ω-6 Polyunsaturated fatty acids (PUFAs) are essential fatty acids involved in various cellular metabolic processes. Their anti-stress activities, including alleviation of oxidative stress, modulation of cyclooxygenase activity, and alterations in membrane phospholipid composition and receptor function, have been extensively studied. However, the precise stress-preventive mechanisms remain unclear. Two critical host defense mechanisms, autophagy and Keap1-Nrf2 system-mediated antioxidation, are linked to metabolic pathways and innate immunity. Dysregulation of these processes is implicated in numerous human diseases. While studies suggest autophagy and antioxidation are altered in certain cancer cell lines in response to PUFAs, the molecular mechanisms governing ω-6 PUFA regulation of autophagy and antioxidation were previously unknown. Recent research indicates PUFAs positively influence AMPK and TOR signaling pathways, with AMPK identified as a novel Nrf2 inducer via ATP-depletion-induced activation. The role of AMPK and TOR pathways in ω-6 PUFA-induced autophagy and antioxidation, however, was not fully understood. Furthermore, the potential interrelationship between autophagy and antioxidation under antioxidant-supplemented conditions, possibly mediated by the P62 and Keap1 complex, warranted investigation. Fish, possessing immune defense systems similar to mammals, provide valuable model organisms for studying human disease pathogenesis. Previous research indicated that ω-6 PUFAs (linoleic acid) influence the antioxidant system of large yellow croaker, but the specific molecular mechanisms remained unclear. This study aimed to investigate how ω-6 PUFAs regulate autophagy and antioxidation and the potential relationship between these two mechanisms, with the ultimate goal of discovering new approaches to improve immune function in human diseases.

Existing literature highlights the multifaceted roles of ω-6 PUFAs in cellular metabolism and stress response. Studies have demonstrated their antioxidant properties, influence on cyclooxygenase activity, and effects on membrane properties. However, a comprehensive understanding of the molecular mechanisms underlying these effects has been lacking. The involvement of autophagy and the Keap1-Nrf2 antioxidant system in various human diseases has been well-established. Previous research has shown alterations in autophagy and antioxidation in response to PUFAs in certain cancer cell lines. Studies have also indicated the positive influence of PUFAs on AMPK and TOR signaling pathways, crucial regulators of both autophagy and antioxidation. The interrelationship between autophagy and the antioxidant system has been suggested in some studies, with evidence suggesting a possible link through the P62 and Keap1 complex. While the effects of ω-3 PUFAs on these pathways have been investigated, research on the specific effects of ω-6 PUFAs remained limited, particularly at the molecular level.

This study employed both in vivo and in vitro approaches. In vivo experiments used large yellow croaker fed diets supplemented with either fish oil (FO, rich in ω-3 PUFAs) or soybean oil (SO, rich in ω-6 PUFAs) for 10 weeks. Transcriptomic analysis was performed on liver samples to identify differentially expressed genes related to autophagy and antioxidation. Antioxidant enzyme activities (CAT, SOD, Gpx) and lipid peroxidation markers (MDA, T-AOC) were also measured. Electron microscopy (EM) was used to visualize autophagosomes, and western blotting assessed LC3 protein levels. In vitro experiments utilized large yellow croaker hepatocytes treated with linoleic acid (LA, representing ω-6 PUFAs) and docosahexaenoic acid (DHA, representing ω-3 PUFAs). Cell viability was assessed using a CCK8 assay. RT-qPCR measured mRNA expression levels of genes related to antioxidation and autophagy. Several methods were used to assess autophagosome formation: acridine orange (AO) staining, monodansylcadaverine (MDC) staining, and LysoTracker Red staining. Western blotting was used to assess protein levels of AMPK, TOR signaling pathway components, and nuclear Nrf2. To investigate the mechanisms, specific activators (AICAR, NK-252, MHY1485, RAPA) and inhibitors (CC, ML385, 3-MA, CQ) of AMPK, TOR, and Nrf2 pathways were used. Immunofluorescence and co-immunoprecipitation (Co-IP) were performed to investigate the interaction between Keap1 and P62. Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assays (EMSAs) were used to assess Nrf2 binding to the P62 promoter. Statistical analyses included independent t-tests and Tukey's test.

In vivo, dietary ω-6 PUFA supplementation increased autophagosome formation (observed via EM) and the LC3-II/LC3-I protein ratio, indicating increased autophagy. However, it decreased the activity of antioxidant enzymes (CAT, SOD, Gpx), total antioxidant capacity (T-AOC), and increased malondialdehyde (MDA) levels, indicating decreased antioxidant ability. In vitro, LA treatment dose-dependently increased autophagosome formation (AO, MDC, and LysoTracker staining) and upregulated the expression of autophagy-related genes (Beclin1, ULK1, ATG101, ATG12, ATG4B, LC3, GABARA, and P62). LA also increased antioxidant ability by increasing the mRNA and protein levels of Nrf2 and other antioxidant genes (SOD1, SOD3, CAT, Gpx). Mechanistically, LA activated AMPK, leading to the inhibition of mTOR and subsequent activation of autophagy and antioxidation. Both TOR-dependent and TOR-independent pathways were involved in this process. Furthermore, a synergistic feedback loop between autophagy and antioxidation was identified. P62 interacted directly with Keap1, promoting Nrf2 nuclear translocation and activation of antioxidant genes. Nrf2 directly bound to the ARE motif in the P62 promoter, leading to increased P62 expression, thus forming a positive feedback loop.

This study provides novel insights into the intricate interplay between ω-6 PUFAs, autophagy, and the antioxidant system. The in vivo and in vitro results revealed distinct but complementary aspects of ω-6 PUFA actions. While in vivo studies highlighted the complex effects of dietary ω-6 PUFAs on antioxidant capacity, in vitro studies clarified the direct mechanistic effects of ω-6 PUFAs on autophagy and antioxidation in hepatocytes. The findings demonstrate that ω-6 PUFAs can activate both autophagy and antioxidation, particularly through AMPK and TOR signaling pathways. The identification of a synergistic feedback loop involving P62 and Keap1 is a significant contribution, enhancing our understanding of the integrated cellular response to ω-6 PUFAs. This coordinated response suggests that ω-6 PUFAs may provide cytoprotective effects by promoting both autophagic clearance of damaged components and enhanced antioxidant defense. The discrepancy between the in vivo and in vitro results underscores the complexity of ω-6 PUFA effects within a whole organism, influenced by multiple factors beyond the cellular level. Further studies should investigate the in vivo effects of ω-6 PUFAs by considering other factors and tissues.

This study demonstrates for the first time that ω-6 PUFAs activate autophagy and antioxidation via AMPK and AMPK-TOR signaling pathways and revealed a synergistic feedback loop between these processes mediated by the P62-Keap1 complex. These findings suggest that ω-6 PUFAs may have therapeutic potential in various pathologies characterized by impaired autophagy and antioxidation. Future research should focus on translating these findings into clinical applications and exploring the potential of ω-6 PUFAs as therapeutic agents.

The study primarily focused on large yellow croaker hepatocytes, which may limit the generalizability of the findings to other cell types and species. The in vivo study used a single dose of ω-6 PUFAs, and future research could investigate a range of doses to determine dose-response relationships. Furthermore, the study focused mainly on the liver, and investigations into other tissues are needed. Finally, the mechanistic studies involved pharmacological interventions, which may not perfectly reflect the natural physiological processes.