The Role of APOE4 in Regulating LRP1 on Aβ25-35-Induced Oxidative Stress and Inflammatory Response in Astrocytes
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摘要:
目的 探讨载脂蛋白E4（apolipoprotein E4，APOE4）调控脂蛋白受体相关蛋白1（lipoprotein receptor-related protein-1，LRP1）对β-淀粉样蛋白（amyloid β-protein，Aβ）25-35诱导的星形胶质细胞氧化应激和炎症反应的作用。 方法 在细胞中转染sh-NC、sh-APOE4、sh-LRP1、pcDNA-NC和pcDNA-LRP1，利用10 μM Aβ25-35诱导人星形胶质细胞24 h构建阿尔兹海默病（Alzheimer's disease，AD）细胞模型，经Western blot、DCFH-DA荧光探针和MDA、SOD和GSH检测试剂盒、TNF-α、IL-6和IL-1β酶联免疫吸附试剂盒探讨APOE4和LRP1对Aβ25-35诱导的星形胶质细胞氧化应激和炎症反应的作用。通过免疫共沉淀和Western blot实验检测APOE4与LRP1的蛋白-蛋白相互作用。 结果 Aβ25-35诱导上调APOE4表达（P < 0.01），促进星形胶质细胞中ROS（ P < 0.001）、MDA（ P < 0.001）和炎性细胞因子TNF-α、IL-6和IL-1β的水平（ P < 0.001），抑制SOD、GSH和LRP1表达（ P < 0.001）。敲降APOE4或过表达LRP1可抑制Aβ 25-35诱导的细胞中ROS、氧化应激和炎性细胞因子（P < 0.05）。APOE4通过蛋白-蛋白相互作用负调控LRP1蛋白表达。同时敲降APOE4和LRP1组细胞中ROS、MDA和炎性细胞因子浓度高于仅敲降APOE4组（ P < 0.05），抑制SOD和GSH浓度（ P < 0.001）。 结论 敲降APOE4通过上调LRP1抑制Aβ25-35诱导的星形胶质细胞的氧化应激和炎症反应。 Abstract:Objective To investigate the effect of APOE4 in regulating LRP1 and on β-amyloid protein Aβ25-35-induced oxidative stress and inflammatory response in astrocytes. Methods Human astrocytes were transfected with sh-NC, sh-APOE4, pcDNA-NC, and pcDNA-LRP1, and then induced with 10 μM Aβ25-35 for 24 h to establish an AD cell model. The roles of APOE4 and LRP1 in Aβ25-35-induced oxidative stress and inflammatory response were assessed using Western blot, flow cytometry, the detection kits of MDA, SOD, and GSH, as well as enzyme-linked immunosorbent assay （ELISA） kits for TNF-α, IL-6, and IL-1β. The protein-protein interaction between APOE4 and LRP1 was detected through co-immunoprecipitation and western blot experiments. Results Aβ25-35 induction upregulated APOE4 expression （P < 0.01）, promoted the levels of ROS （ P < 0.001）, MDA （ P < 0.001）, and inflammatory cytokines TNF-α, IL-6, and IL-1β （ P < 0.001） in astrocytes, while inhibited the expression of SOD, GSH, and LRP1 （ P < 0.001）. Knockdown of APOE4 or overexpression of LRP1 suppressed the Aβ 25-35-induced increase in ROS, oxidative stress markers, and inflammatory cytokines in cells （P < 0.05）. APOE4 negatively regulated LRP1 protein expression through protein-protein interactions. The levels of ROS, MDA, and inflammatory cytokines were higher （ P < 0.05）, and the concentrations of SOD and GSH were lower （ P < 0.001）, in cells with simultaneous knockdown of APOE4 and LRP1 compared to those with APOE4 knockdown alone. Conclusion Knockdown of APOE4 attenuates Aβ25-35-induced oxidative stress and inflammatory response in astrocytes by upregulating LRP1. -
Key words:
- Alzheimer's disease /
- Astrocytes /
- Oxidative stress /
- Inflammatory response
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图 1 敲降APOE4抑制Aβ25-35诱导星形胶质细胞的炎症和氧化应激（ $\bar x \pm s $，n = 3）
A：APOE4的免疫印迹和相对表达统计图；B：转染sh-APOE4的星形胶质细胞中APOE4的免疫印迹和相对表达统计图；C～D：DCFH-DA探针法检测细胞中的ROS水平；E～G：试剂盒分别检测细胞中MDA、SOD和GSH水平；H～J：ELISA检测细胞中的炎性细胞因子TNF-α、IL-6、IL-1β水平；Aβ：Aβ25-35；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 1. Knockdown of APOE4 inhibits Aβ25-35-induced inflammation and oxidative stress in astrocytes （ $\bar x \pm s $，n = 3）
图 2 APOE4调控LRP1蛋白表达（ $\bar x \pm s $，n = 3）
A~B：STRING数据库预测APOE4与LRP1具有蛋白-蛋白相互作用；C~D：CO-IP实验验证APOE4与LRP1的相互作用；E~F：LRP1的免疫印记和相对表达统计图；G~H：LRP1的免疫印记和相对表达统计图；Aβ：Aβ25-35；**P < 0.01；***P < 0.001。
Figure 2. APOE4 regulates LRP1 protein expression （ $\bar x \pm s $，n = 3）
图 3 过表达LRP1抑制星形胶质细胞的炎症和氧化应激（ $\bar x \pm s $，n = 3）
A～B：DCFH-DA探针法检测细胞中的ROS水平；C～E：试剂盒分别检测细胞中MDA、SOD和GSH水平；F～H：ELISA检测细胞中的炎性细胞因子TNF-α、IL-6、IL-1β水平；Aβ：Aβ25-35；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 3. Overexpression of LRP1 inhibits inflammation and oxidative stress in astrocytes （ $\bar x \pm s $，n = 3）
图 4 敲降APOE4通过上调LRP1抑制星形胶质细胞的炎症和氧化应激（ $\bar x \pm s $，n = 3）
A～B：APOE4和LRP1的免疫印记图和蛋白相对表达统计；C～D：DCFH-DA荧光探针法检测细胞中ROS水平；E～G：试剂盒检测MDA、SOD和GSH水平；H～J：ELISA试剂盒检测细胞上清液中TNF-α、IL-6和IL-1β浓度；Aβ：Aβ25-35；*P < 0.05；***P < 0.001。
Figure 4. Knockdown of APOE4 inhibits inflammation and oxidative stress in astrocytes by upregulating LRP1 （ $\bar x \pm s $，n = 3）
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[1] Luo J, Thomassen J Q, Bellenguez C, et al. Genetic associations between modifiable risk factors and Alzheimer disease[J]. JAMA Netw Open, 2023, 6(5): e2313734. doi: 10.1001/jamanetworkopen.2023.13734 [2] De Strooper B, Karran E. The cellular phase of Alzheimer’ s disease[J]. Cell, 2016, 164(4): 603-615. doi: 10.1016/j.cell.2015.12.056 [3] Sofroniew M V. Molecular dissection of reactive astrogliosis and glial scar formation[J]. Trends Neurosci, 2009, 32(12): 638-647. doi: 10.1016/j.tins.2009.08.002 [4] Deng Q, Wu C, Parker E, et al. Microglia and astrocytes in Alzheimer’ s disease: Significance and summary of recent advances[J]. Aging Dis, 2024, 15(4): 1537-1564. [5] Kaur D, Sharma V, Deshmukh R. Activation of microglia and astrocytes: A roadway to neuroinflammation and Alzheimer’ s disease[J]. Inflammopharmacology, 2019, 27(4): 663-677. doi: 10.1007/s10787-019-00580-x [6] Liu C C, Kanekiyo T, Xu H, et al. Apolipoprotein E and Alzheimer disease: Risk, mechanisms and therapy[J]. Nat Rev Neurol, 2013, 9(2): 106-118. doi: 10.1038/nrneurol.2012.263 [7] Kurkinen M. Lecanemab (Leqembi) is not the right drug for patients with Alzheimer’ s disease[J]. Adv Clin Exp Med, 2023, 32(9): 943-947. doi: 10.17219/acem/171379 [8] Shinohara M, Tachibana M, Kanekiyo T, et al. Role of LRP1 in the pathogenesis of Alzheimer’ s disease: Evidence from clinical and preclinical studies[J]. J Lipid Res, 2017, 58(7): 1267-1281. doi: 10.1194/jlr.R075796 [9] Rauch J N, Luna G, Guzman E, et al. LRP1 is a master regulator of tau uptake and spread[J]. Nature, 2020, 580(7803): 381-385. doi: 10.1038/s41586-020-2156-5 [10] Chen K, Martens Y A, Meneses A, et al. LRP1 is a neuronal receptor for α-synuclein uptake and spread[J]. Mol Neurodegener, 2022, 17(1): 57. doi: 10.1186/s13024-022-00560-w [11] Hillen A E J, Burbach J P H, Hol E M. Cell adhesion and matricellular support by astrocytes of the tripartite synapse[J]. Prog Neurobiol, 2018, 165-167: 66-86. [12] Kim M, Choi W, Choi S, et al. In vivo reactive astrocyte imaging in patients with schizophrenia using fluorine 18-labeled THK5351[J]. JAMA Netw Open, 2024, 7(5): e2410684. doi: 10.1001/jamanetworkopen.2024.10684 [13] Verkhratsky A, Zorec R, Rodríguez J J, et al. Astroglia dynamics in ageing and Alzheimer’ s disease[J]. Curr Opin Pharmacol, 2016, 26: 74-79. doi: 10.1016/j.coph.2015.09.011 [14] Preman P, Alfonso-Triguero M, Alberdi E, et al. Astrocytes in Alzheimer’ s disease: Pathological significance and molecular pathways[J]. Cells, 2021, 10(3): 540. doi: 10.3390/cells10030540 [15] Kim S, Chun H, Kim Y, et al. Astrocytic autophagy plasticity modulates Aβ clearance and cognitive function in Alzheimer’ s disease[J]. Mol Neurodegener, 2024, 19(1): 55. doi: 10.1186/s13024-024-00740-w [16] Tang Z, Chen Z, Guo M, et al. NRF2 deficiency promotes ferroptosis of astrocytes mediated by oxidative stress in Alzheimer’ s disease[J]. Mol Neurobiol, 2024, 61(10): 7517-7533. doi: 10.1007/s12035-024-04023-9 [17] Koutsodendris N, Nelson M R, Rao A, et al. Apolipoprotein E and Alzheimer’ s disease: Findings, hypotheses, and potential mechanisms[J]. Annu Rev Pathol, 2022, 17: 73-99. doi: 10.1146/annurev-pathmechdis-030421-112756 [18] Koutsodendris N, Blumenfeld J, Agrawal A, et al. Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits[J]. Nat Aging, 2023, 3(3): 275-296. doi: 10.1038/s43587-023-00368-3 [19] Pires M, Rego A C. Apoe4 and Alzheimer’ s disease pathogenesis-mitochondrial deregulation and targeted therapeutic strategies[J]. Int J Mol Sci, 2023, 24(1): 778. doi: 10.3390/ijms24010778 [20] Xiong M, Wang C, Gratuze M, et al. Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy[J]. Mol Neurodegener, 2023, 18(1): 17. doi: 10.1186/s13024-023-00610-x [21] Litvinchuk A, Suh J H, Guo J L, et al. Amelioration of Tau and ApoE4-linked glial lipid accumulation and neurodegeneration with an LXR agonist[J]. Neuron, 2024, 112(3): 384-403. e8. [22] Zhou J, Zhang L, Peng J, et al. Astrocytic LRP1 enables mitochondria transfer to neurons and mitigates brain ischemic stroke by suppressing ARF1 lactylation[J]. Cell Metab, 2024, 36(9): 2054-2068. e14. [23] Li A, Zhang J, Chen K, et al. Donepezil attenuates inflammation and apoptosis in ulcerative colitis via regulating LRP1/AMPK/NF-κB signaling[J]. Pathol Int, 2023, 73(11): 549-559. doi: 10.1111/pin.13380 [24] Ponchel T, Loeffler E, Ancel J, et al. LRP1 involvement in FHIT-regulated HER2 signaling in non-small cell lung cancer[J]. Eur J Cell Biol, 2025, 104(1): 151475. doi: 10.1016/j.ejcb.2024.151475 [25] Danis C, Dupré E, Bouillet T, et al. Inhibition of tau neuronal internalization using anti-tau single domain antibodies[J]. Nat Commun, 2025, 16(1): 3162. doi: 10.1038/s41467-025-58383-4 [26] Kang S, Lee J, Ali D N, et al. Low to moderate ethanol exposure reduces astrocyte-induced neuroinflammatory signaling and cognitive decline in presymptomatic APP/PS1 mice[J]. Sci Rep, 2024, 14(1): 23989. doi: 10.1038/s41598-024-75202-w [27] Van Gool B, Storck S E, Reekmans S M, et al. LRP1 has a predominant role in production over clearance of aβ in a mouse model of Alzheimer’ s disease[J]. Mol Neurobiol, 2019, 56(10): 7234-7245. doi: 10.1007/s12035-019-1594-2 [28] Zhou R, Wang L, Chen L, et al. Bone marrow-derived GCA+ immune cells drive Alzheimer’ s disease progression[J]. Adv Sci (Weinh), 2023, 10(36): e2303402. doi: 10.1002/advs.202303402 -
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