Background: Oxylipin metabolism plays an essential role in glioma progression and immune modulation in the tumor microenvironment. Lipid metabolic reprogramming has been linked to macrophage remodeling, while the understanding of oxylipins and their catalyzed enzymes lipoxygenases in the regulation of glioma-associated microglia/macrophages (GAMs) remains largely unexplored. Methods: To explore the pathophysiological relevance of oxylipin in human glioma, we performed Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis in human glioma and non-tumor brain tissues. To comprehensively investigate the role of arachidonate lipoxygenase 5 (ALOX5) in glioma, we performed in vivo bioluminescent imaging, immunofluorescence staining and flow cytometry analysis on tumors from orthotopic glioma-bearing mice. We developed an ALOX5-targeted nanobody, and tested its anti-glioma efficacy of combination therapy with α-programmed cell death protein-1 (PD-1). Results: In this study, we found that ALOX5 and its oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) are upregulated in glioma, accumulating programmed death-ligand 1 (PD-L1)⁺ M2-GAMs and orchestrating an immunosuppressive tumor microenvironment. Mechanistically, 5-HETE derived from ALOX5-overexpressing glioma cells, promotes GAMs migration, PD-L1 expression, and M2 polarization by facilitating nuclear translocation of nuclear factor erythroid 2-related factor 2. Additionally, a nanobody targeting ALOX5 is developed that markedly suppresses 5-HETE efflux from glioma cells, attenuates M2 polarization of GAMs, and consequently ameliorates glioma progression. Furthermore, the combination therapy of the ALOX5-targeted nanobody plus α-PD-1 exhibits superior anti-glioma efficacy. Conclusions: Our findings reveal a pivotal role of the ALOX5/5-HETE axis in regulating GAMs and highlight the ALOX5-targeted nanobody as a potential therapeutic agent, which could potentiate immune checkpoint therapy for glioma.

Ultrasound-assisted fermentation (UAF) was evaluated as a calorimetrically calibrated strategy for biotransforming Astragalus membranaceus isoflavones using Lactiplantibacillus plantarum. Water-based calorimetry yielded power-density estimates of 46.51 ± 4.03 and 86.05 ± 4.03 W/L for the fermentation vessel configuration. High-dose UAF accelerated the conversion of isoflavone glucosides into calycosin and formononetin, reaching 99.7 ± 2.4% of the conventional 24 h aglycone level by 18 h with a 50% higher mean formation rate over 10–18 h. No aglycone formation was detected in the sterile ultrasound control under the tested conditions. At the 18 h time-matched sampling point, high-dose UAF yielded higher in vitro antioxidant capacities, stronger DPP-IV and pancreatic lipase inhibition, and greater measured intestinal-phase calycosin recovery (374.80 vs. 314.31 μg/g) than conventional fermentation. At 16 h, extracellular β-glucosidase activity increased dose-dependently from 3.52 ± 0.82 to 11.19 ± 0.45 U/mL (conventional vs. high-dose UAF), whereas intracellular activity remained comparable across treatments. Combined with qualitative SEM and CLSM observations, these findings are consistent with enhanced extracellular enzyme availability and plant matrix disruption, without establishing de novo enzyme synthesis or a specific permeabilisation mechanism.