ALOX15B-GPX4 Axis in Alleviates Depression by Regulating Neuroinflammation and Ferroptosis
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摘要:
目的 探讨花生四烯酸 15-脂氧合酶B型（arachidonate 15-lipoxygenase type B，ALOX15B）在抑郁症中的作用及其潜在分子机制。 方法 构建大鼠慢性不可预知性应激（chronic unpredictable mild stress，CUMS）抑郁症模型，蔗糖偏好、强迫游泳以及悬尾实验检测大鼠的抑郁样行为。通过脂多糖（lipopolysaccharide，LPS）干预BV2细胞构建体外细胞模型。CCK-8法检测细胞活力；流式细胞术检测细胞凋亡率；ELISA试剂盒检测组织和细胞中炎症因子的水平；生化试剂盒检测组织核细胞中Fe2+、丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）、超氧化物歧化酶（superoxide dismutase，SOD）的含量；RT-qPCR和Western blot检测各组组织和细胞中ALOX15B、GPX4、ACSL4、TFR1、FIH1、SLC7A11的表达；免疫荧光染色检测细胞中离子钙结合适配分子1（ionized calcium-binding adaptor molecule 1，IBA-1）的表达。 结果 ALOX15B在CUMS大鼠模型和LPS诱导的BV2细胞模型组均显著上调（P < 0.0001 ），GPX4则显著下调（P <0.0001 ）。敲低ALOX15B能够缓解大鼠的抑郁样行为。敲低ALOX15B组大鼠海马组织中Fe2+、MDA含量显著降低（P <0.0001 ），SOD、GSH含量显著升高（P <0.0001 ），ACSL4、TFR1蛋白的表达显著降低（P < 0.01），FIH1、SLC7A11蛋白表达显著升高（P < 0.001）。敲低ALOX15B能够显著缓解LPS诱导的细胞活力降低、细胞凋亡率升高的现象。此外，敲低ALOX15B组细胞中Fe2+和MDA含量显著降低（P < 0.01）、GSH和SOD的含量升高（P < 0.01）、ACSL4和TFR1蛋白表达显著降低（P < 0.01），FIH1和SLC7A11蛋白表达量显著升高（P < 0.001）。Co-IP实验证实ALOX15B与GPX4之间存在相互作用，且敲低GPX4可逆转ALOX15B沉默所介导的细胞保护效应。结论 敲低ALOX15B能够通过减轻神经炎症并抑制铁死亡，进而缓解抑郁症的进展。 Abstract:Objective To investigate the role of arachidonate 15-lipoxygenase type B （ALOX15B） in depression and its potential molecular mechanisms. Methods A chronic unpredictable mild stress （CUMS） depression model was established in rats. Sucrose preference test, forced swimming test, and tail suspension test were used to detect depressive-like behaviors in rats. An in vitro cell model was established by treating BV2 cells with lipopolysaccharide （LPS）. Cell viability was assessed using the CCK-8 assay, and cell apoptosis was detected by flow cytometry. The levels of inflammatory cytokines in tissues and cells were measured using ELISA kits. The contents of Fe2+, malondialdehyde （MDA）, glutathione （GSH）, and superoxide dismutase （SOD） in tissues and cells were determined using biochemical assay kits. The expression levels of ALOX15B, GPX4, ACSL4, TFR1, FIH1, and SLC7A11 in tissues and cells were detected by RT-qPCR and Western blot. Immunofluorescence staining was performed to detect the expression of ionized calcium-binding adaptor molecule 1 （IBA-1） in cells. Results ALOX15B was significantly upregulated in both the CUMS rat model and the LPS-induced BV2 cell model （P < 0.0001 ）, whereas GPX4 was significantly downregulated （P <0.0001 ）. Knockdown of ALOX15B alleviated depression-like behaviors in rats. In the hippocampal tissues of rats with ALOX15B knockdown, the levels of Fe2+ and MDA were significantly decreased （P <0.0001 ）, while the levels of SOD and GSH were significantly increased （P <0.0001 ）. ACSL4 and TFR1 protein expression were significantly decreased （P < 0.01）, whereas FIH1 and SLC7A11 protein expression were significantly increased （P < 0.001）. ALOX15B knockdown significantly alleviated LPS-induced decreases in cell viability and increases in cell apoptosis rate. In addition, Fe2+ and MDA levels were significantly reduced in ALOX15B-knockdown cells （P < 0.01）, while GSH and SOD levels were increased （P < 0.01）. ACSL4 and TFR1 protein expression were significantly decreased （P < 0.01）, whereas FIH1 and SLC7A11 protein expression were significantly increased （P < 0.001）. Co-immunoprecipitation assays confirmed an interaction between ALOX15B and GPX4, and GPX4 knockdown reversed the cytoprotective effects mediated by ALOX15B silencing.Conclusion ALOX15B knockdown alleviates the progression of depression by attenuating neuroinflammation and inhibiting ferroptosis. -
Key words:
- Ferroptosis /
- Neuroinflammation /
- ALOX15B /
- Depression
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图 1 CUMS模型的构建（$ \bar x \pm s $，n = 6）
A：蔗糖偏好实验检测各组大鼠蔗糖的摄入量；B：强迫游泳实验检测大鼠的抑郁样行为；C：悬尾实验评估大鼠的不动状态；***P < 0.001；****P < 0.0001。
Figure 1. Establishment of the CUMS model （$ \bar x \pm s $，n = 6）
图 2 CUMS模型大鼠脑组织中ALOX15B和GPX4的表达（$ \bar x \pm s $，n = 6）
A～B：RT-qPCR检测各组大鼠海马组织中ALOX15B和GPX4的表达情况；C～E：Western blot检测各组大鼠海马组织中ALOX15B和GPX4的蛋白表达情况；**P < 0.01；****P < 0.0001。
Figure 2. Expression of ALOX15B and GPX4 in brain tissues of CUMS model rats （$ \bar x \pm s $，n = 6）
图 3 敲低ALOX15B缓解CUMS大鼠抑郁样行为（$ \bar x \pm s $，n = 6）
A～B：RT-qPCR检测各组大鼠海马组织中ALOX15B和GPX4的表达情况；C～E：Western blot检测各组大鼠海马组织中ALOX15B和GPX4的蛋白表达情况；F：强迫游泳实验检测大鼠的抑郁样行为；G：悬尾实验评估大鼠的不动状态；H：蔗糖偏好实验检测各组大鼠蔗糖的摄入量；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 3. ALOX15B knockdown alleviates depressive-like behaviors in CUMS rats （$ \bar x \pm s $，n = 6）
图 4 敲低ALOX15B缓解CUMS大鼠的炎症、氧化应激及铁死亡（$ \bar x \pm s $，n = 6）
A～C：ELISA试剂盒检测各组大鼠海马组织中炎症因子TNF-α、IL-6、IL-10的水平；D～G：生化试剂盒检测各组大鼠海马组织中Fe2+、MDA、SOD、GSH的含量；H～L：Western blot检测各组大鼠海马组织中铁死亡相关蛋白ACSL4、TFR1、FIH1、SLC7A11的表达；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 4. ALOX15B knockdown alleviates inflammation，oxidative stress，and ferroptosis in CUMS rats （$ \bar x \pm s $，n = 6）
图 5 敲低ALOX15B缓解LPS诱导的BV2细胞炎症（$ \bar x \pm s $，n = 3）
A～B：RT-qPCR检测各组细胞中ALOX15B和GPX4的表达情况；C～E：Western blot检测各组细胞中ALOX15B和GPX4的蛋白表达情况；F：CCK-8检测BV2细胞活力；G～I：ELISA试剂盒检测各组细胞中炎症因子TNF-α、IL-6、IL-10的水平；J～K：流式细胞术检测细胞凋亡；L：免疫荧光染色检测细胞中IBA-1的表达，40×；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 5. ALOX15B knockdown alleviates LPS-induced inflammation in BV2 cells （$ \bar x \pm s $，n = 3）
图 6 敲低ALOX15B缓解LPS诱导的BV2细胞铁死亡（$ \bar x \pm s $，n = 3）
A～D：生化试剂盒检测细胞中Fe2+、MDA、GSH、SOD的含量；E～I：Western blot检测各组细胞中铁死亡相关蛋白ACSL4、TFR1、FIH1、SLC7A11的表达；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 6. ALOX15B knockdown alleviates LPS-induced ferroptosis in BV2 cells （$ \bar x \pm s $，n = 3）
图 7 ALOX15B与GPX4相互作用
A：Co-IP实验检测ALOX15B与GPX4相互作用关系；B～D：Western blot检测ALOX15B与GPX4的蛋白表达；*P < 0.05；**P < 0.01；****P < 0.0001。
Figure 7. Interaction between ALOX15B and GPX4 （$ \bar x \pm s $，n = 3）
图 8 敲低GPX4逆转敲低ALXO15B对BV2细胞的保护作用
A～B：RT-qPCR检测各组细胞中ALOX15B和GPX4的-表达情况；C～E：Western blot检测各组细胞中ALOX15B和GPX4的蛋白表达情况；F：CCK-8检测BV2细胞活力；G～I：ELISA试剂盒检测各组细胞中炎症因子TNF-α、IL-6、IL-10的水平；J～K：流式细胞术检测细胞凋亡；L～O：生化试剂盒检测细胞中Fe2+、MDA、GSH、SOD的含量；P～T：Western blot检测各组细胞中铁死亡相关蛋白ACSL4、TFR1、FIH1、SLC7A11的表达；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 8. GPX4 knockdownreverses the protective effect of ALOX15B knockdown on BV2 cells
表 1 引物序列
Table 1. Primer sequences
基因 引物序列（5’-3’） ALOX15B F: CCACCCTCTCTTCAAGTCCAC
R: CGATGATTTCAGAGACAAAGCGTGPX4 F: TCACCAAGTTTGGACACCGT
R: ATAGTGGGGCAGGTCCTTCTGAPDH F: GGTCACCAGGGCTGCTTTTA
R: CCCGTTCTCAGCCATGTAGT
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[1] Berk M, Köhler-Forsberg O, Turner M, et al. Comorbidity between major depressive disorder and physical diseases: A comprehensive review of epidemiology, mechanisms and management[J]. World Psychiatry, 2023, 22(3): 366-387. [2] Huang M H, Chen M H, Hsu J W, et al. Inflammation, cognitive dysfunction, and suicidal ideation among patients with major depression[J]. CNS Spectr, 2022, 27(6): 724-730. doi: 10.1017/S1092852921000729 [3] Wu Y, Zhu B, Chen Z, et al. New insights into the comorbidity of coronary heart disease and depression[J]. Curr Probl Cardiol, 2021, 46(3): 100413. [4] Al-Khatib Y, Akhtar M A, Ali Kanawati M, et al. Depression and metabolic syndrome: A narrative review[J]. Cureus, 2022, 14(2): e22153. [5] Cipriani A, Furukawa T A, Salanti G, et al. Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: A systematic review and network meta-analysis[J]. Lancet, 2018, 391(10128): 1357-1366. [6] Irwin C L, Coelho P S, Kluwe-Schiavon B, et al. Non-pharmacological treatment-related changes of molecular biomarkers in major depressive disorder: A systematic review and meta-analysis[J]. Int J Clin Health Psychol, 2023, 23(3): 100367. [7] Collins E, Vieira N, Walsh B, et al. Vagus nerve stimulation (VNS), repetitive transcranial magnetic stimulation (rTMS) or the combination for treating depression[J]. Brain Stimul, 2025, 18(1): 546. [8] Cui L, Li S, Wang S, et al. Major depressive disorder: Hypothesis, mechanism, prevention and treatment[J]. Signal Transduct Target Ther, 2024, 9: 30. [9] Zhou Y, Huang Y, Ye W, et al. Cynaroside improved depressive-like behavior in CUMS mice by suppressing microglial inflammation and ferroptosis[J]. Biomed Pharmacother, 2024, 173: 116425. [10] Tang D, Chen X, Kang R, et al. Ferroptosis: Molecular mechanisms and health implications[J]. Cell Res, 2021, 31(2): 107-125. [11] Moshfeghinia R, Ghahramani M, Naderian R, et al. Pro-inflammatory cytokines level in depressed patients with suicidal behaviour: A systematic review and meta-analysis[J]. Ann Gen Psychiatry, 2025, 24(1): 71. [12] Yi L, Tang J, Yang S, et al. Childhood trauma and the plasma levels of IL-6, TNF-α are risk factors for major depressive disorder and schizophrenia in adolescents: A cross-sectional and case-control study[J]. J Affect Disord, 2022, 305: 227-232. [13] Wang H, He Y, Sun Z, et al. Microglia in depression: An overview of microglia in the pathogenesis and treatment of depression[J]. J Neuroinflammation, 2022, 19(1): 132. [14] Snijders G J L J, Sneeboer M A M, Fernández-Andreu A, et al. Distinct non-inflammatory signature of microglia in post-mortem brain tissue of patients with major depressive disorder[J]. Mol Psychiatry, 2021, 26(7): 3336-3349. [15] Spiteri A G, Wishart C L, Pamphlett R, et al. Microglia and monocytes in inflammatory CNS disease: Integrating phenotype and function[J]. Acta Neuropathol, 2022, 143(2): 179-224. [16] Zhao X, Yin J, Lu T, et al. ALOX15 and ALOX15B regulate autophagy to promote pulmonary arterial hypertension via the PI3K/AKT/mTOR pathway[J]. Eur J Pharmacol, 2025, 1008: 178338. [17] Zhang W, Huang F, Ding X, et al. Identifying ALOX15-initiated lipid peroxidation increases susceptibility to ferroptosis in asthma epithelial cells[J]. Biochim Biophys Acta BBA Mol Basis Dis, 2024, 1870(5): 167176. [18] Cao H, Zuo C, Huang Y, et al. Hippocampal proteomic analysis reveals activation of necroptosis and ferroptosis in a mouse model of chronic unpredictable mild stress-induced depression[J]. Behav Brain Res, 2021, 407: 113261. [19] Liang W, Guo H, Li L, et al. Ferroptosis: A new target for depression prevention and treatment[J]. J Neural Transm, 2025, 132(7): 979-997. [20] Su R, Pan X, Chen Q, et al. Nicotinamide mononucleotide mitigates neuroinflammation by enhancing GPX4-mediated ferroptosis defense in microglia[J]. Brain Res, 2024, 1845: 149197. [21] Hacioglu C. Long-term exposure of sucralose induces neuroinflammation and ferroptosis in human microglia cells via SIRT1/NLRP3/IL-1β/GPx4 signaling pathways[J]. Food Sci Nutr, 2024, 12(11): 9094-9107. [22] Cserép C, Pósfai B, Dénes Á. Shaping neuronal fate: Functional heterogeneity of direct microglia-neuron interactions[J]. Neuron, 2021, 109(2): 222-240. [23] Lu Y, Zheng Y, Sun X, et al. Knockdown of USP22 alleviates LPS-induced microglial inflammation and mouse depressive-like behaviors via KAT2A[J]. Neuropsychopharmacology, 2025, 50(12): 1904-1912. [24] Jia X, Gao Z, Hu H. Microglia in depression: Current perspectives[J]. Sci China Life Sci, 2021, 64(6): 911-925. [25] Cardoso B R, Hare D J, Bush A I, et al. Glutathione peroxidase 4: A new player in neurodegeneration?[J]. Mol Psychiatry, 2017, 22(3): 328-335. [26] Shao L, Dong C, Geng D, et al. Ginkgolide B protects against cognitive impairment in senescence-accelerated P8 mice by mitigating oxidative stress, inflammation and ferroptosis[J]. Biochem Biophys Res Commun, 2021, 572: 7-14. [27] Zhang Z, Wu Y, Yuan S, et al. Glutathione peroxidase 4 participates in secondary brain injury through mediating ferroptosis in a rat model of intracerebral hemorrhage[J]. Brain Res, 2018, 1701: 112-125. [28] Xu Y, Chen J, Zhou L, et al. Gastrodin protects against sepsis-associated encephalopathy by suppressing ferroptosis[J]. Shock, 2025, 63(4): 628-637. [29] 沈晓霞, 赵晓东, 宋永健. SIRT1激动剂调控Nrf2-GPX4铁死亡途径改善冠状动脉病变小鼠心肌功能的作用及机制[J]. 昆明医科大学学报, 2025, 46(5): 55-64. -
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