Purpose. Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common urological disorder. Although ferroptosis is closely associated with inflammation, oxidative stress, and neuropathic pain, its role in CP/CPPS has not yet been elucidated. Therefore, we sought to explore the role and mechanism of ferroptosis in the prostatitis development. Methods. The experimental autoimmune prostatitis (EAP) was established through intradermal immunization of prostate extract. Iron chelator deferoxamine (DFO) and free radical scavenger edaravone (EDA) were applied to evaluate the effects of ferroptosis inhibition on oxidative stress, ferroptosis, inflammation, fibrosis, and mast cell activation in the context of CP/CPPS. Results. Increased generation of lipid peroxidation products (ROS and MDA) and decreased activities of antioxidant enzymes (SOD and CAT) suggested an aberrant oxidative stress status in EAP model. Elevated iron concentration was observed in the EAP model. Meanwhile, we discovered significant biological performances associated with ferroptosis in CP/CPPS, including the downregulation of the system Xc^-/GPX4 axis and the upregulation of the ACSL4/LPCAT3 axis. EAP rats performed serious leukocyte infiltration, advanced inflammatory grade, and abnormal expression of inflammatory mediators. Abundant collagen deposition, enhanced RhoA, ROCK1, and α-SMA protein levels indicated that EAP rats were prone to suffer from stromal fibrosis compared with control group. An elevated number of degranulated mast cells and corresponding marker TPSB2 represented that mast cell-sensitized pain was amplified in the EAP model. Furthermore, reduction of NRF2/HO-1 indicated a vulnerability of EAP towards ferroptosis response. However, application of DFO and EDA had partially reversed the adverse influences mentioned above. Conclusion. We first demonstrated that ferroptosis might be a crucial factor of chronic prostatitis progression. Inhibition of ferroptosis using DFO and EDA represented a promising approach for treating prostatitis by ameliorating inflammation, fibrosis, and mast cell activation.

The adoption of three-dimensional (3D) cell culture systems represents a critical advancement in biomedical research, better mimicking complex 3D tissue environments than traditional two-dimensional (2D) models. However, variability in experimental outcomes has limited their reproducibility and clinical translation. Here, we systematically analyzed 32,000 spheroid images to identify key parameters influencing 3D model reliability. Our large-scale analysis revealed that oxygen levels significantly affect spheroid size and necrosis, while media composition (e.g., glucose and calcium concentrations) and serum levels (0–20%) critically regulate cell viability and structural integrity. For instance, spheroids cultured in 3% oxygen exhibited reduced dimensions and increased necrosis, whereas serum concentrations above 10% promoted dense spheroid formation with distinct necrotic and proliferative zones. By integrating single-cell RNA sequencing and automated image analysis, we uncovered dynamic gene expression patterns linked to spheroid maturation and hypoxia. These findings provide actionable guidelines for standardizing 3D culture protocols, addressing critical reproducibility challenges. Our work establishes a robust framework to enhance the reliability of 3D models in drug testing, personalized medicine, and tumor biology, facilitating their broader adoption in translational research.

Objective: Inflammation plays an important role in the development of benign prostatic hyperplasia (BPH). The aim of the present study was to reference the study of the pathological changes in the prostate gland of rats with experimental autoimmune prostatitis (EAP), for the development of experimental models of BPH.Methods: Experimental autoimmune prostatitis was induced in rats by the intradermal injection of rat prostate antigen with immunoadjuvants. In case of the positive BPH group, BPH was induced by the subcutaneous injection of testosterone propionate. At the end of the 45-day model period, prostate weights were measured, and the histopathological analysis of the prostate glands was performed. The levels of cytokines, TGF-β1/RhoA/ROCK signals, and the oxidative stress status were also examined.Results: Rats from the EAP group had a higher histological score than those from the control group. Compared to the samples from rats in the hormone-induced group, those from the EAP group showed a more pronounced increase in the size of the stromal compartment; this was characterized by the formation of reactive stroma and the deposition of a greater amount of extracellular matrix (ECM). Significant increases in the numbers of CD3-positive cells and CD68-positive cells, as well as a significant upregulation in the cytokine levels, and an increase in the TGF-β1 levels and activation of RhoA/ROCK signaling, were observed in the samples from rats in the EAP group.Conclusion: Chronic inflammation can induce BPH in rats via EAP model method. When performing drug experiments on the stroma compartments of BPH, the use of the EAP model is a recommendation of the authors based on this study.

Lung cancer is the leading cause of cancer-related death globally. Ubiquitin modification plays a crucial role in the regulation of gene expression, and is closely associated with cancer pathogenesis. The aim of our study was to clarify the role and mechanisms of action for HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 (HECW1) in non-small cell lung cancer (NSCLC). Herein, we demonstrate that the expression of HECW1 was significantly increased in NSCLC cell lines and tissues. Upregulation of HECW1 markedly enhanced the proliferation of NSCLC cells, whereas downregulation of HECW1 significantly inhibited proliferation. Moreover, the expression levels of HECW1 positively correlated with the migration and invasiveness of NSCLC cells. Upregulation or downregulation of HECW1 only affected the protein expression levels of SMAD family member 4 (Smad4), but had no effect on the mRNA expression levels. Furthermore, after treatment with MG-132, the relative protein level of Smad4 significantly increased in NSCLC cells. HECW1 promoted the proliferation, migration, and invasiveness of NSCLC cells by inducing the ubiquitination and degradation of Smad4, thus our data provide a novel target for NSCLC treatment.

Background: Among erectile dysfunction (ED) caused by metabolic abnormalities, diabetes mellitus‐induced ED (DMED) progresses rapidly, manifests with severe symptoms, and shows reduced responsiveness to conventional medications. Hyperglycemia in the corpus cavernosum has been linked to the induction of both ferroptosis and oxidative stress, which are mediated by nuclear factor E2 related factor 2 (Nrf2). Hesperidin (Hes), a flavonoid compound, has been revealed to activate Nrf2 in certain diabetic complications, yet the efficacy of Hes on DMED and the specific mechanism remain unclear. Objectives: To elucidate the potential mechanism and efficacy of Hes in regulating Nrf2‐mediated ferroptosis and oxidative stress in DMED. Materials and methods: DMED rats were constructed through the intraperitoneal injection of streptozotocin (STZ), partially supplemented with Hes. In parallel, in vitro research utilized human umbilical vein endothelial cells (HUVECs), with glucose addition to simulating a high glucose (HG) environment, and induced with Hes or ML385 (an Nrf2 inhibitor). Penile tissues and HUVECs were harvested for subsequent analyses. Results: The results of this study indicate that Hes partially reversed the impaired erectile function. The expression of Nrf2, glutathione peroxidase 4 (GPX4), and heme oxygenase‐1 (HO‐1) in the corpus cavernosum elevated after supplementing with Hes, resulted in an inhibition in ferroptosis and oxidative stress. Moreover, the quantity and function of erectile effector cells were restored, and cavernous fibrosis was ameliorated. In HG‐induced HUVECs, Hes ameliorated Nrf2‐mediated ferroptosis and oxidative stress, effects which ML385 partially reversed. Conclusions: Hes exerts a therapeutic effect on DMED rats and a regulatory mechanism on the Nrf2–HO‐1/GPX4 axis, concurrently revitalizing endothelial and smooth muscle cells, and diminishing fibrosis. Our study provides robust preclinical evidence for employing Hes in treating DMED.