从内质网应激的角度探究微囊藻毒素-LR对斑马鱼离体肝细胞脂代谢的影响
Effects of Microcystin-LR on Lipid Metabolism in Zebrafish Liver Cells via Endoplasmic Reticulum Stress Pathway
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摘要: 为了探究微囊藻毒素-LR(MC-LR)对斑马鱼离体肝细胞内质网应激(ERs)和脂质代谢的影响及机制，本实验以斑马鱼肝细胞系(ZFL)为实验材料，使用不同浓度梯度MC-LR(0、10、20、40、80和160 μg·mL-1)分别进行24 h暴露，在明确细胞活力和半致死浓度(49.3 μg·mL-1)的基础上，选定10 μg·mL-1为暴露浓度，研究细胞中总胆固醇(TC)和甘油三酯(TG)的含量及ERs信号分子、下游因子以及与脂质代谢相关的基因表达情况，并利用ERs抑制剂牛磺熊去氧胆酸(TUDCA)进行机制验证。结果表明，和对照组相比，MC-LR(10 μg·mL-1)暴露诱导TC、TG含量显著上升，未折叠蛋白反应(UPR)途径相关基因(包括atf6、eif2ak3、ern1和xbp1s)以及下游脂质代谢相关基因(srebf1、fasn、acaca、scd、srebf2、hmgcra和hmgcs1)的mRNA表达显著上调；而TUDCA处理导致TC、TG含量显著下降，且UPR和脂类合成途径相关基因表达水平显著性下调。相对地，在TUDCA预处理组中，TC、TG含量、UPR和脂类合成途径相关基因表达相对于MC-LR处理组显著下降，但和对照组相比无显著差异。上述结果表明，MC-LR可通过影响肝脏脂质合成相关基因表达对体外ZFL细胞脂质代谢产生影响，其机制是MC-LR会诱导ERs和固醇调控元件结合蛋白(SREBP)活化(srebf1和srebf2)，进而驱动下游脂质和胆固醇代谢合成基因(fasn、acaca、scd、hmgcs1和hmgcra)的上调，最终导致肝脏脂质的蓄积。TUDCA预暴露组相应检测指标的恢复进一步验证了ERs在MC-LR引起的斑马鱼肝脏脂质代谢异常中的作用。本研究的发现为MC-LR肝毒性提供了机制上的见解，并由此可外推到MC对人健康的潜在影响。Abstract: To investigate the effects of MC-LR on endoplasmic reticulum stress (ERs) and lipid metabolism in vitro liver cells and its mechanism, we selected ZFL (zebrafish liver) cells as experimental model and expose them to MC-LR (0, 10, 20, 40, 80, 160 μg·mL-1) for 24 h. After determining the 24 h-LC50 for MC-LR (49.3 μg·mL-1), the 10 μg·mL-1 of MC-LR was set as exposure concentration and tauroursodeoxycholic acid (TUDCA) was used as an inhibitor to verify the role of ERs in lipid metabolism. The results showed that the contents of intracellular total cholesterol (TC) and total triglycerides (TG) were increased significantly in the MC-LR (10 μg·mL-1) treatment group compared to the control group, and transcriptional levels of unfolded protein response (UPR) pathways related genes including atf6, eif2ak3, ern1, and xbp1s as well as downstream lipid metabolism-related genes including srebf1, fasn, acaca, scd, srebf2, hmgcra and hmgcs1 were significantly up-regulated. In contrast, the contents of TC and TG were decreased significantly in the TUDCA treatment group compared to the control group, and the expression levels of genes related to UPR pathways and lipid synthesis were significantly downregulated. In the TUDCA pretreatment group, the contents of TC and TG and expression levels of genes involved with UPR pathways and lipid synthesis returned to the control levels, but significantly decreased compared with the MC-LR treatment group. This finding indicates that MC-LR mainly affected lipid metabolism of in vitro ZFL cells by interfering with the expression of lipid synthesis-related genes (fasn, acaca, scd, hmgcs1 and hmgcra). The mechanism was that the ERs caused by MC-LR further upregulated the expression of the steroid regulatory element protein factor srebf1 and srebf2 as well as genes related to lipid synthesis. The exposure of TUDCA and the recovery of corresponding test indicators further verified the role of ERs in the abnormal lipid metabolism of zebrafish liver caused by MC-LR. In conclusion, the results of this study provided a mechanistic insight for the hepatotoxicity of MC-LR, and could be extrapolated to the potential impacts of MCs on human health.
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Key words:
- microcystin-LR /
- ZFL cell line /
- endoplasmic reticulum stress /
- lipid metabolism /
- TUDCA
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van Apeldoorn M E, van Egmond H P, Speijers G J A, et al. Toxins of cyanobacteria[J]. Molecular Nutrition & Food Research, 2007, 51(1): 7-60 汪洋, 李樾, 冯悦, 等. 蓝藻毒素的类型及其产毒基因[J]. 生态学杂志, 2017, 36(2): 517-523 Wang Y, Li Y, Feng Y, et al. Research progress on cyanobacterial toxins and the cyanotoxin synthetase gene[J]. Chinese Journal of Ecology, 2017, 36(2): 517-523(in Chinese)
侯杰. 微囊藻毒素-LR对斑马鱼生殖和生长发育的影响及其机制[D]. 武汉: 华中农业大学, 2017: 1-15 Hou J, Li L, Xue T, et al. Hepatic positive and negative antioxidant responses in zebrafish after intraperitoneal administration of toxic microcystin-LR[J]. Chemosphere, 2015, 120: 729-736 Lin W, Hou J, Guo H H, et al. The synergistic effects of waterborne microcystin-LR and nitrite on hepatic pathological damage, lipid peroxidation and antioxidant responses of male zebrafish[J]. Environmental Pollution, 2018, 235: 197-206 Li L, Xie P, Lei H H, et al. Renal accumulation and effects of intraperitoneal injection of extracted microcystins in omnivorous crucian carp (Carassius auratus)[J]. Toxicon: Official Journal of the International Society on Toxinology, 2013, 70: 62-69 Qiu T, Xie P, Liu Y, et al. The profound effects of microcystin on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in rat[J]. Toxicology, 2009, 257(1-2): 86-94 Hou J, Li L, Wu N, et al. Reproduction impairment and endocrine disruption in female zebrafish after long-term exposure to MC-LR: A life cycle assessment[J]. Environmental Pollution, 2016, 208(Pt B): 477-485 Yang L P, Guo H H, Kuang Y, et al. Neurotoxicity induced by combined exposure of microcystin-LR and nitrite in male zebrafish (Danio rerio): Effects of oxidant-antioxidant system and neurotransmitter system[J]. Comparative Biochemistry and Physiology Toxicology & Pharmacology, 2022, 253: 109248 Lin W, Hou J, Guo H H, et al. Dualistic immunomodulation of sub-chronic microcystin-LR exposure on the innate-immune defense system in male zebrafish[J]. Chemosphere, 2017, 183: 315-322 Nishiwaki-Matsushima R, Fujiki H, Harada K I, et al. The role of arginine in interactions of microcystins with protein phosphatases 1 and 2a[J]. Bioorganic & Medicinal Chemistry Letters, 1992, 2(7): 673-676 Mezhoud K, Praseuth D, Puiseux-Dao S, et al. Global quantitative analysis of protein expression and phosphorylation status in the liver of the medaka fish (Oryzias latipes) exposed to microcystin-LR I. Balneation study[J]. Aquatic Toxicology, 2008, 86(2): 166-175 Ding W X, Shen H M, Ong C N. Critical role of reactive oxygen species and mitochondrial permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes[J]. Hepatology, 2000, 32(3): 547-555 Prieto A I, Pichardo S, Jos Á, et al. Time-dependent oxidative stress responses after acute exposure to toxic cyanobacterial cells containing microcystins in tilapia fish (Oreochromis niloticus) under laboratory conditions[J]. Aquatic Toxicology, 2007, 84(3): 337-345 Ding W X, Ong C N. Role of oxidative stress and mitochondrial changes in cyanobacteria-induced apoptosis and hepatotoxicity[J]. FEMS Microbiology Letters, 2003, 220(1): 1-7 Žegura B, Zajc I, Lah T T, et al. Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis[J]. Toxicon, 2008, 51(4): 615-623 Amado L L, Monserrat J M. Oxidative stress generation by microcystins in aquatic animals: Why and how[J]. Environment International, 2010, 36(2): 226-235 Li L, Xie P, Chen J. In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins[J]. Toxicon, 2005, 46(5): 533-545 Zhang F, Lee J, Liang S, et al. Cyanobacteria blooms and non-alcoholic liver disease: Evidence from a county level ecological study in the United States[J]. Environmental Health: A Global Access Science Source, 2015, 14: 41 Zhao Y Y, Xue Q J, Su X M, et al. Microcystin-LR induced thyroid dysfunction and metabolic disorders in mice[J]. Toxicology, 2015, 328: 135-141 He J, Li G Y, Chen J, et al. Prolonged exposure to low-dose microcystin induces nonalcoholic steatohepatitis in mice: A systems toxicology study[J]. Archives of Toxicology, 2017, 91(1): 465-480 Duan Y F, Zeng S, Lu Z J, et al. Responses of lipid metabolism and lipidomics in the hepatopancreas of Pacific white shrimp Litopenaeus vannamei to microcystin-LR exposure[J]. Ecotoxicology and Environment Safety, 2021, 228: 113030 Zhang Z Y, Zhang X X, Wu B, et al. Comprehensive insights into microcystin-LR effects on hepatic lipid metabolism using cross-omics technologies[J]. Journal of Hazardous Materials, 2016, 315: 126-134 He J, Chen J, Wu L Y, et al. Metabolic response to oral microcystin-LR exposure in the rat by NMR-based metabonomic study[J]. Journal of Proteome Research, 2012, 11(12): 5934-5946 Alverca E, Andrade M, Dias E, et al. Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosa extract on a kidney cell line[J]. Toxicon, 2009, 54(3): 283-294 Babour A, Bicknell A A, Tourtellotte J, et al. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance[J]. Cell, 2010, 142(2): 256-269 Hotamisligil G S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease[J]. Cell, 2010, 140(6): 900-917 Cai F, Liu J, Li C R, et al. Critical role of endoplasmic reticulum stress in cognitive impairment induced by microcystin-LR[J]. International Journal of Molecular Sciences, 2015, 16(12): 28077-28086 Huang P, Zheng Y F, Xu L H. Oral administration of cyanobacterial bloom extract induced the altered expression of the PP2A, Bax, and Bcl-2 in mice[J]. Environmental Toxicology, 2008, 23(6): 688-693 Christen V, Meili N, Fent K. Microcystin-LR induces endoplasmatic reticulum stress and leads to induction of NFκB, interferon-alpha, and tumor necrosis factor-alpha[J]. Environmental Science & Technology, 2013, 47(7): 3378-3385 Colgan S M, Tang D M, Werstuck G H, et al. Endoplasmic reticulum stress causes the activation of sterol regulatory element binding protein-2[J]. The International Journal of Biochemistry & Cell Biology, 2007, 39(10): 1843-1851 Kammoun H L, Chabanon H, Hainault I, et al. GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice[J]. The Journal of Clinical Investigation, 2009, 119(5): 1201-1215 Tocher D R. Metabolism and functions of lipids and fatty acids in teleost fish[J]. Reviews in Fisheries Science, 2003, 11(2): 107-184 Deng T F, Xie J K, Ge H T, et al. Tauroursodeoxycholic acid (TUDCA) enhanced intracytoplasmic sperm injection (ICSI) embryo developmental competence by ameliorating endoplasmic reticulum (ER) stress and inhibiting apoptosis[J]. Journal of Assisted Reproduction and Genetics, 2020, 37(1): 119-126 Vettorazzi J F, Kurauti M A, Soares G M, et al. Bile acid TUDCA improves insulin clearance by increasing the expression of insulin-degrading enzyme in the liver of obese mice[J]. Scientific Reports, 2017, 7(1): 14876 Sozio M S, Liangpunsakul S, Crabb D. The role of lipid metabolism in the pathogenesis of alcoholic and nonalcoholic hepatic steatosis[J]. Seminars in Liver Disease, 2010, 30(4): 378-390 Li X Y, Chung I K, Kim J I, et al. Subchronic oral toxicity of microcystin in common carp (Cyprinus carpio L.) exposed to Microcystis under laboratory conditions[J]. Toxicon: Official Journal of the International Society on Toxinology, 2004, 44(8): 821-827 Li L, Xie P, Li S X, et al. Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix[J]. Comparative Biochemistry and Physiology Toxicology & Pharmacology, 2007, 146(3): 357-367 Lee A H, Iwakoshi N N, Glimcher L H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response[J]. Molecular and Cellular Biology, 2003, 23(21): 7448-7459 Kuo Y T, Lin T H, Chen W L, et al. Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells[J]. European Journal of Pharmacology, 2012, 692(1-3): 10-18 Horton J D. Sterol regulatory element-binding proteins: Transcriptional activators of lipid synthesis[J]. Biochemical Society Transactions, 2002, 30(Pt 6): 1091-1095 Passeri M J, Cinaroglu A, Gao C, et al. Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation[J]. Hepatology, 2009, 49(2): 443-452 Lhoták S, Sood S, Brimble E, et al. ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death[J]. American Journal of Physiology Renal Physiology, 2012, 303(2): F266-F278 Lee J S, Mendez R, Heng H H, et al. Pharmacological ER stress promotes hepatic lipogenesis and lipid droplet formation[J]. American Journal of Translational Research, 2012, 4(1): 102-113 Kim J Y, Garcia-Carbonell R, Yamachika S, et al. ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P[J]. Cell, 2018, 175(1): 133-145.e15 -
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