Microsporidia such as Encephalitozoon hellem are obligate intracellular human parasites that remain genetically intractable, limiting functional characterization of their proteomes. Structural studies based on homology-based modeling and the use of deep learning algorithms of microsporidian proteins also remain limited because most have little to no sequence similarity to proteins with solved structures. To address these limitations, we developed an approach that incorporates cross-linking mass spectrometry (XL-MS) data into structure prediction. XL-MS data provides upper bound distance constraints that can be incorporated into protein deep-learning based modeling and subsequent docking. Using this approach, we generated a model for two interacting E. hellem spore wall proteins Spore Wall Protein 1B (Swp1b) and Endospore Protein 1 (EnP1), with no clear homologs outside of microsporidia, and which contain several disordered regions. These proteins are extremely abundant spore wall proteins of microsporidia and previously were not known to interact with one another. The resulting model not only is consistent with the experimental crosslinks used to generate the model but was subsequently confirmed by independently generated XL-MS data. The described AlphaLink-Modeller framework for structure prediction is particularly well suited to proteins with limited homology and/or substantial flexible regions, given they adopt a defined structural state within a biological context, thereby extending integrative modeling approaches to previously inaccessible targets.

Microsporidia are known intracellular pathogens that infect nearly all animals and deeply manipulate host mitochondrial homeostasis for survival. Here, we report a novel mechanism by which the human-pathogenic Encephalitozoon hellem modulates the mitophagy machinery of its host. We identified the secreted protein EhPTP4 as a key effector in disrupting selective degradation processes in the infected cells. EhPTP4 is found to localize within the nucleus of infected cells, where it induces increased expression of endoplasmic reticulum-associated degradation (ERAD) pathway components, including HSPA5, HERPUD1, and PDIA4. This induction enhances protein ubiquitination in host cells and leads to the degradation of BNIP3L, a critical regulator of mitophagy. Investigation into the molecular interaction network revealed that EhPTP4 interacts with host corepressor RCOR1 and histone H3. This interaction modulates histone acetylation, specifically at H3K14ac sites, thereby further influencing the expression of a key ERAD gene, HERPUD1. This study uncovers a sophisticated strategy by which microsporidia manipulates both ER stress response and the histone acetylation to suppress mitophagy. These findings provide new insights into the mechanisms of microsporidian pathogenesis.

Abstract Background Toxoplasma gondii , an obligate intracellular protozoan parasite, infects almost one-third of the world’s population and all warm-blooded animals, posing a substantial threat to public health. Accordingly, the development of effective vaccines against T. gondii has become an urgent priority. In this study, we constructed a multi-epitope chimeric antigen T-SGR targeting three key protective antigens of T. gondii (SAG1, GRA7, and ROP16), and developed both a messenger RNA (mRNA) lipid nanoparticle (LNP) vaccine and a recombinant protein vaccine based on T-SGR. The immunogenicity and protective effects were further evaluated in C57BL/6 mice. Methods The T-SGR mRNA–LNP vaccine was prepared via in vitro transcription followed by LNP encapsulation, while the T-SGR protein vaccine was obtained via prokaryotic expression and purification. Mice were administered a two-dose immunization regimen. Serum levels of specific IgG, IgG1, and IgG2a antibodies and cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA). T lymphocyte subsets and lymphocyte proliferation were assessed by flow cytometry and Cell Counting Kit-8 (CCK-8) assay. Protective efficacy was evaluated by monitoring survival rates after challenge with highly virulent T. gondii RH strain tachyzoites and moderately virulent ME49 strain tachyzoites. Results Both T-SGR mRNA and protein vaccines induced robust humoral and cellular immune responses in mice. Notably, the IgG antibody titer induced by the mRNA–LNP vaccine was significantly higher than that of the protein vaccine ( P < 0.05). Both vaccines drove a Th1-biased immune response, as evidenced by markedly higher IgG2a levels relative to IgG1. Compared with the phosphate-buffered saline (PBS) control group, both vaccine groups significantly promoted splenocyte proliferation ( P < 0.05). The mRNA vaccine induced significantly higher secretion of IFN-γ, IL-10, IL-12, and IL-2 than the protein vaccine. Both vaccines conferred significant protection against T. gondii infection and prolonged mouse survival. Strikingly, the T-SGR mRNA–LNP vaccine provided 100% protection against the T. gondii ME49 strain, outperforming the recombinant protein vaccine. Conclusions We successfully developed a multi-epitope T-SGR mRNA–LNP vaccine and a recombinant protein vaccine against T. gondii . The T-SGR mRNA–LNP vaccine elicited stronger humoral and cellular immune responses and conferred superior protective efficacy, representing a promising candidate vaccine against toxoplasmosis.

Toxoplasma gondii is a parasitic protozoan that poses a significant threat to both human and livestock. Currently, effective control measures remain elusive, largely due to the unclear mechanisms underlying T. gondii infection and the host immune response. This study aims to elucidate the role of tripartite motif-containing 26 (TRIM26) in modulating the host immune response to T. gondii via regulating tumor necrosis factor receptor-associated factor 6 (TRAF6). Our findings revealed that T. gondii infection significantly upregulates TRIM26 expression in murine macrophages. Notably, TRIM26 acts as a novel regulator of TRAF6 by reducing K48-linked polyubiquitination of TRAF6, which in turn promotes the expression of downstream cytokines. This indicates that TRIM26 plays a critical role in the host immune response against T. gondii. Furthermore, in vivo investigations demonstrated that TRIM26 expression is upregulated during T. gondii infection. Additionally, Trim26−/− mice exhibited significantly reduced proportions of macrophages, inflammatory monocytes in ascites, and T cells in the spleen following infection. These knockout mice also exhibited reduced survival rates, decreased levels of IL-12, IFN-γ, and TNF-α, more severe organ pathological damage, and markedly higher parasite burdens compared to wild-type mice. Collectively, this study highlights the role of TRIM26 in regulating TRAF6 ubiquitination, enriches our understanding of the mechanism of TRAF6 modification. Additionally, it elucidates the significance of TRIM26 in the host immune response to T. gondii, and providing new insights and potential targets for the prevention and treatment of toxoplasmosis.

Microsporidia, as opportunistic parasitic pathogens, constitute a formidable threat to human health. Although the regulatory circuitry of the nucleus-targeted effector EnP1 remains highly intricate and only partially characterized, our study identifies histone H2A as a novel binding partner of EnP1. Furthermore, we demonstrate that both EnP1 overexpression and microsporidia infection induce monoubiquitination of H2A (H2Aub) through downregulation of BAP1 expression. Subsequent mechanistic analyses revealed that elevated H2Aub levels positively correlate with enhanced microsporidian proliferation, whereas attenuation of H2Aub markedly suppresses pathogen expansion. Furthermore, EnP1 orchestrates the enrichment of H2Aub at the SLC7A11 promoter, driving its transcriptional upregulation. Collectively, these findings underscore that EnP1 modulates the ferroptosis state of host cells through H2Aub-mediated epigenetic reprogramming, ultimately facilitating pathogen propagation. This study endeavors to elucidate the critical survival strategies of microsporidia within host cells mediated by EnP1 and to unravel the multifaceted interplay between these pathogens and their hosts.

The surfaces of microsporidian spores are frequently adorned with filamentous appendages of unknown origin and function. Although some studies suggest that these structures may be host-acquired, the absence of identified parasite-encoded components has hindered our understanding of their biogenesis and role in infection. Here, we applied surface shaving proteomics to profile the surface-exposed proteins of Ameson portunus -a microsporidian pathogen causing severe myopathy in portunid crabs. Our analysis identified 120 candidate surface proteins. Nineteen of these were highly enriched by both direct shaving and SDS-assisted methods, representing a high-confidence surfome. Among these, a previously uncharacterized protein, designated 8-2.11, was confirmed via immunofluorescence assay and immunoelectron microscopy. It was expressed early in development stage and specifically localized to the spore wall and hair-like projections (HLPs) of microsporidia. Notably, polyclonal antibodies against recombinant 8-2.11 recognized a native protein in spores, specifically labeled the HLP structures, and showed no cross-reactivity with host cells. Our results provide the first evidence of a parasite-encoded protein that is integral to HLP formation, challenging the prevailing hypothesis that these surface filaments are solely host-derived. This study establishes surface shaving as a powerful tool for microsporidian research and highlights 8-2.11 as a promising candidate for future functional studies on spore surface biology and host-parasite interactions.

Malaria caused by Plasmodium falciparum remains a public health issue, yet direct targets of antimalarial drugs remain elusive. Membrane proteins in Plasmodium are potential drug targets and may contribute to pathophysiological processes in malaria. Recent studies show that the serpentine receptor SR10 is essential for coordinating host rhythms during parasite development. In this study, we found that antimalarial drugs including chloroquine (CQ), dihydroartemisinin (DHA), piperaquine-tetraphosphatetetrahydrate (PIP-TT), and primaquine diphosphate (PQ) are PfSR10 agonists that induce coupling with human Gi/Gq proteins, confirmed through biochemical reconstitution and cryo-EM analysis. Using proteomic profiling, we also identified bradykinin as an endogenous agonist activating PfSR10. Ligand binding and conformational changes were characterized via mutagenesis and FlAsH-BRET assays. These results establish PfSR10 as a receptor for both antimalarials and host peptides, highlighting its dual role in drug action and host-parasite communication, with broad implications in understanding malaria pathogenesis and developing new therapeutics.

The sterile alpha and HEAT/Armadillo motif (SARM) is the fifth Toll-like receptor (TLR) adaptor protein containing the Toll/interleukin-1 receptor (TIR) domain, which is highly enriched in the brain. Toxoplasma gondii (T. gondii) is an obligate intracellular parasitic protozoan that causes zoonotic toxoplasmosis, resulting in threats to human health, such as brain damage. Previous studies have shown that SARM plays crucial roles in cell death and triggers specific transcription programs of innate immunity in response to cell stress, viral, and bacterial infections. However, whether SARM is involved in T. gondii infection remains unclear. In this report, quantitative real-time polymerase chain reaction (qPCR), western blot, flow cytometry, ethynyldeoxyuridine (EdU) assay, and enzyme-linked immunosorbent assay (ELISA) were used to explore the relationship between SARM and T. gondii. Here, we showed that T. gondii infection increased the expression of SARM in vitro and in vivo. SARM induced cell apoptosis during T. gondii infection, activating the mitochondrial apoptotic pathway, the endoplasmic reticulum stress (ER) pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway, and prompting the production of reactive oxygen species (ROS). Furthermore, SARM participated in the regulation of the inflammatory response through the nod-like receptor pyrin domain 3 (NLRP3) inflammasome signaling pathway during T. gondii in vitro infection. These results elucidate the relationship between SARM and T. gondii infection, suggesting that SARM may represent a potential target for T. gondii control.

The protozoan parasite T. gondii employs intricate mechanisms to exploit host cells while sustaining their viability, yet its interaction with ferroptosis - an iron-dependent cell death driven by lipid peroxidation - remains poorly defined. Here, we show T. gondii infection induces ferroptotic hallmarks in RAW264.7 macrophages, including elevated lactate dehydrogenase release, labile Fe2 + accumulation, reactive oxygen species (ROS) generation, and lipid peroxidation. Molecular analyses revealed infection-induced downregulation of ferroptosis suppressor GPX4 and upregulation of pro-ferroptotic ACSL4 in macrophages and mice. Mechanistically, the SLC7A11/GPX4 axis governed parasite growth: knockdown of these genes promoted T. gondii replication, whereas overexpression restricted proliferation. Pharmacological studies showed ferroptosis inhibitor Fer-1 suppressed intracellular parasite proliferation. Notably, GPX4 inhibitor RSL3 exhibited context-dependent effects: pre-infection treatment enhanced replication, while post-infection administration inhibited growth. Direct RSL3 exposure induced time-dependent growth arrest in extracellular tachyzoites, associated with disrupted transcriptomes, increased lipid ROS, and downregulated parasite antioxidant genes (TgPRX2, TgTPX1/2, TgNXN), indicating redox homoeostasis impairment. In vivo murine studies corroborated this biphasic effect: therapeutic RSL3 administration post-infection significantly reduced parasite burdens across multiple organs (spleen, liver, kidney, brain) and improved survival rates, while prophylactic pretreatment exacerbated disease progression. We propose RSL3 exerts direct parasiticidal effects via oxidative damage but also enables early nutrient acquisition from ferroptosis-compromised host cells. These findings establish ferroptosis as a critical node in T. gondii pathogenesis, highlighting the parasite's hijacking of host iron-lipid metabolism. The dual role of ferroptosis regulators underscores the host-pathogen metabolic complexity and positions the SLC7A11/GPX4 axis as a promising therapeutic target.

Toxoplasma gondii is a globally widespread pathogen of significant veterinary and medical importance, causing abortion or congenital disease in humans and other warm-blooded animals. Nevertheless, the current treatment options are restricted and sometimes result in toxic side effects. Hence, it is essential to discover drugs that demonstrate potent anti-Toxoplasma activity. Herein, we found that vorinostat, a pan-HDAC inhibitor, exhibited an IC50 value of 260.1 nM against the T. gondii RH strain and a selectivity index (SI) > 800 with respect to HFF cells. Vorinostat disrupted the entire lytic cycle of T. gondii in vitro. Proteome analysis indicated that vorinostat remarkably perturbed the protein expression of T. gondii, and proteins involved in “DNA replication” and “membrane” were significantly dysregulated. Furthermore, we found that vorinostat significantly enhanced ROS production and induced parasite apoptosis. Importantly, vorinostat could prolong survival in a murine model. Our findings reveal that vorinostat is effective against T. gondii both in vitro and in vivo, suggesting its potential as a therapeutic option for human toxoplasmosis.

Microsporidia are obligate intracellular parasites that infect a wide variety of hosts, including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming the hollow polar tube that transports the sporoplasm components including nucleus into the host cell to achieve the invasion. Due to the complicated topological changes occurring in this process, the formation of sporoplasm is unclear. Here, electron microscopy observation and DiI staining confirmed that during spore germination, a large number of vesicles derived from the polaroplast, nucleus and other cytoplasm were transported out via the polar tube. Meanwhile, the posterior vacuole and plasma membrane remained in the empty spore coat. In addition, there was no DiI-labeled membrane around the nucleus in mature spores, whereas a DiI-labeled limit membrane wrapping nucleus was found at the tip of the extruded polar tube, suggesting that the membrane of sporoplasm was formed outside the mature spore. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were located at the polaroplast in mature spores, in the extruded polar tube and on the sporoplasm membrane, which indicated that the polaroplast transported via the polar tube finally became the limiting membrane of the sporoplasm. Golgi-tracker green and Golgi marker protein syntaxin 6 were also found the same model, which was consistent with the transported polaroplast derived from Golgi transformed into the novel sporoplasm membrane during spore germination. Importance Microsporidia, obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection of microsporidia is its unique invasion apparatus which includes the polar filament, polaroplast and posterior vacuole. When the spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that will transport the sporoplasm components including nucleus into a host cell to achieve the invasion. Knowledge of structure difference between polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting elongated nucleus and other sporoplasm components. And we confirm that the transported polaroplast finally transforms into the novel sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of polar tube and origin of the sporoplasm membrane, which serve as foundations for clarifying the microsporidian infection mechanism.

Toxoplasma gondii is an important protozoan pathogen, which can cause severe diseases in the newborns and immunocompromised individuals. Developing an effective vaccine against Toxoplasma infection is a critically important global health priority. Immunofluorescence staining analysis revealed that TgSAG2 and TgSRS2 are membrane associated and displayed on the surface of the parasite. Immunizations with pBud-SAG2, pBud-SRS2 and pBud-SAG2-SRS2 DNA vaccines significantly increased the production of specific IgG antibodies. Immunization with pBud-SAG2-SRS2 elicited cellular immune response with higher concentrations of IFN-γ and IL-4 compared to the control group. Antigen-specific lymphocyte proliferations in the pBud-SRS2 and pBud-SAG2-SRS2 groups were significantly higher compared to that in the control group. Furthermore, 30 % of mice immunized with pBud-SAG2-SRS2 survived after the challenge infection with virulent T. gondii RH tachyzoites. This study revealed that immunization with pBud-SAG2-SRS2 induced potent immune responses, and has the potential as a promising vaccine candidate for the control of T. gondii infection.

Microsporidia are prolific producers of effector molecules, encompassing both proteins and nonproteinaceous effectors, such as toxins, small RNAs, and small peptides. These secreted effectors play a pivotal role in the pathogenicity of microsporidia, enabling them to subvert the host's innate immunity and co-opt metabolic pathways to fuel their own growth and proliferation. However, the genomes of microsporidia, despite falling within the size range of bacteria, exhibit significant reductions in both structural and physiological features, thereby affecting the repertoire of secretory effectors to varying extents. This review focuses on recent advances in understanding how microsporidia modulate host cells through the secretion of effectors, highlighting current challenges and proposed solutions in deciphering the complexities of microsporidial secretory effectors.

Microsporidia comprise a large phylum of single-cell and obligate intracellular parasites that can infect a wide range of invertebrate and vertebrate hosts including humans. These fungal-related parasites are characterized by a highly reduced genome, a strong energy dependence on their host, but also by their unique invasion organelle known as the polar tube which is coiled within the resistant spore. Upon appropriate environmental stimulation, the long hollow polar tube (ranging from 50 to 500 μm in length) is extruded at ultra-fast speeds (300 μm/s) from the spore acting as a harpoon-like organelle to transport and deliver the infectious material or sporoplasm into the host cell. To date, seven polar tube proteins (PTPs) with distinct localizations along the extruded polar tube have been described. For example, the specific location of PTP4 and PTP7 at the tip of the polar tube supports their role in interacting with cellular receptor(s). This chapter provides a brief overview on the current understanding of polar tube structure and dynamics of extrusion, primarily through recent advancements in cryo-tomography and 3D reconstruction. It also explores the various mechanisms used for host cell invasion. Finally, recent studies on the structure and maturation of sporoplasm and its moving through the tube are discussed.

Background Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. Methods The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. Results We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 μM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 μM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii , and genes involved in “ribosome biogenesis (GO:0042254)” and “ribosome (GO:0005840)” were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. Conclusions Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis. Graphical Abstract

Ameson portunus is an intracellular pathogen that infects marine crabs Portunus trituberculatus and Scylla paramamosain, causing significant economic losses. However, research into this important parasite has been limited due to the absence of an in vitro culture system. To address this challenge, we developed an in vitro cultivation model of A. portunus using RK13 cell line in this study. The fluorescent labeling assay indicated a high infection rate (similar to 60 %) on the first day post-infection and quantitative PCR (qPCR) detection demonstrated successful infection as early as six hours post-inoculation. Fluorescence in situ hybridization (FISH) and qPCR were used for the detection of A. portunus infected cells. The FISH probe we designed allowed detection of A. portunus in infected cells and qPCR assay provided accurate quantification of A. portunus in the samples. Transmission electron microscopy (TEM) images revealed that A. portunus could complete its entire life cycle and produce mature spores in RK13 cells. Additionally, we have identified novel life cycle characteristics during the development of A. portunus in RK 13 cells using TEM. These findings contribute to our understanding of new life cycle pathways of A. portunus. The establishment of an in vitro culture model for A. portunus is critical as it provides a valuable tool for understanding the molecular and immunological events that occur during infection. Furthermore, it will facilitate the development of effective treatment strategies for this intracellular pathogen.

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1’s translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell’s resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

ABSTRACT:The most common sites of distant metastases are lung, bone, pleura, and mediastinum in malignant phyllodes breast tumors. However, small bowel metastasis from malignant phyllodes breast tumors is rare. We reported that using CT and FDG PET/CT imaging we identified a case with small bowel metastasis from breast cancer. PET/CT scan showed that high 18 F-FDG uptake occurred in the duodenum and jejunum. Histopathology and immunohistochemistry analyses further confirmed that malignant phyllodes tumors are derived from the breast.

微管功能的多样性受到翻译后修饰(PTM)的影响,其中通过去除α-微管蛋白末端的酪氨酸所产生的去酪氨酸化修饰作用在妇科肿瘤转移中发挥着重要作用.近来,微管蛋白羧肽酶(TCP)的鉴定引起了人们对这一修饰作用的广泛关注.本文综述了微管蛋白去酪氨酸化修饰的过程及功能,探讨了该修饰对妇科肿瘤转移和微管靶向药物治疗的影响,为妇科肿瘤靶向治疗提供新的思路.

Vasohibin1 (VASH1) is a kind of vasopressor, produced by negative feedback from vascular endothelial growth factor A (VEGFA). Anti-angiogenic therapy targeting VEGFA is currently the first-line treatment for advanced ovarian cancer (OC), but there are still many adverse effects. Regulatory T cells (Tregs) are the main lymphocytes mediating immune escape function in the tumor microenvironment (TME) and have been reported to influence the function of VEGFA. However, whether Tregs are associated with VASH1 and angiogenesis in TME in OC is unclear. We aimed to explore the relationship between angiogenesis and immunosuppression in the TME of OC. We validated the relationship between VEGFA, VASH1, and angiogenesis in ovarian cancer and their prognostic implications. The infiltration level of Tregs and its marker forkhead box protein 3 (FOXP3) were explored in relation to angiogenesis-related molecules. The results showed that VEGFA and VASH1 were associated with clinicopathological stage, microvessel density and poor prognosis of ovarian cancer. Both VEGFA and VASH1 expression were associated with angiogenic pathways and there was a positive correlation between VEGFA and VASH1 expression. Tregs correlated with angiogenesis-related molecules and indicated that high FOXP3 expression is harmful to the prognosis. Gene set enrichment analysis (GSEA) predicted that angiogenesis, IL6/JAK/STAT3 signaling, PI3K/AKT/mTOR signaling, TGF-β signaling, and TNF-α signaling via NF-κB may be common pathways for VEGFA, VASH1, and Tregs to be involved in the development of OC. These findings suggest that Tregs may be involved in the regulation of tumor angiogenesis through VEGFA and VASH1, providing new ideas for synergistic anti-angiogenic therapy and immunotherapy in OC.