Background Neuropathic pain (NP) remains one of the most challenging problems in the world. This study aims to investigate the role of aurora kinase B (AURKB) and developmentally downregulated protein 4-like (NEDD4L) on NP and its underlying mechanisms. Methods BV2 microglial cells were stimulated with lipopolysaccharide (LPS). Protein expression was assessed using western blot. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-10 productions were examined using enzyme-linked immunosorbent assay (ELISA) assays. M1-like pro-inflammatory microglial activation in vitro was induced using LPS/IFN-γ. Ratio of CD86+ cells was analyzed using flow cytometry. Additionally, the relationship between NEDD4L and AURKB was analyzed using Ubibrowser database. Protein blotting analysis was performed after inhibiting protein synthesis and degradation to assess the stability of the AURKB protein, and the ubiquitination status was evaluated after treatment with the proteasome inhibitor MG132. For in vivo confirmation, a rat model of neuropathic pain was established via spinal nerve ligation (SNL) to evaluate the functional effects of NEDD4L overexpression on pain behaviors and inflammatory markers. Results Silencing AURKB inhibited LPS-induced inflammation and pro-inflammatory microglial activation in BV2 microglial cells. Furthermore, NEDD4L reduced AURKB protein abundance by promoting its degradation. NEDD4L overexpression suppressed the inflammatory response and pro-inflammatory microglial activation process in LPS-induced BV2 microglial cells. Mechanistically, AURKB overexpression reversed OE-NEDD4L-induced inhibitory effects on inflammatory response and pro-inflammatory microglial activation process in LPS-induced BV2 microglial cells. In the rat SNL model, NEDD4L overexpression inhibited pain and inflammatory responses, downregulated iNOS/AURKB, upregulated ARG1/NEDD4L, confirming its anti‑nociceptive and anti‑inflammatory function in vivo. Conclusion In LPS-induced BV2 microglial cells, NEDD4L affects AURKB stability via ubiquitination to control inflammatory response and the pro-inflammatory microglial activation state. The rat model confirms that NEDD4L overexpression protects against neuropathic pain, supporting the translational relevance of this axis.
Background The incidence of coronary heart disease (CHD), a complication of atherosclerosis (AS), is higher in postmenopausal women. Estrogen receptor α (ERα, encoded by ESR1) expression is decreased in this population, yet the molecular mechanism responsible for its downregulation remains poorly understood. Objectives To investigate how tripartite motif containing 37 (TRIM37) regulates ERα in oxidized low-density lipoprotein (ox-LDL)-stimulated HUVECs, and to explore whether manipulating this axis alters macrophage lipid-related readouts upon co-culture with small extracellular-vesicle-enriched preparations. Methods Human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL served as an in vitro model. Bioinformatic prediction, Western blot, GST pull-down, co-immunoprecipitation (Co-IP), cycloheximide (CHX) chase, and ubiquitination assays were employed to investigate the TRIM37-ERα interaction. Endothelial injury was assessed by CCK-8, flow cytometry, and tube formation assays. Oxidative stress, endoplasmic reticulum (ER) stress and inflammation were evaluated by measuring reactive oxygen species (ROS), antioxidant enzymes, ER stress markers, and inflammatory factors. Endothelial-derived extracellular vesicles were characterized by transmission electron microscopy, Western blot, and nanoparticle tracking analysis. Fluorescent labeling and lipid content assays were used to assess whether co-incubation with endothelial-derived extracellular vesicle-enriched preparations was associated with changes in macrophage lipid-related readouts. Results ox-LDL upregulated TRIM37 expression and downregulated ERα protein levels in HUVECs (P<0.01). TRIM37 was associated with ERα degradation via the ubiquitin-proteasome pathway. TRIM37 knockdown prolonged ERα half-life, reduced its ubiquitination level, and reversed ox-LDL-induced ERα downregulation. Furthermore, TRIM37 knockdown alleviated ox-LDL-induced metabolic activity loss, apoptosis increase, and tube-forming capacity impairment, while also inhibiting ROS accumulation, restoring antioxidant enzyme activities, and downregulating ER stress markers and inflammatory factors. All these protective effects were linked to ERα upregulation. Enriched extracellular vesicles from HUVEC supernatants exhibited classic characteristics in morphology, size, and markers, and displayed prominent fluorescence association with macrophages. Co-incubation with extracellular vesicle-enriched preparations from TRIM37-silenced endothelial cells was associated with lower macrophage triglyceride (TG) (P<0.001), total cholesterol (TC) (P<0.01), and LDL-C-like cholesterol readouts (P<0.001), and higher HDL-C-like cholesterol readouts (P<0.001). However, co-silencing of ESR1 reversed these associations. Conclusion In ox-LDL-stimulated HUVECs, TRIM37 was associated with ubiquitination-dependent ERα degradation and aggravated endothelial injury/inflammation. Extracellular vesicle-enriched preparations from TRIM37-silenced cells were associated with altered macrophage lipid readouts, but EV necessity remains unproven. This candidate pathway warrants validation in estrogen-deficient and EV-depleted/inhibited models.
Background Hepatocellular carcinoma (HCC) remains a leading cause of cancer‑related mortality worldwide. Cancer stem‑like cells contribute to HCC metastasis and recurrence partly through epithelial‑mesenchymal transition (EMT). This study aimed to investigate whether curcumol, an active compound from *Rhizoma Curcumae*, inhibits hepatocellular carcinoma stem‑like cells (HCSLCs) by repressing EMT and the Jak2/Stat3 signaling axis. Methods UPLC‑MS/MS was applied to characterize chemical constituents of Rhizoma Curcumae. HCSLCs were enriched by serum‑free spheroid culture combined with EpCAM‑based immunomagnetic positive selection. CCK‑8, wound‑healing and Transwell assays were performed to evaluate cell viability, migration and invasion. Western blot (WB) was used to detect EMT‑ and Jak2/Stat3‑related protein expression and phosphorylation. TMT‑based phosphoproteomics was conducted to profile phosphorylation modification; a nude‑mouse xenograft model was used for in‑vivo validation with oral curcumol at 50 mg/kg and 100 mg/kg. Results Curcumol exerted concentration‑dependent cytotoxicity against HCSLCs, among which HepG2‑EpCAM cells displayed the highest sensitivity. Transwell assays showed that increased curcumol concentration reduced the number of migratory and invasive HepG2‑EpCAM cells. In nude‑mouse xenograft assays, dose‑dependent tumour‑growth inhibition was observed in curcumol‑treated groups. The high‑dose curcumol group displayed smaller tumour mass, together with elevated tumour‑cell apoptosis compared with the vehicle control; statistically significant differences in apoptosis were detected by TUNEL staining (*P*<0.05). Western blot demonstrated that curcumol up‑regulated the epithelial marker E‑cadherin, down‑regulated the mesenchymal marker N‑cadherin, and decreased Jak2 and Stat3 phosphorylation in HepG2‑EpCAM cells. Phosphoproteomics analysis identified 889 up‑regulated and 248 down‑regulated phosphorylation sites upon curcumol treatment; these differential modification sites map to a total of 365 unique non‑redundant phosphoproteins. At the total‑protein abundance level, 633 up‑regulated and 177 down‑regulated proteins were quantified. Conclusion Curcumol suppresses malignant phenotypes of HCSLCs via inhibiting EMT through blockade of the Jak2/Stat3 signalling pathway. These findings provide experimental evidence supporting the potential application of curcumol in HCC therapy targeting liver cancer stem‑like cells.
Background Osteosarcoma (OS) is an aggressive malignant bone tumor with a high risk of metastasis. This study aimed to develop a histone deacetylase (HDAC)-related prognostic signature and explore its associations with immune features and predicted drug sensitivity. Methods Transcriptomic and clinical data from TARGET-OS were used as the training cohort, while GSE21257 and GSE39055 were used for external validation. HDAC-related co-expression modules were identified using ssGSEA and WGCNA, and a prognostic signature was constructed using Cox regression. Model stability was assessed by regularized regression, bootstrap resampling, repeated five-fold cross-validation, and clinical Cox analyses. Immune infiltration and predicted drug sensitivity were evaluated computationally. PSD3 was preliminarily validated in SJSA-1 cells. Results A high HDAC score was associated with metastasis and poor survival. A four-gene signature comprising CASK, NFX1, PSD3, and SAV1 stratified overall survival in TARGET-OS and GSE21257. Repeated five-fold cross-validation yielded a mean C-index of 0.713, and RiskScore remained associated with overall survival after adjustment for available clinical variables (HR = 3.112, 95% CI: 1.866–5.190, P < 0.001). In GSE39055, the high-risk group had shorter recurrence-free survival (P = 0.03), with AUCs of 0.71 at 3–5 years. Higher RiskScore was associated with lower inferred infiltration of CD8⁺ T cells, macrophages, and natural killer cells, higher inferred MDSC scores, and lower predicted IC50 values for 15 compounds that remained significant after false discovery rate correction, whereas TIDE scores did not differ significantly between risk groups. PSD3 knockdown inhibited SJSA-1 cell proliferation, invasion, and migration. Conclusion The four-gene HDAC-related signature was associated with prognosis and immune microenvironment features in OS. Its robustness and predicted drug-sensitivity associations require further prospective and experimental validation.
Objective Self-assembling peptide nanoparticles (SAPNs) offer a promising platform for multivalent antigen display, mimicking the repetitive patterns of microbial surfaces to induce robust immune responses. Here, we propose a novel SAPN-based vaccine targeting Helicobacter pylori (H. pylori), uniquely utilizing a native trimeric coiled-coil domain (Ccrp1142) derived from H. pylori as the oligomerization scaffold. The vaccine design incorporates immunodominant epitopes from UreB and HspA proteins, a mucosal targeting motif (CTGKSC) and a pentameric Trp-zipper domain to drive nanoparticle formation. Methods In silico structural modeling, molecular dynamics simulations, and docking analyses revealed favorable structural properties of the vaccine construct and a potential interaction with Toll-like receptor 2 (TLR2). Immunoinformatics analysis predicted strong antigenicity and non-allergenicity. Results These findings were reinforced by immune simulation results, which suggested the induction of robust humoral and cellular immune responses. Additionally, codon optimization and in silico cloning indicated the construct’s potential suitability for recombinant production. Conclusion Although experimental validation is required to confirm the safety and immunogenicity profile of the proposed vaccine construct, this study presents a pathogen-specific SAPN vaccine with potential for mucosal immunization against H. pylori.
