Lipoxygenase-Derived Oxylipins are Elevated in MDD Patients and Stress-Induced Depressed Mice: Inhibition of Lipoxygenases as a Therapeutic Strategy

Lipoxygenase-Derived Oxylipins are Elevated in MDD Patients and Stress-Induced Depressed Mice: Inhibition of Lipoxygenases as a Therapeutic Strategy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lipoxygenase-Derived Oxylipins are Elevated in MDD Patients and Stress-Induced Depressed Mice: Inhibition of Lipoxygenases as a Therapeutic Strategy Xiaohong Ma, Jinxue Wei, Yikai Dou, Liansheng Zhao, Min Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9366044/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Oxylipins are bioactive lipid metabolites that may bridge immune dysregulation and depressive symptomatology. However, systematic alterations of oxylipin networks in MDD and their therapeutic implications remain unclear. In this study, a plasma oxylipidomics analysis was performed in 154 patients with MDD and 134 healthy controls (HC). Findings were validated in a chronic restraint stress (CRS) mouse model of depression, followed by pharmacological inhibition of lipoxygenase (LOX) pathways using ML-355 and zileuton. Behavioral tests and immunofluorescence analysis of microglial activation were conducted to assess therapeutic effects. MDD patients exhibited significantly elevated plasma levels of 22 oxylipins compared to HCs, including oxylipins derived from lipoxygenase (LOX), cytochrome P450 (CYP450), cyclooxygenase (COX), and non-enzymatic pathways. Notably, non-responders to antidepressant treatment displayed higher baseline levels of 20 oxylipins than both responders and HCs, and baseline oxylipin levels negatively correlated with Hamilton Depression Rating Scale (HAMD) score reduction rates. In CRS mice, the LOX pathway was activated, as evidenced by increased LOX levels in the blood and brain, as well as elevated plasma levels of LOX-derived oxylipins. Pharmacological inhibition of 12-lipoxygenase (12-LOX) with ML-355 significantly alleviated depressive-like and anxiety-like behaviors and reversed stress-induced microglial activation in the hippocampus and medial prefrontal cortex. The 5-LOX inhibitor zileuton reduced microglial activation in a region-dependent manner but did not significantly improve behavioral outcomes. These findings reveal elevated LOX-derived oxylipins as potential biomarkers predicting poor antidepressant response in MDD. Targeting the LOX pathway, particularly 12-LOX, represents a promising therapeutic strategy for depression by ameliorating neuroinflammatory processes. Biological sciences/Biochemistry Health sciences/Diseases/Psychiatric disorders/Depression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Major depressive disorder (MDD) is one of the leading causes of disability worldwide, imposing a substantial and growing global health burden. According to the Global Burden of Disease 2021 study, depressive disorders account for a major proportion of years lived with disability, particularly among adolescents and young adults, and their burden has continued to rise over recent decades ( 1 ). Despite its high prevalence and profound functional impairment, MDD is a biologically heterogeneous disorder characterized by diverse symptom profiles and variable treatment responses. Increasing evidence indicates that inflammation plays a pivotal role in the pathophysiology of MDD, featuring systemic immune activation marked by elevated peripheral pro-inflammatory cytokines and central neuroinflammation involving microglial activation and blood-brain barrier disruption. Notably, elevated inflammatory markers predict resistance to conventional antidepressants, whereas anti-inflammatory agents, as monotherapy or adjunctive therapy, demonstrate therapeutic potential in inflammation-associated MDD ( 2 , 3 ). Collectively, these observations underscore immune-targeted interventions as a viable precision medicine strategy for patients with inflammation-related major depressive disorder. Given the established role of inflammation in MDD pathophysiology, understanding the specific mediators driving inflammatory processes is essential. Oxylipins represent one such class of bioactive lipid metabolites that may bridge immune dysregulation and depressive symptomatology. These molecules are derived from the oxidation of polyunsaturated fatty acids (PUFAs) via either enzymatic or non-enzymatic mechanisms ( 4 ). The enzymatic pathways are mediated by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases (CYP450) that catalyze PUFA oxidation, while non-enzymatic pathways are driven by reactive oxygen species (ROS)-induced lipid peroxidation ( 5 , 6 ). Oxylipins act as key inflammatory mediators and are widely recognized as reliable biomarkers, as their elevated levels directly reflect the extent of lipid damage and oxidative stress in biological systems ( 5 , 7 – 9 ). Moreover, these lipid mediators can be transported in circulation via lipoprotein particles, thereby enabling systemic signaling and modulating their contributions to neurological diseases ( 10 ). Recent advances in profiling techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have enabled comprehensive analysis of oxylipin networks in clinical research, underscoring their potential as biomarkers and therapeutic targets ( 11 ). Previous studies have reported alterations in specific oxylipin profiles in MDD, including decreased 14,15-EET and increased 14,15-DHET ( 12 ), differential oxidized phosphatidylcholines in rTMS treatment responders versus non-responders ( 13 ), as well as changes in sEH-derived metabolites in seasonal depression ( 14 ) and diabetes-associated depression ( 15 , 16 ). However, these investigations were limited by narrow coverage of oxylipin species and small sample sizes, precluding systematic characterization of oxylipin network alterations in MDD. In this study, we investigated oxylipin profile alterations in patients with MDD and tested the therapeutic potential of targeting the LOX pathway in a mouse model of depression. We found that multiple oxylipins and their precursors, particularly those enriched in LOX and CYP450 pathways, were elevated in MDD patients, with LOX-derived metabolites showing the strongest association with treatment resistance. Based on these clinical findings, we examined the LOX pathway in a chronic restraint stress mouse model and found altered plasma levels of LOX-derived oxylipins and increased LOX expression across peripheral tissues and brain. Furthermore, pharmacological inhibition of 12-LOX ameliorated stress-induced depressive-like behaviors and microglial activation in mice. These findings implicate dysregulated LOX-mediated oxylipin metabolism in MDD pathophysiology and support targeting 12-LOX as a potential therapeutic strategy for depression. Methods Participants A total of 154 patients with major depressive disorder (MDD) aged 16–55 years were recruited from the Mental Health Center of West China Hospital, Sichuan University, between 2021 and 2023.Of the 154 MDD patients, 74 patients completed the 8-week follow-up assessment and were included in the treatment response analysis. All patients were required to meet the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria for MDD and had no comorbid psychiatric or other medical disorders. Patients who had used any antidepressants or antipsychotics within 2 weeks prior to recruitment were excluded. The inclusion and exclusion criteria were consistent with those reported in previous publications ( 17 ). Healthy Han Chinese volunteers (n = 134) aged 16–60 years were recruited via online advertisements. Both MDD patients and healthy controls (HCs) with neurodegenerative diseases, major endocrine or metabolic disorders, as well as pregnant or breastfeeding individuals, were excluded from the study. Each participant spent approximately 2 hours completing all assessments. The research protocol was approved by the Institutional Ethics Committee of West China Hospital, Sichuan University (Approval Number: [2016] 170). All participants were fully informed about the study procedures and provided written informed consent. The Hamilton Depression Scale (HAMD) and the Hamilton Anxiety Scale (HAMA) were used to assess depressive symptoms and anxiety symptoms in all the patients with MDD. The HAMD comprises five subscales: retardation (items 1, 7, 8, 14), cognitive disturbance (items 2, 3, 9), sleep disturbance (items 4, 5, 6), somatization (items 10, 11, 12, 15,17), and weight (item 16). The HAMA comprises two subscales: psychological anxiety (items 1–5) and somatic anxiety (items 6–14). Plasma sample collection Peripheral blood samples were collected from all participants on the day of enrollment using ethylenediaminetetraacetic acid (EDTA)-coated anticoagulant tubes. Immediately after collection, the blood samples were centrifuged at 2000 × g for 10 minutes at 4℃ to separate the components. Subsequently, the upper plasma layer was carefully aspirated using a sterile pipette, transferred to a new RNase/DNase-free cryovial, and immediately stored at − 80℃ until subsequent experimental analysis. Oxylipidomics analysis in plasma The quantitative determination of oxylipins in plasma was based on a previous report and was subsequently optimized ( 18 ). Briefly, oxylipins in plasma were extracted via solid - phase extraction (SPE). The extracts were analyzed using ultra - performance liquid chromatography (UPLC) coupled with AB SCIEX 6500 + mass spectrometry in multiple reaction monitoring (MRM) mode. Oxylipin identification and validation were primarily achieved by comparing the retention times of chromatographic peaks with those of standard substances. Relative quantification of oxylipins was performed through peak integration preprocessing using SCIEX OS (v2.0, AB Sciex, Foster, ND, USA), followed by analysis using R software (v3.5.1). A total of 120 species of lipids, including 11 PUFAs and 109 their derived oxidized lipids were quantified in this study (Table S1 ). Experimental animals and chronic restraint stress model Male C57BL/6J mice were obtained from Jicui Yaokang Biotechnology Co., Ltd (Chengdu, China). Mice were maintained under standard laboratory conditions with controlled temperature and a 12 h light/dark cycle. All procedures were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (Approval No. 20250714003). Chronic restraint stress (CRS) was employed to induce depression-like behaviors. Mice were placed individually into 50 mL centrifuge tubes for 4 h per day for 28 consecutive days. During the 14-day drug treatment period, restraint procedures were maintained in all stressed groups to ensure sustained stress exposure. Animals were randomly allocated into five groups (n = 7–8): Control (saline), CRS + saline, CRS + ML355 (10 mg/kg), CRS + Zileuton (10 mg/kg), and CRS + Fluoxetine (10 mg/kg). All treatments were administered once daily by intraperitoneal injection for 14 days (Zileuton: 5-LOX inhibitor; ML-355: 12-LOX inhibitor). Sucrose preference test The sucrose preference test (SPT) was performed to assess anhedonia-like behavior in mice. The experiment consisted of an adaptation phase and a formal testing phase. During the adaptation phase, mice were individually housed and provided with two bottles containing 2% (w/v) sucrose solution for 24 h. Subsequently, one bottle was replaced with pure water for an additional 24 h to allow acclimatization to both liquids. After adaptation, mice were deprived of food and water for 12 h. Following deprivation, each mouse was given free access to two pre-weighed bottles containing 100 mL of 2% (w/v) sucrose solution and 100 mL of water, respectively. The testing period lasted 12 h. At the end of the test, bottles were weighed to determine fluid consumption. Sucrose preference was calculated using the following formula: Sucrose preference (%) = sucrose consumption / (sucrose consumption + water consumption) × 100%. Open field test The open field test (OFT) was conducted to evaluate locomotor activity and anxiety-like behavior [21]. Approximately 24 h before testing, mice were transferred to the behavioral testing room for 2 h of habituation. On the test day, animals were allowed to acclimate to the environment for an additional 30 min prior to the experiment.The test was performed in a white plexiglass arena (50 cm × 50 cm × 40 cm) under dim illumination (approximately 60 lux). Each mouse was gently placed in the same corner of the apparatus and allowed to freely explore the arena for 10 min. The central zone was defined as a 25 cm × 25 cm square located in the middle of the field. Behavior was recorded and analyzed using the EthoVision XT video tracking system (Noldus, Netherlands). The parameters measured included total distance traveled, average movement velocity, number of entries into the central area, and time spent in the central area.After each trial, the apparatus was cleaned with 75% ethanol and allowed to dry to eliminate olfactory cues. Elevated plus maze The elevated plus maze (EPM) was used to evaluate anxiety-like behavior in mice. Animals were habituated to the behavioral testing room for at least 1 h before the experiment. The EPM apparatus consisted of two open arms (30 cm × 6 cm) and two closed arms (30 cm × 6 cm × 15 cm), elevated 50 cm above the floor. At the beginning of the test, each mouse was placed in the central platform of the maze and allowed to freely explore for 5 min.The time spent in the open and closed arms was recorded and analyzed. After each trial, the maze was carefully cleaned with 75% ethanol to remove residual odors before the next animal was tested. Tail suspension test The tail suspension test (TST) was conducted to evaluate despair-like behavior in mice. Each mouse was suspended individually by adhesive tape placed approximately 1 cm from the tip of the tail on the edge of a suspension shelf (height 30 cm, diameter 21 cm). The head of the mouse was positioned approximately 5 cm above the surface to prevent grasping or support. The total testing time was 6 min. The first 2 min were regarded as an adaptation period. Immobility time during the final 4 min was automatically recorded using the EthoVision XT video tracking system (Noldus, Netherlands).Immobility was defined as the absence of voluntary body movement while hanging passively. Forced swimming test The forced swimming test (FST) was performed to evaluate despair-like behavior in mice. Each mouse was individually placed in a transparent plastic cylinder (30 cm in height, 15 cm in diameter) containing water at a depth of 15 cm maintained at 25 ± 1 ℃. The total duration of the test was 6 min. The first 2 min served as an adaptation period, and the cumulative immobility time during the last 4 min was automatically recorded. Immobility was defined as floating passively in the water without active struggling or climbing, except for minimal movements required to keep the head above the water surface. Behavior was recorded and analyzed using the EthoVision XT video tracking system (Noldus, Netherlands). After each trial, the water was replaced to avoid interference with subsequent animals. Tested mice were dried and kept separately from untested animals after the experiment. Immunofluorescence staining Immunofluorescence staining was performed after completion of behavioral tests. Mice were anesthetized with 2,2,2-tribromoethanol (10%, 0.2 ml/10 g, i.p.), and three animals per group were randomly selected. Following transcardial perfusion with 0.9% saline and 4% paraformaldehyde (pH 7.4), brains were collected, post-fixed, dehydrated, and coronally sectioned at 30 µm using a Leica CM1950 cryostat. Sections were blocked in 5% donkey serum containing 0.3% Triton X-100 for 1 h and incubated overnight at 4℃ with primary antibodies against IBA-1(66827-1-IG, 1:200 dilution, Proteintech, China) and NeuN (26975-1-AP, 1:200 dilution, Proteintech, China). After washing, sections were incubated with fluorescent secondary antibodies for 2 h at room temperature and counterstained with DAPI. Fluorescence images were acquired using a Zeiss LSM 710 confocal microscope at 200× magnification. Two to three randomly selected fields from the prefrontal cortex and hippocampus were analyzed per animal. ImageJ software was used to quantify the mean fluorescence intensity of IBA-1 staining, which reflects microglial activation levels. All analyses were performed by an investigator blinded to group allocation. Statistical analysis Statistical analyses were performed using R (version 4.3.1). The chi-square test was used to compare sex distribution between groups. Independent-samples t-tests were applied to compare age, BMI, number of episodes, illness duration, and HAMD/HAMA total scores between the two groups. One-way ANOVA followed by Tukey's multiple comparison test was used to compare lipid levels among healthy controls, responders, and non-responders, as well as to evaluate group differences in behavioral and microglia data. Spearman's correlation analysis was performed to assess associations between lipid levels and scores of HAMD or HAMA. To control for multiple comparisons in the correlation analysis, the false discovery rate (FDR) method was applied, and adjusted p values were reported. Results Demographic characteristics A total of 154 MDD patients (42 males, 112 females) and 134 healthy controls (44 males, 90 females) were included in this study. There were no significant differences of sex (χ² = 0.81, p = 0.37), age ( t = -0.34, p = 0.74) and body mass index (BMI, t = -0.76, p = 0.45) between MDD and HC. The onset age, number of episodes, current disease duration, total score of HAMD and total score of HAMA of patients with MDD were 1.67 (1.45), 9.61 (14.55), 21.65 (4.67), 21.75 (7.18), respectively (Table 1). Plasma oxylipin profile in patients with MDD For clinical samples, after excluding metabolites with a missing-value proportion exceeding 50%, a total of 41 lipid species were included in subsequent statistical analyses. Among them, 22 lipid species exhibited differential expression between healthy controls (HC) and patients with major depressive disorder (MDD). Relative to HC, only 9,12,13-TriHOME was downregulated in MDD patients, while the remaining 21 differential lipid species were upregulated. Of these differential lipids, three belong to precursor polyunsaturated fatty acids (PUFAs): arachidonic acid (ARA), eicosapentaenoic acid (EPA), and adrenic acid. The remaining upregulated lipids are oxylipins derived from five PUFAs (ARA, EPA, docosahexaenoic acid (DHA), dihomo-γ-linolenic acid (DGLA), and linolenic acid (LA)). Specifically, 8 of these oxylipins are generated via the lipoxygenase (LOX) pathway (12-HETE, 5-HETE, 15-HETE, 15-HETrE, 14s-HDoHE, 4-HDHA, 12-HEPE, and 9,12,13-TriHOME), 8 via the cytochrome P450 (CYP450) pathway (5,6-diHETrE, 14,15-DiHET, 11,12-DiHET, 16,17-DiHDPA, 13,14-DiHDPA, 10,11-DiHDPA, 22-HDHA, and 17,18-DiHETE), 1 via the cyclooxygenase (COX) pathway (11-HETE), and 2 via the non-enzymatic pathway (16-HDHA and 11-HDHA) (Fig. 1 ). Relationship between plasma lipid levels and antidepressant treatment effects in MDD Patients with a ≥ 50% reduction in HAMD score at week 8 were classified as treatment responders, while those with a reduction rate < 50% were defined as non-responders. Among the 154 MDD patients, follow-up data at week 8 were available for 74 individuals (responders: n = 39; non-responders: n = 35; Table S2). There were no significant between-group differences in sex, age, BMI, age of onset, number of episodes, or duration of the current depressive episode (Table S2). At baseline (W0), there were no significant differences in total HAMD ( p = 1.00) and HAMA scores ( p = 0.73) between responders and non-responders (Figure S1 A). At week 8 (W8), responders showed significantly lower scores than non-responders in both total HAMD and HAMA scores (both p < 0.001, Figure S1 B). In responders, both the total HAMD and HAMA scores at week 8 (W8) were significantly lower than those at W0 (both p < 0.001, Figure S1 C). In non-responders, the total HAMD score at W8 was lower than that at W0 ( p < 0.001), whereas no significant change was noted in the total HAMA score between W0 and W8 ( p = 0.11, Figure S1 D). To determine whether differential treatment responses were associated with plasma oxylipin profiles, we compared baseline lipid levels among responders, non-responders, and HCs. At W0, 20 lipids differed significantly across the three groups. All of these lipids were elevated in non-responders compared to both HCs and responders, and 13 of them were also higher in responders than in HCs. These differential lipids included PUFAs (ALA, ARA, and adrenic acid) and oxylipins from the LOX pathway (12-HETE, 5-HETE, 15-HETE, 15-HETrE, 14s-HDoHE, 4-HDHA, and 12-HEPE), the CYP450 pathway (5,6-diHETrE, 14,15-DiHET, 11,12-DiHET, 13,14-DiHDPA, 10,11-DiHDPA, and 22-HDHA), the COX pathway (11-HETE), and the non-enzymatic pathway (16-HDHA, 11-HDHA, and 8-HDHA) (Fig. 2 A and Fig. 2 B left panel). In contrast, no significant differences in lipid levels were observed among the three groups at W8 (Fig. 2 A, Fig. 2 B right panel). To further clarify the association between baseline lipid profiles and treatment outcomes, we analyzed correlations between lipid levels and HAMD/HAMA scores. Lipid levels showed no significant correlation with either the total scores or subscales of HAMD/HAMA at both W0 and W8 (Figure S2, Table S3). At W0, 16 lipids were negatively correlated with the HAMD score reduction rate. These lipids included PUFAs (ALA, EPA, GLA, and adrenic acid) and oxylipins from the LOX pathway (12-HETE, 5-HETE, 15-HETE, 15-HETrE, and 4-HDHA), the CYP450 pathway (14,15-DiHET, 11,12-DiHET, and 13,14-DiHDPA), the COX pathway (11-HETE and 13-HODE), and the non-enzymatic pathway (16-HDHA and 11-HDHA) (Fig. 3 , upper panel). However, no such correlations were detected at W8 (Fig. 3 , lower panel). Alteration of LOX pathway in CRS-induced mouse model of depression Given that LOX-derived oxylipins exhibited the most robust elevation in MDD patients and showed the strongest association with antidepressant efficacy, while the roles of CYP450 and COX pathways have been well documented in previous publications, we prioritized the less characterized LOX pathway for mechanistic investigation in our mouse model of depression. The plasma LOX-derived oxylipins profile was analyzed in CRS-induced mouse model of depression. Compared with control (CON) mice, CRS mice showed nominal increases in seven LOX-derived oxylipins, i.e.12-HETE, 15-HETE, 9,12,13-TriHOME, LXA4, 9-HOTrE, 13-HOTrE, 7-HDHA (Fig. 4 A). However, these differences did not remain significant after FDR correction for multiple comparisons. To explore the sources of LOX-derived oxylipins, various tissues including the liver, spleen, lung, kidney, small intestine, colon, blood and brain were collected from a murine model of CRS-induced depression. The mouse LOX family is currently known to comprise seven members: Alox15, Alox15b, Alox12, Alox12b, Aloxe3, Alox5 and Alox5ap ( 19 ); these seven enzymes were therefore selected for expression analysis. The results demonstrated that the mRNA expression levels of Alox5ap and Alox12 were significantly higher in the blood of CRS mice compared with CON mice. In the brain, CRS mice exhibited markedly elevated expression of Alox5, Alox12, Alox15b, and Aloxe3 relative to CON mice. No significant differences in the expression of these genes were observed in the other analyzed tissues (Fig. 4 B). Due to the limited sample size and exploratory nature of this study, nominal p -values are reported without adjustment for multiple comparisons. Inhibition of LOX alleviates CRS-induced depressive-like behaviors in mice We further evaluated the effects of LOX inhibitors on alleviating depressive- and anxiety-like behaviors by administering the 5-LOX inhibitor zileuton (ZLT) and the 12-LOX inhibitor ML-355 to mice with chronic restraint stress-induced depressive-like behaviors (Fig. 5 A). After 2 weeks of treatment, ML-355 significantly improved depressive-like and anxiety-like behaviors. It effectively ameliorated anhedonia (Fig. 5 B), increased the total distance traveled in the open field test (Fig. 5 C), and prolonged the time spent in the open arms of the elevated plus maze (Fig. 5 D). ML-355 also reduced the immobility time in the tail suspension test and forced swim test, reflecting an attenuation of despair-like behavior (Figs. 5 E and 5 F). Although ZLT showed a tendency to alleviate depressive-like behaviors, the difference was not statistically significant. Similarly, the standard antidepressant fluoxetine (FLX) did not produce significant improvements in depressive- and anxiety-like behaviors after 2 weeks of administration. Inhibition of LOX suppresses microglia activation in depressive-like mice induced by CRS Because microglia undergo pronounced structural and functional alterations in response to chronic stress and are likely the target cells of LOX-derived oxylipins, we evaluated microglial activation in the hippocampal subregions (DG, CA3, CA1) (Fig. 6 A) under CRS and pharmacological interventions. Compared with the CON group, CRS significantly increased IBA-1 fluorescence intensity in all three hippocampal subregions (all p 0.05) in DG, CA3 and CA1 (all p < 0.0001, Fig. 6 B, 6 C, 6 D). In contrast, ZLT significantly reduced IBA-1 expression in the DG and CA3 (both p < 0.0001) but not to control levels (both p 0.05). Fluoxetine (FLX) failed to reduce IBA-1 expression in the DG relative to CRS ( p < 0.0001) and only marginally reduced it in CA3 and CA1 (both p < 0.05) ( Fig. 6 B, 6 C, 6 D). Representative images are shown in Fig. 6 E (DG), Figure S3 (CA3), and Figure S4 (CA1). To further investigate regional effects, we examined IBA-1 expression in the mPFC. CRS significantly increased IBA-1 intensity in both Cg1 and PrL (both p 0.05). ZLT significantly attenuated IBA-1 expression in Cg1 and PrL (both p < 0.0001) but not to control levels. Similarly, FLX reduced IBA-1 levels in the mPFC compared with CRS ( p < 0.01 and p < 0.001, respectively) but remained elevated relative to controls. Representative images are shown in Figure S5D (Cg1) and S5E (PrL). Collectively, ML-355 consistently reversed CRS-induced microglial activation across the hippocampus and mPFC, whereas ZLT exerted partial, region-dependent effects, and FLX showed limited efficacy in suppressing stress-induced microglial activation. Discussion This study systematically investigated alteration of oxylipin profiles in patients with MDD. Using targeted oxylipidomics analysis, we found significantly elevated levels of multiple oxylipins in MDD patients compared to healthy controls, with the most pronounced changes observed in LOX and CYP450 pathway metabolites. Notably, higher baseline oxylipin levels predicted poorer antidepressant treatment response, as non-responders exhibited elevated levels of 20 oxylipins relative to both responders and healthy controls. In parallel, CRS mice displayed increased LOX pathway activity and elevated plasma LOX-derived oxylipins. Pharmacological inhibition of 12-LOX with ML-355 ameliorated depressive-like behaviors and reversed stress-induced microglial activation in the hippocampus and medial prefrontal cortex. These findings identify dysregulated LOX-derived oxylipin metabolism as a potential biomarker for treatment resistance and support targeting the LOX pathway, especial 12-LOX pathway, as a therapeutic strategy for depression. Our finding of elevated oxylipin levels in MDD patients aligns with previous reports of altered oxylipin profiles in depression ( 14 , 15 ). By using a broader targeted oxylipidomics approach, the present study moves beyond single-metabolite observations and instead highlights a coordinated disturbance across multiple lipid pathways. This broader network-level pattern is important because MDD is unlikely to be explained by a single inflammatory mediator; rather, it may arise from a maladaptive inflammatory-metabolic state in which several lipid signaling systems are shifted simultaneously. Our current results along with these studies, support the involvement of PUFA oxidation pathways in MDD pathophysiology. The elevation of LOX- and CYP450-derived oxylipins observed here is consistent with the established role of inflammatory and oxidative stress processes in depression. Given that oxylipins serve as direct indicators of lipid peroxidation and immune activation, their increased levels likely reflect underlying inflammatory dysregulation in MDD. A key finding of this study is that baseline oxylipin levels predict antidepressant treatment outcomes, independent of baseline symptom severity. While we observed no correlation between oxylipin levels and baseline HAMD or HAMA scores, multiple oxylipins showed significant negative correlations with HAMD score reduction rates following 8 weeks of treatment. This dissociation, where oxylipins predict treatment response but not initial severity, suggests that these lipid mediators may specifically influence treatment resistance mechanisms rather than general depressive symptomatology. This pattern mirrors findings with other inflammatory biomarkers, wherein elevated inflammation predicts poor antidepressant response despite variable associations with baseline depression severity ( 2 ). Oxylipins, as central mediators of inflammatory signaling, may thus provide a mechanistic link between immune dysregulation and treatment resistance. These results support further investigation of LOX-derived oxylipins as biomarkers to stratify patients for personalized treatment approaches, including anti-inflammatory adjunctive therapies. Lipoxygenases (LOXs) play critical roles in regulating microglial functions through multiple mechanisms. In pathological conditions, LOXs metabolize arachidonic acid to generate pro-inflammatory lipid mediators such as leukotrienes and HETEs, thereby activating microglia, promoting their polarization toward a pro-inflammatory phenotype, and exacerbating neuroinflammation ( 20 – 22 ). Additionally, 5-LOX (ALOX5) and 15-LOX (ALOX15) catalyze the peroxidation of polyunsaturated fatty acids, leading to the accumulation of lipid peroxides that drive microglial ferroptosis, where elevated LOX expression in microglia triggers inflammatory cascades and non-cell-autonomous neuronal death ( 22 – 24 ). In the present study, systemic pharmacological inhibition of 12-LOX with ML-355 significantly ameliorated CRS-induced depressive-like and anxiety-like behaviors, accompanied by reduced microglial activation in the hippocampus and medial prefrontal cortex. These findings suggest that 12-LOX-derived oxylipins promote microglial activation and sustain pro-inflammatory signaling, thereby contributing to stress-induced neuronal dysfunction and depressive behaviors. Notably, the 5-LOX inhibitor zileuton showed only region-dependent effects on microglial activation without significant behavioral improvement, indicating that 12-LOX may be the predominant isoform mediating stress-induced neuroinflammation in depression. This isoform-specific effect aligns with evidence that different LOX pathways have distinct roles in microglial regulation: while 5-LOX is associated with ferroptosis and acute inflammatory responses, 12-LOX appears to be more critically involved in sustained microglial activation and synaptic dysfunction ( 21 , 24 ). Furthermore, beyond acute neuroinflammation, persistent LOX pathway activation may have long-term consequences through trained immunity, a process of innate immune cell reprogramming that leads to heightened inflammatory responses upon subsequent challenges. Recent studies have implicated LOX-derived oxylipins as key mediators of trained immunity ( 25 ), and given that trained immunity has been proposed as a mechanism underlying recurrent depressive episodes ( 26 ), our findings raise the speculative possibility that chronic 12-LOX activation may sensitize microglia to stress re-exposure, potentially contributing to illness chronicity or relapse vulnerability. This hypothesis warrants direct investigation in future studies using recurrence-based animal models. Collectively, these results provide in vivo evidence that pharmacological inhibition of 12-LOX exerts antidepressant effects by attenuating microglial activation and neuroinflammatory processes, highlighting the therapeutic potential of targeting this pathway in depression. This study has several limitations. First, the single-center design and modest sample size limit generalizability, particularly given the ethnically homogeneous (Han Chinese) cohort. Future validation in independent, multi-ethnic samples is warranted. Second, while our findings suggest an inflammatory mechanism, we did not measure conventional inflammatory biomarkers (e.g., IL-6, TNF-α, CRP), limiting our ability to confirm the specific pathways linking oxylipins to microglial activation. Third, the mouse study focused exclusively on the LOX pathway and did not assess CYP450 or COX alterations observed in patients, precluding direct cross-pathway comparisons and a comprehensive understanding of oxylipin dysregulation in stress-induced depression. Fourth, pharmacokinetic validation of ML-355 brain penetration was not performed, and whether peripheral 12-LOX inhibition adequately reduces central oxylipin levels to mediate the observed behavioral effects remains unknown. Fifth, oxylipin levels were not assessed post-treatment, limiting direct evidence that behavioral improvements resulted from reduced LOX pathway activity. Sixth, the elevation of LOX pathway oxylipins in CRS mice did not withstand correction for multiple comparisons given the limited sample size (n = 6 per group) and should be interpreted as exploratory. In conclusion, this study demonstrates elevated plasma oxylipin levels in MDD patients, with the most pronounced elevations observed in treatment-resistant patients, and identifies 12-LOX as a potential therapeutic target using a translational approach. These findings implicate dysregulated LOX-mediated lipid metabolism in depression pathophysiology and provide preclinical evidence supporting the development of 12-LOX inhibitors as novel antidepressant strategies. Future research should investigate the dynamic regulation of oxylipins across MDD subtypes and disease stages, and evaluate the clinical efficacy of targeted LOX inhibition in treatment-resistant depression. Declarations Acknowledgement This work was supported by the Ministry of Science and Technology of the People’s Republic of China (2022ZD0211700), the 135 Project from West China Hospital of Sichuan University (2023HXFH006), the National Natural Science Foundation of China (82571768), the China Postdoctoral Science Foundation (2024M762243, 2025M771987), the Postdoctoral Fellowship Program of CPSF (GZC20251514), the Postdoctoral Fellowship Program of CPSF (GZC20251341), the Sichuan Science and Technology Program (2025ZNSFSC0781, 2025ZNSFSC1563, 2025ZNSFSC1649, 2024NSFSC1559). Conflict of Interest We declare no competing financial interests. References Wang Z, Dou Y, Yang X, Guo X, Ma X, Zhou B, et al. Global, regional, and national burden of mental disorders among adolescents and young adults, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021. Transl Psychiatry. 2025;15(1):397. PubMed PMID: 41073427. 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Mashima R, Okuyama T. The role of lipoxygenases in pathophysiology; new insights and future perspectives. Redox Biol. 2015;6:297–310. PubMed PMID: 26298204. PMCID: PMC4556770. Epub 2015/08/25. eng. Fu L, Luo T, Hao Z, Pan Y, Xin W, Zhang L, et al. Exploring novel roles of lipid droplets and lipid metabolism in regulating inflammation and blood-brain barrier function in neurological diseases. Front Neurosci. 2025;19:1603292. PubMed PMID: 40880849. PMCID: PMC12380697. Epub 2025/08/29 20:45. eng. Chen S, Zou H. Lipoxygenase Metabolism: Critical Pathways in Microglia-mediated Neuroinflammation and Neurodevelopmental Disorders. Neurochem Res. 2022;47(11):3213–20. PubMed PMID: 35674930. Epub 2022/06/09. eng. Liddell JR, Hilton JBW, Kysenius K, Billings JL, Nikseresht S, McInnes LE, et al. Microglial ferroptotic stress causes non-cell autonomous neuronal death. Mol Neurodegener. 2024;19(1):14. PubMed PMID: 38317225. PMCID: PMC10840184. Epub 2024/02/06. eng. Tang S, Zhang J, Chen J, Zhou Z, Lin Q. Ferroptosis in neurodegenerative diseases: potential mechanisms of exercise intervention. Front Cell Dev Biol. 2025;13:1622544. PubMed PMID: 40661149. PMCID: PMC12256474. Epub 2025/07/15. eng. Gao S, Zhou L, Lu J, Fang Y, Wu H, Xu W, et al. Cepharanthine Attenuates Early Brain Injury after Subarachnoid Hemorrhage in Mice via Inhibiting 15-Lipoxygenase-1-Mediated Microglia and Endothelial Cell Ferroptosis. Oxid Med Cell Longev. 2022;2022:4295208. PubMed PMID: 35186185. PMCID: PMC8850040. Epub 2022/02/22. eng. Ferreira AV, Alarcon-Barrera JC, Domínguez-Andrés J, Bulut Ö, Kilic G, Debisarun PA, et al. Fatty acid desaturation and lipoxygenase pathways support trained immunity. Nat Commun. 2023;14(1):7385. PubMed PMID: 37968313. PMCID: PMC10651900 interest. The funders had no role in the design of the study; in the collection, analyzes, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Epub 2023/11/16. eng. Xu G, Yuan M, He H, Yi J, Li X, Yan H, et al. NLRP3-mediated trained immunity of microglia is involved in the recurrence-like episode of depressive disorders. Mol Psychiatry. 2026;31(4):1958–69. PubMed PMID: 41249553. Epub 2025/11/18. eng. Table 1 Table 1 is not available with this version. Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files supplementary.docx supplementary figures and tables Cite Share Download PDF Status: Under Review Version 1 posted Review # 3 received at journal 12 May, 2026 Review # 1 received at journal 11 May, 2026 Review # 2 received at journal 10 May, 2026 Reviewer # 3 agreed at journal 28 Apr, 2026 Reviewer # 2 agreed at journal 28 Apr, 2026 Reviewer # 1 agreed at journal 28 Apr, 2026 Reviewers invited by journal 28 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 10 Apr, 2026 Unknown event 10 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9366044","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":631041226,"identity":"0b82eb1d-ba92-47bf-b4a6-3ff44eba684f","order_by":0,"name":"Xiaohong Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYJCCA2DEwHwAziVWC1sC8VqgyngMiNOi237G8MCPijty5vxrPn/42cYgx3cjgfFzAR4tZmfSEg72nHlmbDnj7TbJ3jYGY8kbCczSM/BpOZB84ABv2+HEDTfObmNmbGMAMhLYmHnwaTn/sOHgX7CWM48/A7XUE9ZyI/nAYbAt53sYpIFaEgwIa3mWcFjmzGFjgxtsZpI95yQMZ5552CyN32E5xh/fVByWMzh/+PGHH2U28nzHkw9+xqcFASQSwCQQMzYQpYGBgf8AkQpHwSgYBaNgxAEAAxRY2QYLMpQAAAAASUVORK5CYII=","orcid":"","institution":"Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Ma","suffix":""},{"id":631041227,"identity":"76d74e15-4832-4378-a48e-38d4343a03a7","order_by":1,"name":"Jinxue Wei","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinxue","middleName":"","lastName":"Wei","suffix":""},{"id":631041228,"identity":"ac33a1a9-ab99-4071-91a6-b4729d67a62d","order_by":2,"name":"Yikai Dou","email":"","orcid":"https://orcid.org/0000-0001-6210-3206","institution":"West China Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yikai","middleName":"","lastName":"Dou","suffix":""},{"id":631041229,"identity":"49e711c9-0aeb-46cc-a2b1-75b9fc489a23","order_by":3,"name":"Liansheng Zhao","email":"","orcid":"https://orcid.org/0000-0002-3008-5135","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Liansheng","middleName":"","lastName":"Zhao","suffix":""},{"id":631041230,"identity":"56946af0-ea2c-491b-922f-b06db49d6ab9","order_by":4,"name":"Min Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Wang","suffix":""},{"id":631041231,"identity":"f12d8d3e-8abd-4f14-9f96-813333af4ddb","order_by":5,"name":"Yushun Yan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yushun","middleName":"","lastName":"Yan","suffix":""},{"id":631041232,"identity":"6b542a6d-e277-40ce-b33f-e841fe76cfa7","order_by":6,"name":"Ge Liang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Liang","suffix":""},{"id":631041233,"identity":"ca07b048-f4de-4bec-b258-48e07fecfa11","order_by":7,"name":"Wen Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zheng","suffix":""},{"id":631041234,"identity":"f19a2820-aaba-4947-8a78-402abf19c710","order_by":8,"name":"Rong-Jun Ni","email":"","orcid":"https://orcid.org/0000-0001-7421-136X","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Rong-Jun","middleName":"","lastName":"Ni","suffix":""},{"id":631041235,"identity":"89e3371b-54f4-4773-8a7f-0adc08dd872a","order_by":9,"name":"Xiao Yang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Yang","suffix":""},{"id":631041236,"identity":"d69c2056-7e3a-401d-8f42-5afdfea3d920","order_by":10,"name":"Meng Gong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Gong","suffix":""}],"badges":[],"createdAt":"2026-04-09 09:11:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9366044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9366044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108942750,"identity":"71046769-a992-43ae-8d78-37e2d4643ab8","added_by":"auto","created_at":"2026-05-11 05:42:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594034,"visible":true,"origin":"","legend":"\u003cp\u003eIncreased plasma oxylipin levels in patients with MDD in comparison to HC.\u003c/p\u003e\n\u003cp\u003e(A) Plasma levels of oxylipin that were significantly differ between MDD and HC and their PUFA precursors were presented in the schematic diagram of oxylipins biosynthesis through cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases (CYP) and non-enzymatic pathways. Oxylipins are color - coded to correspond with their precursor fatty acids. (B) Heatmap presentation of comparison of plasma oxylipins between patients with MDD and HC. Significant levels were indicated using asterisks: *, 0.05; **, 0.01; ***, 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/c0a7aa1dc6281fdf500fecc5.png"},{"id":108942749,"identity":"332e83bd-0954-47be-8b61-8d5f57e1fcbc","added_by":"auto","created_at":"2026-05-11 05:42:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":618312,"visible":true,"origin":"","legend":"\u003cp\u003eDifference of oxylipin levels between responders and non-responders\u003c/p\u003e\n\u003cp\u003e(A) Plasma levels of oxylipin that were significantly differ between responders, non-responders and HCs and their PUFA precursors at W0 or W8 were presented in a schematic diagram. Oylipins are grouped by their biosynthetic enzymes: non-enzymatic reactions, COX, LOX, and CYP450 and are color-coded to correspond with their precursor PUFA. (B) Heatmap presentation of comparison of plasma oxylipins between responders, non-responders and HC. Significant levels were indicated using asterisks: *, 0.05; **, 0.01; ***, 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/458f5756d0dcf0410de881bb.png"},{"id":108942713,"identity":"5f5af9f3-1a29-4d37-a03b-b7c7a2a871f3","added_by":"auto","created_at":"2026-05-11 05:42:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":258663,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between plasma oxylipin levels and reduction rate of HAMD\u003c/p\u003e\n\u003cp\u003eHeatmap presentation of spearman correlation between lipids and HAMD score reduction rate. Significant levels were indicated using asterisks: *, 0.05; **, 0.01; ***, 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/8b5f8144148495ec3fe3b8b2.png"},{"id":108942719,"identity":"0cfbb8ea-4d33-4d92-bc53-57b5867e9632","added_by":"auto","created_at":"2026-05-11 05:42:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":362392,"visible":true,"origin":"","legend":"\u003cp\u003eAlteration of LOX pathway in CRS-induced mouse model of depression\u003c/p\u003e\n\u003cp\u003e(A) Plasma levels of LOX-derived oxylipin that were nominally differ between CRS mice and CON mice and their PUFA precursors were presented in a schematic diagram. Oylipins are color-coded to correspond with their precursor PUFA. (B) Relative expression levels of LOX isoforms in tissues of mice. Significant levels were indicated using asterisks: *, 0.05; **, 0.01; ***, 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/aebf6f10557d600cade295c1.png"},{"id":108942717,"identity":"4a870fd2-be3f-4452-8330-46c8d0b55adf","added_by":"auto","created_at":"2026-05-11 05:42:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":497220,"visible":true,"origin":"","legend":"\u003cp\u003eML‑355 significantly alleviates chronic restraint stress-induced depressive-like behaviors in mice\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the timeline for intervention and evaluation in CRS-induced depressive mice. (B) Sucrose preference test (preference%). (C) Open field test (total distance). (D)Elevated plus maze (cumulative duration in open arm). (E) Tail suspension test (immobility duration). (F) Forced swimming test (immobility duration). N=7 mice per group in (B)~(F). Statistical significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/31328ca4dc57fbbb0fe5afcc.png"},{"id":108942718,"identity":"8e18cf3e-4e65-42e4-95c9-020fab3834a3","added_by":"auto","created_at":"2026-05-11 05:42:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1044920,"visible":true,"origin":"","legend":"\u003cp\u003eML-355 inhibits chronic restraint stress-induced microglial activation (IBA-1) in the hippocampus\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of coronal section of the hippocampus with regional segmentation. (B-D) Quantitative analysis of relative fluorescence intensity of IBA-1 (microglial activation) in the hippocampal DG region, CA3 region and CA1 region among different groups. (E) Representative images of microglial activation (IBA-1) in the hippocampal DG region across treatment groups. White scale bar = 200 μm, yellow scale bar= 50μm. N=6~8 per group in (B)~(D). Statistical significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001,****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/9ae572971871bcc53442a25e.png"},{"id":109067807,"identity":"8f9cb271-e045-4f80-b59e-5b791e8bc797","added_by":"auto","created_at":"2026-05-12 10:01:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3196646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/15166adf-fd36-4446-9d97-a5d6d5072719.pdf"},{"id":108942748,"identity":"c940393f-0f98-4e94-b578-7ff669f5b101","added_by":"auto","created_at":"2026-05-11 05:42:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4244370,"visible":true,"origin":"","legend":"supplementary figures and tables","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9366044/v1/559e8e8b52c18b592ae97325.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Lipoxygenase-Derived Oxylipins are Elevated in MDD Patients and Stress-Induced Depressed Mice: Inhibition of Lipoxygenases as a Therapeutic Strategy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder (MDD) is one of the leading causes of disability worldwide, imposing a substantial and growing global health burden. According to the Global Burden of Disease 2021 study, depressive disorders account for a major proportion of years lived with disability, particularly among adolescents and young adults, and their burden has continued to rise over recent decades (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite its high prevalence and profound functional impairment, MDD is a biologically heterogeneous disorder characterized by diverse symptom profiles and variable treatment responses. Increasing evidence indicates that inflammation plays a pivotal role in the pathophysiology of MDD, featuring systemic immune activation marked by elevated peripheral pro-inflammatory cytokines and central neuroinflammation involving microglial activation and blood-brain barrier disruption. Notably, elevated inflammatory markers predict resistance to conventional antidepressants, whereas anti-inflammatory agents, as monotherapy or adjunctive therapy, demonstrate therapeutic potential in inflammation-associated MDD (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Collectively, these observations underscore immune-targeted interventions as a viable precision medicine strategy for patients with inflammation-related major depressive disorder.\u003c/p\u003e \u003cp\u003eGiven the established role of inflammation in MDD pathophysiology, understanding the specific mediators driving inflammatory processes is essential. Oxylipins represent one such class of bioactive lipid metabolites that may bridge immune dysregulation and depressive symptomatology. These molecules are derived from the oxidation of polyunsaturated fatty acids (PUFAs) via either enzymatic or non-enzymatic mechanisms (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The enzymatic pathways are mediated by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases (CYP450) that catalyze PUFA oxidation, while non-enzymatic pathways are driven by reactive oxygen species (ROS)-induced lipid peroxidation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Oxylipins act as key inflammatory mediators and are widely recognized as reliable biomarkers, as their elevated levels directly reflect the extent of lipid damage and oxidative stress in biological systems (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Moreover, these lipid mediators can be transported in circulation via lipoprotein particles, thereby enabling systemic signaling and modulating their contributions to neurological diseases (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Recent advances in profiling techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have enabled comprehensive analysis of oxylipin networks in clinical research, underscoring their potential as biomarkers and therapeutic targets (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Previous studies have reported alterations in specific oxylipin profiles in MDD, including decreased 14,15-EET and increased 14,15-DHET (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), differential oxidized phosphatidylcholines in rTMS treatment responders versus non-responders (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), as well as changes in sEH-derived metabolites in seasonal depression (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and diabetes-associated depression (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, these investigations were limited by narrow coverage of oxylipin species and small sample sizes, precluding systematic characterization of oxylipin network alterations in MDD.\u003c/p\u003e \u003cp\u003eIn this study, we investigated oxylipin profile alterations in patients with MDD and tested the therapeutic potential of targeting the LOX pathway in a mouse model of depression. We found that multiple oxylipins and their precursors, particularly those enriched in LOX and CYP450 pathways, were elevated in MDD patients, with LOX-derived metabolites showing the strongest association with treatment resistance. Based on these clinical findings, we examined the LOX pathway in a chronic restraint stress mouse model and found altered plasma levels of LOX-derived oxylipins and increased LOX expression across peripheral tissues and brain. Furthermore, pharmacological inhibition of 12-LOX ameliorated stress-induced depressive-like behaviors and microglial activation in mice. These findings implicate dysregulated LOX-mediated oxylipin metabolism in MDD pathophysiology and support targeting 12-LOX as a potential therapeutic strategy for depression.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eParticipants\u003c/p\u003e \u003cp\u003eA total of 154 patients with major depressive disorder (MDD) aged 16\u0026ndash;55 years were recruited from the Mental Health Center of West China Hospital, Sichuan University, between 2021 and 2023.Of the 154 MDD patients, 74 patients completed the 8-week follow-up assessment and were included in the treatment response analysis. All patients were required to meet the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria for MDD and had no comorbid psychiatric or other medical disorders. Patients who had used any antidepressants or antipsychotics within 2 weeks prior to recruitment were excluded. The inclusion and exclusion criteria were consistent with those reported in previous publications (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Healthy Han Chinese volunteers (n\u0026thinsp;=\u0026thinsp;134) aged 16\u0026ndash;60 years were recruited via online advertisements. Both MDD patients and healthy controls (HCs) with neurodegenerative diseases, major endocrine or metabolic disorders, as well as pregnant or breastfeeding individuals, were excluded from the study. Each participant spent approximately 2 hours completing all assessments. The research protocol was approved by the Institutional Ethics Committee of West China Hospital, Sichuan University (Approval Number: [2016] 170). All participants were fully informed about the study procedures and provided written informed consent. The Hamilton Depression Scale (HAMD) and the Hamilton Anxiety Scale (HAMA) were used to assess depressive symptoms and anxiety symptoms in all the patients with MDD. The HAMD comprises five subscales: retardation (items 1, 7, 8, 14), cognitive disturbance (items 2, 3, 9), sleep disturbance (items 4, 5, 6), somatization (items 10, 11, 12, 15,17), and weight (item 16). The HAMA comprises two subscales: psychological anxiety (items 1\u0026ndash;5) and somatic anxiety (items 6\u0026ndash;14).\u003c/p\u003e \u003cp\u003ePlasma sample collection\u003c/p\u003e \u003cp\u003ePeripheral blood samples were collected from all participants on the day of enrollment using ethylenediaminetetraacetic acid (EDTA)-coated anticoagulant tubes. Immediately after collection, the blood samples were centrifuged at 2000 \u0026times; g for 10 minutes at 4℃ to separate the components. Subsequently, the upper plasma layer was carefully aspirated using a sterile pipette, transferred to a new RNase/DNase-free cryovial, and immediately stored at \u0026minus;\u0026thinsp;80℃ until subsequent experimental analysis.\u003c/p\u003e \u003cp\u003eOxylipidomics analysis in plasma\u003c/p\u003e \u003cp\u003eThe quantitative determination of oxylipins in plasma was based on a previous report and was subsequently optimized (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Briefly, oxylipins in plasma were extracted via solid - phase extraction (SPE). The extracts were analyzed using ultra - performance liquid chromatography (UPLC) coupled with AB SCIEX 6500\u0026thinsp;+\u0026thinsp;mass spectrometry in multiple reaction monitoring (MRM) mode. Oxylipin identification and validation were primarily achieved by comparing the retention times of chromatographic peaks with those of standard substances. Relative quantification of oxylipins was performed through peak integration preprocessing using SCIEX OS (v2.0, AB Sciex, Foster, ND, USA), followed by analysis using R software (v3.5.1). A total of 120 species of lipids, including 11 PUFAs and 109 their derived oxidized lipids were quantified in this study (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExperimental animals and chronic restraint stress model\u003c/p\u003e \u003cp\u003eMale C57BL/6J mice were obtained from Jicui Yaokang Biotechnology Co., Ltd (Chengdu, China). Mice were maintained under standard laboratory conditions with controlled temperature and a 12 h light/dark cycle. All procedures were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (Approval No. 20250714003). Chronic restraint stress (CRS) was employed to induce depression-like behaviors. Mice were placed individually into 50 mL centrifuge tubes for 4 h per day for 28 consecutive days. During the 14-day drug treatment period, restraint procedures were maintained in all stressed groups to ensure sustained stress exposure. Animals were randomly allocated into five groups (n\u0026thinsp;=\u0026thinsp;7\u0026ndash;8): Control (saline), CRS\u0026thinsp;+\u0026thinsp;saline, CRS\u0026thinsp;+\u0026thinsp;ML355 (10 mg/kg), CRS\u0026thinsp;+\u0026thinsp;Zileuton (10 mg/kg), and CRS\u0026thinsp;+\u0026thinsp;Fluoxetine (10 mg/kg). All treatments were administered once daily by intraperitoneal injection for 14 days (Zileuton: 5-LOX inhibitor; ML-355: 12-LOX inhibitor).\u003c/p\u003e \u003cp\u003eSucrose preference test\u003c/p\u003e \u003cp\u003eThe sucrose preference test (SPT) was performed to assess anhedonia-like behavior in mice. The experiment consisted of an adaptation phase and a formal testing phase. During the adaptation phase, mice were individually housed and provided with two bottles containing 2% (w/v) sucrose solution for 24 h. Subsequently, one bottle was replaced with pure water for an additional 24 h to allow acclimatization to both liquids. After adaptation, mice were deprived of food and water for 12 h. Following deprivation, each mouse was given free access to two pre-weighed bottles containing 100 mL of 2% (w/v) sucrose solution and 100 mL of water, respectively. The testing period lasted 12 h. At the end of the test, bottles were weighed to determine fluid consumption. Sucrose preference was calculated using the following formula: Sucrose preference (%) = sucrose consumption / (sucrose consumption\u0026thinsp;+\u0026thinsp;water consumption) \u0026times; 100%.\u003c/p\u003e \u003cp\u003eOpen field test\u003c/p\u003e \u003cp\u003eThe open field test (OFT) was conducted to evaluate locomotor activity and anxiety-like behavior [21]. Approximately 24 h before testing, mice were transferred to the behavioral testing room for 2 h of habituation. On the test day, animals were allowed to acclimate to the environment for an additional 30 min prior to the experiment.The test was performed in a white plexiglass arena (50 cm \u0026times; 50 cm \u0026times; 40 cm) under dim illumination (approximately 60 lux). Each mouse was gently placed in the same corner of the apparatus and allowed to freely explore the arena for 10 min. The central zone was defined as a 25 cm \u0026times; 25 cm square located in the middle of the field. Behavior was recorded and analyzed using the EthoVision XT video tracking system (Noldus, Netherlands). The parameters measured included total distance traveled, average movement velocity, number of entries into the central area, and time spent in the central area.After each trial, the apparatus was cleaned with 75% ethanol and allowed to dry to eliminate olfactory cues.\u003c/p\u003e \u003cp\u003eElevated plus maze\u003c/p\u003e \u003cp\u003eThe elevated plus maze (EPM) was used to evaluate anxiety-like behavior in mice. Animals were habituated to the behavioral testing room for at least 1 h before the experiment. The EPM apparatus consisted of two open arms (30 cm \u0026times; 6 cm) and two closed arms (30 cm \u0026times; 6 cm \u0026times; 15 cm), elevated 50 cm above the floor. At the beginning of the test, each mouse was placed in the central platform of the maze and allowed to freely explore for 5 min.The time spent in the open and closed arms was recorded and analyzed. After each trial, the maze was carefully cleaned with 75% ethanol to remove residual odors before the next animal was tested.\u003c/p\u003e \u003cp\u003eTail suspension test\u003c/p\u003e \u003cp\u003eThe tail suspension test (TST) was conducted to evaluate despair-like behavior in mice. Each mouse was suspended individually by adhesive tape placed approximately 1 cm from the tip of the tail on the edge of a suspension shelf (height 30 cm, diameter 21 cm). The head of the mouse was positioned approximately 5 cm above the surface to prevent grasping or support. The total testing time was 6 min. The first 2 min were regarded as an adaptation period. Immobility time during the final 4 min was automatically recorded using the EthoVision XT video tracking system (Noldus, Netherlands).Immobility was defined as the absence of voluntary body movement while hanging passively.\u003c/p\u003e \u003cp\u003eForced swimming test\u003c/p\u003e \u003cp\u003eThe forced swimming test (FST) was performed to evaluate despair-like behavior in mice. Each mouse was individually placed in a transparent plastic cylinder (30 cm in height, 15 cm in diameter) containing water at a depth of 15 cm maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃. The total duration of the test was 6 min. The first 2 min served as an adaptation period, and the cumulative immobility time during the last 4 min was automatically recorded. Immobility was defined as floating passively in the water without active struggling or climbing, except for minimal movements required to keep the head above the water surface. Behavior was recorded and analyzed using the EthoVision XT video tracking system (Noldus, Netherlands). After each trial, the water was replaced to avoid interference with subsequent animals. Tested mice were dried and kept separately from untested animals after the experiment.\u003c/p\u003e \u003cp\u003eImmunofluorescence staining\u003c/p\u003e \u003cp\u003eImmunofluorescence staining was performed after completion of behavioral tests. Mice were anesthetized with 2,2,2-tribromoethanol (10%, 0.2 ml/10 g, i.p.), and three animals per group were randomly selected. Following transcardial perfusion with 0.9% saline and 4% paraformaldehyde (pH 7.4), brains were collected, post-fixed, dehydrated, and coronally sectioned at 30 \u0026micro;m using a Leica CM1950 cryostat. Sections were blocked in 5% donkey serum containing 0.3% Triton X-100 for 1 h and incubated overnight at 4℃ with primary antibodies against IBA-1(66827-1-IG, 1:200 dilution, Proteintech, China) and NeuN (26975-1-AP, 1:200 dilution, Proteintech, China). After washing, sections were incubated with fluorescent secondary antibodies for 2 h at room temperature and counterstained with DAPI. Fluorescence images were acquired using a Zeiss LSM 710 confocal microscope at 200\u0026times; magnification. Two to three randomly selected fields from the prefrontal cortex and hippocampus were analyzed per animal. ImageJ software was used to quantify the mean fluorescence intensity of IBA-1 staining, which reflects microglial activation levels. All analyses were performed by an investigator blinded to group allocation.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using R (version 4.3.1). The chi-square test was used to compare sex distribution between groups. Independent-samples t-tests were applied to compare age, BMI, number of episodes, illness duration, and HAMD/HAMA total scores between the two groups. One-way ANOVA followed by Tukey's multiple comparison test was used to compare lipid levels among healthy controls, responders, and non-responders, as well as to evaluate group differences in behavioral and microglia data. Spearman's correlation analysis was performed to assess associations between lipid levels and scores of HAMD or HAMA. To control for multiple comparisons in the correlation analysis, the false discovery rate (FDR) method was applied, and adjusted \u003cem\u003ep\u003c/em\u003e values were reported.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eDemographic characteristics\u003c/p\u003e \u003cp\u003eA total of 154 MDD patients (42 males, 112 females) and 134 healthy controls (44 males, 90 females) were included in this study. There were no significant differences of sex (χ\u0026sup2; = 0.81, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.37), age ( t = -0.34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.74) and body mass index (BMI, t = -0.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.45) between MDD and HC. The onset age, number of episodes, current disease duration, total score of HAMD and total score of HAMA of patients with MDD were 1.67 (1.45), 9.61 (14.55), 21.65 (4.67), 21.75 (7.18), respectively (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003ePlasma oxylipin profile in patients with MDD\u003c/p\u003e \u003cp\u003eFor clinical samples, after excluding metabolites with a missing-value proportion exceeding 50%, a total of 41 lipid species were included in subsequent statistical analyses. Among them, 22 lipid species exhibited differential expression between healthy controls (HC) and patients with major depressive disorder (MDD). Relative to HC, only 9,12,13-TriHOME was downregulated in MDD patients, while the remaining 21 differential lipid species were upregulated. Of these differential lipids, three belong to precursor polyunsaturated fatty acids (PUFAs): arachidonic acid (ARA), eicosapentaenoic acid (EPA), and adrenic acid. The remaining upregulated lipids are oxylipins derived from five PUFAs (ARA, EPA, docosahexaenoic acid (DHA), dihomo-γ-linolenic acid (DGLA), and linolenic acid (LA)). Specifically, 8 of these oxylipins are generated via the lipoxygenase (LOX) pathway (12-HETE, 5-HETE, 15-HETE, 15-HETrE, 14s-HDoHE, 4-HDHA, 12-HEPE, and 9,12,13-TriHOME), 8 via the cytochrome P450 (CYP450) pathway (5,6-diHETrE, 14,15-DiHET, 11,12-DiHET, 16,17-DiHDPA, 13,14-DiHDPA, 10,11-DiHDPA, 22-HDHA, and 17,18-DiHETE), 1 via the cyclooxygenase (COX) pathway (11-HETE), and 2 via the non-enzymatic pathway (16-HDHA and 11-HDHA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRelationship between plasma lipid levels and antidepressant treatment effects in MDD\u003c/p\u003e \u003cp\u003ePatients with a\u0026thinsp;\u0026ge;\u0026thinsp;50% reduction in HAMD score at week 8 were classified as treatment responders, while those with a reduction rate\u0026thinsp;\u0026lt;\u0026thinsp;50% were defined as non-responders. Among the 154 MDD patients, follow-up data at week 8 were available for 74 individuals (responders: n\u0026thinsp;=\u0026thinsp;39; non-responders: n\u0026thinsp;=\u0026thinsp;35; Table S2). There were no significant between-group differences in sex, age, BMI, age of onset, number of episodes, or duration of the current depressive episode (Table S2). At baseline (W0), there were no significant differences in total HAMD (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.00) and HAMA scores (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.73) between responders and non-responders (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). At week 8 (W8), responders showed significantly lower scores than non-responders in both total HAMD and HAMA scores (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). In responders, both the total HAMD and HAMA scores at week 8 (W8) were significantly lower than those at W0 (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In non-responders, the total HAMD score at W8 was lower than that at W0 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas no significant change was noted in the total HAMA score between W0 and W8 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.11, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo determine whether differential treatment responses were associated with plasma oxylipin profiles, we compared baseline lipid levels among responders, non-responders, and HCs. At W0, 20 lipids differed significantly across the three groups. All of these lipids were elevated in non-responders compared to both HCs and responders, and 13 of them were also higher in responders than in HCs. These differential lipids included PUFAs (ALA, ARA, and adrenic acid) and oxylipins from the LOX pathway (12-HETE, 5-HETE, 15-HETE, 15-HETrE, 14s-HDoHE, 4-HDHA, and 12-HEPE), the CYP450 pathway (5,6-diHETrE, 14,15-DiHET, 11,12-DiHET, 13,14-DiHDPA, 10,11-DiHDPA, and 22-HDHA), the COX pathway (11-HETE), and the non-enzymatic pathway (16-HDHA, 11-HDHA, and 8-HDHA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eB left panel). In contrast, no significant differences in lipid levels were observed among the three groups at W8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eB right panel).\u003c/p\u003e \u003cp\u003eTo further clarify the association between baseline lipid profiles and treatment outcomes, we analyzed correlations between lipid levels and HAMD/HAMA scores. Lipid levels showed no significant correlation with either the total scores or subscales of HAMD/HAMA at both W0 and W8 (Figure S2, Table S3). At W0, 16 lipids were negatively correlated with the HAMD score reduction rate. These lipids included PUFAs (ALA, EPA, GLA, and adrenic acid) and oxylipins from the LOX pathway (12-HETE, 5-HETE, 15-HETE, 15-HETrE, and 4-HDHA), the CYP450 pathway (14,15-DiHET, 11,12-DiHET, and 13,14-DiHDPA), the COX pathway (11-HETE and 13-HODE), and the non-enzymatic pathway (16-HDHA and 11-HDHA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e, upper panel). However, no such correlations were detected at W8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e, lower panel).\u003c/p\u003e \u003cp\u003eAlteration of LOX pathway in CRS-induced mouse model of depression\u003c/p\u003e \u003cp\u003eGiven that LOX-derived oxylipins exhibited the most robust elevation in MDD patients and showed the strongest association with antidepressant efficacy, while the roles of CYP450 and COX pathways have been well documented in previous publications, we prioritized the less characterized LOX pathway for mechanistic investigation in our mouse model of depression. The plasma LOX-derived oxylipins profile was analyzed in CRS-induced mouse model of depression. Compared with control (CON) mice, CRS mice showed nominal increases in seven LOX-derived oxylipins, i.e.12-HETE, 15-HETE, 9,12,13-TriHOME, LXA4, 9-HOTrE, 13-HOTrE, 7-HDHA (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, these differences did not remain significant after FDR correction for multiple comparisons. To explore the sources of LOX-derived oxylipins, various tissues including the liver, spleen, lung, kidney, small intestine, colon, blood and brain were collected from a murine model of CRS-induced depression. The mouse LOX family is currently known to comprise seven members: Alox15, Alox15b, Alox12, Alox12b, Aloxe3, Alox5 and Alox5ap (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e); these seven enzymes were therefore selected for expression analysis. The results demonstrated that the mRNA expression levels of Alox5ap and Alox12 were significantly higher in the blood of CRS mice compared with CON mice. In the brain, CRS mice exhibited markedly elevated expression of Alox5, Alox12, Alox15b, and Aloxe3 relative to CON mice. No significant differences in the expression of these genes were observed in the other analyzed tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Due to the limited sample size and exploratory nature of this study, nominal \u003cem\u003ep\u003c/em\u003e-values are reported without adjustment for multiple comparisons.\u003c/p\u003e \u003cp\u003eInhibition of LOX alleviates CRS-induced depressive-like behaviors in mice\u003c/p\u003e \u003cp\u003eWe further evaluated the effects of LOX inhibitors on alleviating depressive- and anxiety-like behaviors by administering the 5-LOX inhibitor zileuton (ZLT) and the 12-LOX inhibitor ML-355 to mice with chronic restraint stress-induced depressive-like behaviors (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After 2 weeks of treatment, ML-355 significantly improved depressive-like and anxiety-like behaviors. It effectively ameliorated anhedonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), increased the total distance traveled in the open field test (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and prolonged the time spent in the open arms of the elevated plus maze (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). ML-355 also reduced the immobility time in the tail suspension test and forced swim test, reflecting an attenuation of despair-like behavior (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Although ZLT showed a tendency to alleviate depressive-like behaviors, the difference was not statistically significant. Similarly, the standard antidepressant fluoxetine (FLX) did not produce significant improvements in depressive- and anxiety-like behaviors after 2 weeks of administration.\u003c/p\u003e \u003cp\u003eInhibition of LOX suppresses microglia activation in depressive-like mice induced by CRS\u003c/p\u003e \u003cp\u003eBecause microglia undergo pronounced structural and functional alterations in response to chronic stress and are likely the target cells of LOX-derived oxylipins, we evaluated microglial activation in the hippocampal subregions (DG, CA3, CA1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) under CRS and pharmacological interventions. Compared with the CON group, CRS significantly increased IBA-1 fluorescence intensity in all three hippocampal subregions (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). ML-355 restored IBA-1 levels to control levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in DG, CA3 and CA1 (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In contrast, ZLT significantly reduced IBA-1 expression in the DG and CA3 (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but not to control levels (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the CA1, ZLT treatment normalized IBA-1 levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Fluoxetine (FLX) failed to reduce IBA-1 expression in the DG relative to CRS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and only marginally reduced it in CA3 and CA1 (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) ( Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Representative images are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eE (DG), Figure S3 (CA3), and Figure S4 (CA1).\u003c/p\u003e \u003cp\u003eTo further investigate regional effects, we examined IBA-1 expression in the mPFC. CRS significantly increased IBA-1 intensity in both Cg1 and PrL (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). ML-355 normalized IBA-1 expression in both regions to control levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). ZLT significantly attenuated IBA-1 expression in Cg1 and PrL (both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but not to control levels. Similarly, FLX reduced IBA-1 levels in the mPFC compared with CRS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) but remained elevated relative to controls. Representative images are shown in Figure S5D (Cg1) and S5E (PrL).\u003c/p\u003e \u003cp\u003eCollectively, ML-355 consistently reversed CRS-induced microglial activation across the hippocampus and mPFC, whereas ZLT exerted partial, region-dependent effects, and FLX showed limited efficacy in suppressing stress-induced microglial activation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study systematically investigated alteration of oxylipin profiles in patients with MDD. Using targeted oxylipidomics analysis, we found significantly elevated levels of multiple oxylipins in MDD patients compared to healthy controls, with the most pronounced changes observed in LOX and CYP450 pathway metabolites. Notably, higher baseline oxylipin levels predicted poorer antidepressant treatment response, as non-responders exhibited elevated levels of 20 oxylipins relative to both responders and healthy controls. In parallel, CRS mice displayed increased LOX pathway activity and elevated plasma LOX-derived oxylipins. Pharmacological inhibition of 12-LOX with ML-355 ameliorated depressive-like behaviors and reversed stress-induced microglial activation in the hippocampus and medial prefrontal cortex. These findings identify dysregulated LOX-derived oxylipin metabolism as a potential biomarker for treatment resistance and support targeting the LOX pathway, especial 12-LOX pathway, as a therapeutic strategy for depression.\u003c/p\u003e \u003cp\u003eOur finding of elevated oxylipin levels in MDD patients aligns with previous reports of altered oxylipin profiles in depression (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). By using a broader targeted oxylipidomics approach, the present study moves beyond single-metabolite observations and instead highlights a coordinated disturbance across multiple lipid pathways. This broader network-level pattern is important because MDD is unlikely to be explained by a single inflammatory mediator; rather, it may arise from a maladaptive inflammatory-metabolic state in which several lipid signaling systems are shifted simultaneously. Our current results along with these studies, support the involvement of PUFA oxidation pathways in MDD pathophysiology. The elevation of LOX- and CYP450-derived oxylipins observed here is consistent with the established role of inflammatory and oxidative stress processes in depression. Given that oxylipins serve as direct indicators of lipid peroxidation and immune activation, their increased levels likely reflect underlying inflammatory dysregulation in MDD.\u003c/p\u003e \u003cp\u003eA key finding of this study is that baseline oxylipin levels predict antidepressant treatment outcomes, independent of baseline symptom severity. While we observed no correlation between oxylipin levels and baseline HAMD or HAMA scores, multiple oxylipins showed significant negative correlations with HAMD score reduction rates following 8 weeks of treatment. This dissociation, where oxylipins predict treatment response but not initial severity, suggests that these lipid mediators may specifically influence treatment resistance mechanisms rather than general depressive symptomatology. This pattern mirrors findings with other inflammatory biomarkers, wherein elevated inflammation predicts poor antidepressant response despite variable associations with baseline depression severity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Oxylipins, as central mediators of inflammatory signaling, may thus provide a mechanistic link between immune dysregulation and treatment resistance. These results support further investigation of LOX-derived oxylipins as biomarkers to stratify patients for personalized treatment approaches, including anti-inflammatory adjunctive therapies.\u003c/p\u003e \u003cp\u003eLipoxygenases (LOXs) play critical roles in regulating microglial functions through multiple mechanisms. In pathological conditions, LOXs metabolize arachidonic acid to generate pro-inflammatory lipid mediators such as leukotrienes and HETEs, thereby activating microglia, promoting their polarization toward a pro-inflammatory phenotype, and exacerbating neuroinflammation (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Additionally, 5-LOX (ALOX5) and 15-LOX (ALOX15) catalyze the peroxidation of polyunsaturated fatty acids, leading to the accumulation of lipid peroxides that drive microglial ferroptosis, where elevated LOX expression in microglia triggers inflammatory cascades and non-cell-autonomous neuronal death (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In the present study, systemic pharmacological inhibition of 12-LOX with ML-355 significantly ameliorated CRS-induced depressive-like and anxiety-like behaviors, accompanied by reduced microglial activation in the hippocampus and medial prefrontal cortex. These findings suggest that 12-LOX-derived oxylipins promote microglial activation and sustain pro-inflammatory signaling, thereby contributing to stress-induced neuronal dysfunction and depressive behaviors. Notably, the 5-LOX inhibitor zileuton showed only region-dependent effects on microglial activation without significant behavioral improvement, indicating that 12-LOX may be the predominant isoform mediating stress-induced neuroinflammation in depression. This isoform-specific effect aligns with evidence that different LOX pathways have distinct roles in microglial regulation: while 5-LOX is associated with ferroptosis and acute inflammatory responses, 12-LOX appears to be more critically involved in sustained microglial activation and synaptic dysfunction (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Furthermore, beyond acute neuroinflammation, persistent LOX pathway activation may have long-term consequences through trained immunity, a process of innate immune cell reprogramming that leads to heightened inflammatory responses upon subsequent challenges. Recent studies have implicated LOX-derived oxylipins as key mediators of trained immunity (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and given that trained immunity has been proposed as a mechanism underlying recurrent depressive episodes (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), our findings raise the speculative possibility that chronic 12-LOX activation may sensitize microglia to stress re-exposure, potentially contributing to illness chronicity or relapse vulnerability. This hypothesis warrants direct investigation in future studies using recurrence-based animal models. Collectively, these results provide in vivo evidence that pharmacological inhibition of 12-LOX exerts antidepressant effects by attenuating microglial activation and neuroinflammatory processes, highlighting the therapeutic potential of targeting this pathway in depression.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the single-center design and modest sample size limit generalizability, particularly given the ethnically homogeneous (Han Chinese) cohort. Future validation in independent, multi-ethnic samples is warranted. Second, while our findings suggest an inflammatory mechanism, we did not measure conventional inflammatory biomarkers (e.g., IL-6, TNF-α, CRP), limiting our ability to confirm the specific pathways linking oxylipins to microglial activation. Third, the mouse study focused exclusively on the LOX pathway and did not assess CYP450 or COX alterations observed in patients, precluding direct cross-pathway comparisons and a comprehensive understanding of oxylipin dysregulation in stress-induced depression. Fourth, pharmacokinetic validation of ML-355 brain penetration was not performed, and whether peripheral 12-LOX inhibition adequately reduces central oxylipin levels to mediate the observed behavioral effects remains unknown. Fifth, oxylipin levels were not assessed post-treatment, limiting direct evidence that behavioral improvements resulted from reduced LOX pathway activity. Sixth, the elevation of LOX pathway oxylipins in CRS mice did not withstand correction for multiple comparisons given the limited sample size (n\u0026thinsp;=\u0026thinsp;6 per group) and should be interpreted as exploratory.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates elevated plasma oxylipin levels in MDD patients, with the most pronounced elevations observed in treatment-resistant patients, and identifies 12-LOX as a potential therapeutic target using a translational approach. These findings implicate dysregulated LOX-mediated lipid metabolism in depression pathophysiology and provide preclinical evidence supporting the development of 12-LOX inhibitors as novel antidepressant strategies. Future research should investigate the dynamic regulation of oxylipins across MDD subtypes and disease stages, and evaluate the clinical efficacy of targeted LOX inhibition in treatment-resistant depression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Science and Technology of the People\u0026rsquo;s Republic of China (2022ZD0211700), the 135 Project from West China Hospital of Sichuan University (2023HXFH006), the National Natural Science Foundation of China (82571768), the China Postdoctoral Science Foundation (2024M762243, 2025M771987), the Postdoctoral Fellowship Program of CPSF (GZC20251514), the Postdoctoral Fellowship Program of CPSF (GZC20251341), the Sichuan Science and Technology Program (2025ZNSFSC0781, 2025ZNSFSC1563, 2025ZNSFSC1649, 2024NSFSC1559).\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eWe declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang Z, Dou Y, Yang X, Guo X, Ma X, Zhou B, et al. 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PubMed PMID: 37968313. PMCID: PMC10651900 interest. The funders had no role in the design of the study; in the collection, analyzes, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Epub 2023/11/16. eng.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu G, Yuan M, He H, Yi J, Li X, Yan H, et al. NLRP3-mediated trained immunity of microglia is involved in the recurrence-like episode of depressive disorders. Mol Psychiatry. 2026;31(4):1958\u0026ndash;69. PubMed PMID: 41249553. Epub 2025/11/18. eng.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9366044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9366044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxylipins are bioactive lipid metabolites that may bridge immune dysregulation and depressive symptomatology. However, systematic alterations of oxylipin networks in MDD and their therapeutic implications remain unclear. In this study, a plasma oxylipidomics analysis was performed in 154 patients with MDD and 134 healthy controls (HC). Findings were validated in a chronic restraint stress (CRS) mouse model of depression, followed by pharmacological inhibition of lipoxygenase (LOX) pathways using ML-355 and zileuton. Behavioral tests and immunofluorescence analysis of microglial activation were conducted to assess therapeutic effects. MDD patients exhibited significantly elevated plasma levels of 22 oxylipins compared to HCs, including oxylipins derived from lipoxygenase (LOX), cytochrome P450 (CYP450), cyclooxygenase (COX), and non-enzymatic pathways. Notably, non-responders to antidepressant treatment displayed higher baseline levels of 20 oxylipins than both responders and HCs, and baseline oxylipin levels negatively correlated with Hamilton Depression Rating Scale (HAMD) score reduction rates. In CRS mice, the LOX pathway was activated, as evidenced by increased LOX levels in the blood and brain, as well as elevated plasma levels of LOX-derived oxylipins. Pharmacological inhibition of 12-lipoxygenase (12-LOX) with ML-355 significantly alleviated depressive-like and anxiety-like behaviors and reversed stress-induced microglial activation in the hippocampus and medial prefrontal cortex. The 5-LOX inhibitor zileuton reduced microglial activation in a region-dependent manner but did not significantly improve behavioral outcomes. These findings reveal elevated LOX-derived oxylipins as potential biomarkers predicting poor antidepressant response in MDD. Targeting the LOX pathway, particularly 12-LOX, represents a promising therapeutic strategy for depression by ameliorating neuroinflammatory processes.\u003c/p\u003e","manuscriptTitle":"Lipoxygenase-Derived Oxylipins are Elevated in MDD Patients and Stress-Induced Depressed Mice: Inhibition of Lipoxygenases as a Therapeutic Strategy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 05:40:08","doi":"10.21203/rs.3.rs-9366044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-12T14:01:53+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-11T14:29:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-10T10:15:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-28T20:15:13+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-28T14:12:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-28T12:02:04+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-28T11:59:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T15:27:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-13T15:24:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2026-04-10T13:13:03+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-04-10T10:01:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d71651ca-5f26-4d13-86a5-62e87711b47c","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-12T14:01:53+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-11T14:29:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-10T10:15:23+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67165647,"name":"Biological sciences/Biochemistry"},{"id":67165648,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2026-05-11T05:40:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 05:40:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9366044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9366044","identity":"rs-9366044","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}