, FOS silencing inhibited mast cell migration, cytokine secretion, including C-C motif chemokine ligand 2 (CCL2), interleukin-33 (IL-33), and stem cell factor (SCF), as well as histamine release, accompanied by reduced Treg polarization and decreased expression of transforming growth factor-β and forkhead box P3. FOS silencing inhibited mast cell activation and Treg cell polarization in HP-induced gastritis, suggesting its promising value as an intervention point in HP-driven gastritis.

Ulcerative colitis (UC) is a chronic inflammatory disease of the gastrointestinal tract, characterized by immune dysregulation, genetic susceptibility, and environmental factors such as diet and psychosocial stress. The etiology of UC is complex, involving multiple interrelated factors that drive its pathogenesis and clinical progression. This cross-sectional study investigated the potential role of ribosomal protein L19 (RPL19) mRNA in UC. A total of 40 patients with UC and 29 healthy controls (HC) were prospectively enrolled from the Department of Gastroenterology, Affiliated Hospital of Jining Medical University, between November 2021 and November 2023. RPL19 mRNA levels in the intestinal mucosa of UC patients were quantified using quantitative PCR analysis. Subsequently, the associations of RPL19 mRNA expression with disease severity and with the levels of key inflammatory cytokines were statistically assessed. RPL19 mRNA expressions were significantly regulated in UC patients compared to HC. It exhibited a significant inverse correlation with the levels of both interleukin-2 (IL-2) and interleukin-4 (IL-4). Importantly, RPL19 mRNA levels also correlated with key clinical disease activity indices. This study demonstrates that mucosal RPL19 mRNA expression levels are significantly elevated in UC patients and correlate positively with endoscopic and histological disease severity. These findings identify RPL19 as a potential biomarker for reflecting local disease activity in UC.

Epithelial-mesenchymal transition (EMT) represents a critical process that facilitates metastatic dissemination and immune evasion in colorectal cancer (CRC); however, the molecular factors that connect EMT to modifications in the immune microenvironment remain poorly elucidated. In this investigation, we identify AJUBA as an essential regulator that mediates the association between EMT and immune modulation in CRC. By integrating multi-cohort transcriptomic datasets (The Cancer Genome Atlas (TCGA)-CRC, GSE18105, GSE22598, GSE89076, and GSE110224) with single-cell RNA-seq data (GSE132465), we applied machine learning and deep learning methodologies to comprehensively identify EMT-associated genes demonstrating prognostic significance. AJUBA validation was performed at mRNA and protein levels in a cohort of 90 CRC patient samples using quantitative PCR, Western blotting, and immunohistochemical (IHC) approaches. Functional analyses involved siRNA-mediated knockdown experiments, coupled with evaluations of cell proliferation (CCK-8 assay), migration and invasion (transwell assay), clonogenic capacity (colony formation assay), and in vivo tumor growth in xenograft models. Immune infiltration was assessed via ssGSEA and CIBERSORT algorithms, and spatial transcriptomics data (GSE225857) were used to delineate AJUBA expression within tumor microdomains. Across multiple CRC cohorts, AJUBA exhibited marked upregulation and showed distinct enrichment in epithelial cells with activated EMT characteristics. Spatial transcriptomic profiling demonstrated AJUBA colocalization with cancer-associated fibroblasts (CAFs) within immune-excluded niches. Enhanced AJUBA expression exhibited a positive correlation with heightened infiltration of M2 macrophages and activation of VEGF/NOTCH signaling cascades. In vivo, AJUBA knockdown led to suppressed tumor growth, reduced Ki-67 proliferation indices, and diminished M2 macrophage abundance. Clinically, elevated AJUBA expression correlated with advanced nodal metastasis and served as an independent predictor of poor overall survival (OS; HR = 4.809, 95% CI: 2.385-9.695, p < 0.001). AJUBA functions as a key regulator that links EMT to immune modulation, promoting macrophage polarization and facilitating immune evasion in CRC. Through its coupling of EMT activation with proangiogenic signaling, AJUBA represents both a prognostic biomarker and a promising therapeutic target for alleviating immune exclusion in metastatic CRC.

The mechanism of Yiqi-Wenyang-Tiaoshen decoction (YWT) in treating cisplatin-induced acute kidney injury (AKI) remains unknown. This study identifies the key components of YWT and explores its therapeutic potential and mechanisms in a cisplatin-induced AKI rat model. UPLC-ESI-MS/MS was utilized for the identification of compounds present in both the aqueous extract of YWT and serum samples. The overlapping components were recognized as active constituents, followed by a network pharmacological analysis. A rat model of cisplatin-induced AKI was established, and comprehensive pathological analyses including HE, PAS, and electron microscopy, as well as biochemical assessments of serum Cre, BUN, IL-6, and TNF-α levels, were conducted. Western blotting was utilized to evaluate the expression levels of Caspase-3, Caspase-9, BAX, Bcl-2, and LC3 Ⅱ/Ⅰ. Using UPLC-ESI-MS/MS, we identified 182 compounds in the aqueous extract of YWT, 34 of which are confirmed to be absorbable into the bloodstream. Network pharmacological analysis suggests that YWT primarily acts by inhibiting apoptosis and activating autophagy. In the rat model, YWT significantly ameliorated renal pathology and electron microscopic features. Additionally, YWT mitigated body weight loss and renal hypertrophy while lowering serum creatinine and blood urea nitrogen levels. YWT alleviates AKI by suppressing apoptosis-related proteins such as Caspase-3, Caspase-9, and BAX, enhancing Bcl-2 expression, increasing the LC3 Ⅱ/Ⅰ ratio, and reducing p62, a marker of autophagy. This study confirms the therapeutic efficacy of YWT in cisplatin-induced AKI, potentially linked to its ability to inhibit apoptosis, activate autophagy, and mitigate mitochondrial damage.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, closely associated with neuroinflammation, immune dysregulation, and gut microbiota imbalance. Gut microbiota-derived metabolites may modulate key targets involved in MS pathogenesis. This study integrated network pharmacology, machine learning (ML), and single-cell transcriptome analysis to identify MS-related differentially expressed genes (DEGs) and potential targets of gut microbial metabolites. Feature contributions were evaluated using the SHapley Additive exPlanations (SHAP) method, and causal relationships were validated via Mendelian randomization (MR). Single-cell analysis, molecular docking, and assessments of drug-likeness and toxicity were also performed. Caspase-3 (CASP3) was identified as a core target interacting with multiple gut microbial metabolites, including L-isoleucine, aromatic lactic acid derivatives, 3-hydroxyphenethyl alcohol, and D-xylose, potentially regulating neuroimmune responses via TNF, MAPK, IL-17, and galectin pathways. Specific microbial taxa, such as Akkermansia, Bacteroides, and Bifidobacterium, were closely associated with these metabolites. The metabolites exhibited favorable drug-likeness and low predicted toxicity, indicating potential therapeutic value. Gut microbial dysbiosis and its metabolites play a significant role in MS onset and progression, providing a theoretical basis for identifying therapeutic targets and gut-CNS axis interventions. Experimental validation is needed to confirm mechanisms and translational potential.