根皮素抑制LPS诱导的BV2小胶质细胞氧化应激反应

doi:10.3969/j.issn.1006-7795.2022.01.017

摘要/Abstract

摘要： 目的探讨根皮素(phloretin, PHL)对脂多糖(lipopolysaccharide, LPS)导致的BV2小胶质细胞的氧化应激损伤的抑制作用及可能的分子机制。方法利用LPS建立BV2小胶质细胞的氧化应激损伤模型,对该模型予以不同浓度的PHL 预处理。利用MTS方法检测细胞活力。ELISA法检测氧化应激相关产物一氧化氮(nitric oxide,NO)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)含量及超氧化物歧化酶(superoxide dismutase,SOD)的活性。双荧光素酶活性检验方法检测抗氧化响应元件荧光素酶报告基因质粒(antioxidant reaction element luciferase reporter plasmid,ARE-LUC)报告基因转录活性。Western blotting法检测磷酸化核因子-E2相关因子2(nuclear factor E2-related factor 2,Nrf2)和血红蛋白加氧酶-1(heme oxygenase-1,HO-1)蛋白表达。结果与对照组相比,LPS处理后的BV2小胶质细胞活力显著降低,氧化应激产物NO和MDA水平明显升高,GSH含量和SOD活性明显下降,但Nrf2磷酸化水平、ARE-LUC报告基因转录活性和HO-1蛋白表达略有增高。与模型组比较,高剂量PHL(20 μmol/L)预处理明显改善了LPS导致的BV2小胶质细胞活力下降,降低NO和MDA的含量,增高GSH的含量和SOD的活性,且进一步增加了Nrf2的磷酸化水平,上调ARE-LUC的转录活性和HO-1蛋白的表达。结论 PHL可以显著抑制LPS导致的BV2小胶质细胞的氧化应激损伤,Nrf2/ARE通路可能是根皮素发挥抑制BV2小胶质细胞氧化应激损伤作用的途径之一。

关键词: 根皮素, BV2小胶质细胞, 氧化应激, 核因子-E2相关因子, 抗氧化响应元件

Abstract: Objective To explore the inhibition effect of phloretin (PHL) on oxidative stress response in lipopolysaccharide (LPS)-stimulated BV2 microglia and its related molecular mechanism. Methods LPS was used to establish BV2 microglia oxidative stress damage cell model, and the cell model was pretreated with different concentrations of PHL. MTS assay was used to examine the cell survival rate of BV2 microglia. ELISA kits were used to detect oxidative stress related products such as nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activity. Luciferase reporter gene system was used to determine the transcriptional activity of antioxidant reaction element luciferase reporter plasmid (ARE-LUC). Western blotting was used to detect the level of phosphorylated nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein. Results LPS treatment significantly decreased the viability of BV2 microglia, the level of GSH and the activity of SOD, but increased the level of NO, MDA. However, the pretreatment of PHL remarkably increased the viability of BV2 microglia and the level of GSH and SOD, but decreased the level of NO and MDA. Meanwhile, the pretreatment of PHL further increased the level of phosphorylated Nrf2, the activity of ARE-LUC reporter gene and the level of HO-1. Conclusion PHL can significantly inhibit the oxidative stress response of BV2 microglia induced by LPS, and the inhibition effect might be mediated activating the Nrf2/ARE pathway.

Key words: phloretin, BV2 microglia, oxidative stress, nuclear factor E2-related factor, antioxidant response element

中图分类号:

R739.41

李琛琛, 徐君美, 贺芊芊, 李嘉嬴, 李志恒, 徐志卿, 杨予涛. 根皮素抑制LPS诱导的BV2小胶质细胞氧化应激反应[J]. 首都医科大学学报, 2022, 43(1): 99-105.

Li Chenchen, Xu Junmei, He Qianqian, Li Jiaying, Li Zhiheng, Xu Zhiqing, Yang Yutao. Phloretin inhibits oxidative stress response in LPS-stimulated BV2 microglia[J]. Journal of Capital Medical University, 2022, 43(1): 99-105.

参考文献

[1] Jia Z Y, Huang X Q, Wu Q Z, et al. High-field magnetic resonance imaging of suicidality in patients with major depressive disorder[J]. Am J Psychiatry, 2010, 167(11): 1381-1390.
[2] GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the global burden of disease study 2016[J]. Lancet, 2017, 390(10100): 1211-1259.
[3] 秦碧勇, 戴立磊, 汪键, 等. 抑郁症患者自杀风险与共病数量、抑郁程度的相关性研究[J]. 重庆医学, 2016, 45(13): 1810-1812.
[4] Rapaport M H, Clary C, Fayyad R, et al. Quality-of-life impairment in depressive and anxiety disorders[J]. Am J Psychiatry, 2005, 162(6): 1171-1178.
[5] Wohleb E S, Hanke M L, Corona A W, et al. β-adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat[J]. J Neurosci, 2011, 31(17): 6277-6288.
[6] Stein D J, Vasconcelos M F, Albrechet-Souza L, et al. Microglial over-activation by social defeat stress contributes to anxiety- and depressive-like behaviors[J]. Front Behav Neurosci, 2017, 11: 207.
[7] Kudlow P, Cha D S, Carvalho A F, et al. Nitric oxide and major depressive disorder: pathophysiology and treatment implications[J]. Curr Mol Med, 2016, 16(2): 206-215.
[8] Grinberg Y Y, Dibbern M E, Levasseur V A, et al. Insulin-like growth factor-1 abrogates microglial oxidative stress and TNF-α responses to spreading depression[J]. J Neurochem, 2013, 126(5): 662-672.
[9] Brites D, Fernandes A. Neuroinflammation and depression: microglia activation, extracellular microvesicles and microRNA dysregulation[J]. Front Cell Neurosci, 2015, 9: 476.
[10] Fedoce A D G, Ferreira F, Bota R G, et al. The role of oxidative stress in anxiety disorder: cause or consequence?[J]. Free Radic Res, 2018, 52(7): 737-750.
[11] Ramirez K, Sheridan J F. Antidepressant imipramine diminishes stress-induced inflammation in the periphery and central nervous system and related anxiety- and depressive-like behaviors[J]. Brain Behav Immun, 2016, 57: 293-303.
[12] 肖雪. 帕罗西汀联合参松养心胶囊治疗老年冠心病合并抑郁症疗效及对患者氧化／抗氧化应激平衡和生活质量的影响[J]. 现代中西医结合杂志, 2017, 26(5): 481-484.
[13] Fu K Q, Feng C, Shao L Z, et al. Tanshinone ⅡA exhibits anti-inflammatory and antioxidative effects in LPS-stimulated bovine endometrial epithelial cells by activating the Nrf2 signaling pathway[J]. Res Vet Sci, 2021, 136: 220-226.
[14] Das R, Balmik A A, Chinnathambi S. Melatonin reduces GSK3β-mediated tau phosphorylation, enhances Nrf2 nuclear translocation and anti-inflammation[J]. ASN Neuro, 2020, 12: 1759091420981204.
[15] Goodfellow M J, Borcar A, Proctor J L, et al. Transcriptional activation of antioxidant gene expression by Nrf2 protects against mitochondrial dysfunction and neuronal death associated with acute and chronic neurodegeneration[J]. Exp Neurol, 2020, 328: 113247.
[16] Abuelezz S A, Hendawy N. Insights into the potential antidepressant mechanisms of cilostazol in chronically restraint rats: impact on the Nrf2 pathway[J]. Behav Pharmacol, 2018, 29(1): 28-40.
[17] Tian L, Sun S S, Cui L B, et al. Repetitive transcranial magnetic stimulation elicits antidepressant- and anxiolytic-like effect via nuclear factor-E2-related factor 2-mediated anti-inflammation mechanism in rats[J]. Neuroscience, 2020, 429: 119-133.
[18] Morris G, Walker A J, Walder K, et al. Increasing Nrf2 activity as a treatment approach in neuropsychiatry[J]. Mol Neurobiol, 2021, 58(5): 2158-2182.
[19] 高贵元, 黄捷, 刘丹, 等. 抑郁症的发病机制及抗抑郁药物的研究进展[J]. 中国医药导报, 2021, 18(1): 52-55, 70.
[20] Nørr L, Bennedsen B, Fedder J, et al. Use of selective serotonin reuptake inhibitors reduces fertility in men[J]. Andrology, 2016, 4(3): 389-394.
[21] Ahmadimanesh M, Balarastaghi S, Rashedinia M, et al. A systematic review on the genotoxic effect of serotonin and norepinephrine reuptake inhibitors (SNRIs) antidepressants[J]. Psychopharmacology, 2020, 237(7): 1909-1915.
[22] 魏丽娜, 赵静, 罗仓学, 等. 根皮素的制备、结构修饰及生理活性研究进展[J]. 食品与发酵工业, 2019, 45(17): 278-285.
[23] Ren D Y, Liu Y F, Zhao Y, et al. Hepatotoxicity and endothelial dysfunction induced by high choline diet and the protective effects of phloretin in mice[J]. Food Chem Toxicol, 2016, 94: 203-212.
[24] Alvariño R, Alonso E, Abbasov M E, et al. Gracilin A derivatives target early events in Alzheimer's disease: in vitro effects on neuroinflammation and oxidative stress[J]. ACS Chem Neurosci, 2019, 10(9): 4102-4111.
[25] Zhang G J, Yang G Q, Liu J. Phloretin attenuates behavior deficits and neur-oinflammatory response in MPTP induced Parkinson's disease in mice[J]. Life Sci, 2019, 232: 116600.
[26] Ying Y, Jin J Y, Ye L, et al. Phloretin prevents diabetic cardiomyopathy by dissociating Keap1/Nrf2 complex and inhibiting oxidative stress[J]. Front Endocrinol, 2018, 9: 774.
[27] 李兵, 张婵, 林芳, 等. 葛根素通过Nrf2/ARE通路调控小神经胶质BV-2细胞的氧化应激损伤[J]. 天津中医药, 2018, 35(12): 943-946.
[28] Kwon Y W, Cheon S Y, Park S Y, et al. Tryptanthrin suppresses the activation of the LPS-treated BV2 microglial cell line via Nrf2/HO-1 antioxidant signaling[J]. Front Cell Neurosci, 2017, 11: 18.
[29] Joshi G, Johnson J A. The Nrf2-ARE pathway: a valuable therapeutic target for the treatment of neurodegenerative diseases[J]. Recent Pat CNS Drug Discov, 2012, 7(3): 218-229.