连二亚硫酸钠诱导大鼠肝细胞氧化应激模型的建立

doi:10.16431/j.cnki.1671-7236.2023.06.005

摘要/Abstract

摘要： 【目的】探讨连二亚硫酸钠（Na₂S₂O₄）诱导大鼠肝细胞（BRL-3A）氧化应激模型的制备方法，以期建立一种稳定的BRL-3A细胞氧化应激模型。【方法】利用含Na₂S₂O₄的培养基造成细胞缺氧，更换正常培养基对BRL-3A细胞进行复氧培养，造成细胞氧化应激损伤。将BRL-3A细胞分为5组，空白对照组（0 mmol/L Na₂S₂O₄）在细胞培养箱中正常培养，试验组给予含不同浓度（0.1、1、5、10 mmol/L） Na₂S₂O₄的培养基进行缺氧培养4 h、复氧培养2 h的处理后，采用CCK-8法检测细胞存活率。采用荧光探针DCFH-DA法检测细胞中活性氧（ROS）的释放，利用试剂盒检测细胞培养液中乳酸脱氢酶（LDH）活性，以及BRL-3A细胞中丙二醛（MDA）、谷胱甘肽（GSH）含量及超氧化物歧化酶（SOD）活性。【结果】BRL-3A细胞经过缺氧/复氧处理后，与空白对照组相比，随着Na₂S₂O₄浓度的增加，试验组BRL-3A细胞存活率逐渐下降（P<0.01），ROS释放量逐渐增高（P<0.01），细胞培养液中LDH活性及细胞内MDA含量呈浓度依赖性增高（P<0.01），细胞内GSH含量及SOD活性呈浓度依赖性降低（P<0.05；P<0.01）。【结论】利用含5 mmol/L Na₂S₂O₄的培养基对BRL-3A细胞进行缺氧培养4 h、复氧培养2 h的处理后，可以建立稳定的BRL-3A细胞氧化应激模型。

关键词: BRL-3A细胞; 连二亚硫酸钠(Na₂S₂O₄); 氧化应激

Abstract: 【Objective】 This study was aimed to explore the preparation method of rat hepatocyte (BRL-3A) oxidative stress model induced by sodium hydrosulfite (Na₂S₂O₄), in order to establish a stable oxidative stress model of BRL-3A cells.【Method】 The medium containing Na₂S₂O₄ was used to cause hypoxia of cells, and the normal medium was replaced to reoxygenate BRL-3A cells, thus causing oxidative stress damage of cells.BRL-3A cells were divided into 5 groups, and the control group was cultured normally in a cell incubator (0 mmol/L Na₂S₂O₄), the experiment group was treated with different concentrations (0.1, 1, 5 and 10 mmol/L) of Na₂S₂O₄ for 4 h of hypoxia and 2 h of reoxygenation, then the cell viabiliaty rate was detected by CCK-8 assay.The fluorescent probe DCFH-DA method was used to detect the release of reactive oxygen species (ROS) in cells.The activity of lactate dehydrogenase (LDH) in cell culture medium, the contents of malondialdehyde (MDA) and glutathione (GSH), and the activity of superoxide dismutase (SOD) in BRL-3A cells were detected by kit.【Result】 After hypoxia/reoxygenation treament, compared with blank control group, with the increase of Na₂S₂O₄ concentration, the viability rate of BRL-3A cells was gradually decreased (P<0.01), the release of ROS was gradually increased (P<0.01), the activity of LDH and the content of MDA were increased in a concentration dependent manner (P<0.01), the content of GSH and the activity of SOD were decreased in a concentration dependent manner (P<0.05 or P<0.01).【Conclusion】 This study showed that a stable oxidative stress model of BRL-3A cells could be established which was induced by 5 mmol/L Na₂S₂O₄ for hypoxia of 4 h and reoxygenation of 2 h.

Key words: BRL-3A cells; sodium hydrosulfite (Na₂S₂O₄); oxidative stress

中图分类号:

S852.21

孙晨, 戈胜, 宁夏青, 王佳琦, 张暖暖, 王晓然, 张士霞. 连二亚硫酸钠诱导大鼠肝细胞氧化应激模型的建立[J]. 中国畜牧兽医, 2023, 50(6): 2217-2223.

SUN Chen, GE Sheng, NING Xiaqing, WANG Jiaqi, ZHANG Nuannuan, WANG Xiaoran, ZHANG Shixia. Oxidative Stress Model of BRL-3A Cells Induced by Sodium Hydrosulfite[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2217-2223.

参考文献

[1] ALKADI H.A review on free radicals and antioxidants[J].Infectious Disorders-Drug Targets, 2020, 20(1):16-26.
[2] RUART M, CHAVARRIA L, CAMPRECIÓS G, et al.Impaired endothelial autophagy promotes liver fibrosis by aggravating the oxidative stress response during acute liver injury[J].Journal of Hepatology, 2019, 70(3):458-469.
[3] GETZIN T, GUELER F, HARTLEBEN B, et al.Gd-EOB-DTPA-enhanced MRI for quantitative assessment of liver organ damage after partial hepatic ischaemia reperfusion injury:Correlation with histology and serum biomarkers of liver cell injury[J].European Radiology, 2018, 28(10):4455-4464.
[4] LI L, ZHAO J, GE C, et al.Dehydroepiandrosterone rehabilitate BRL-3A cells oxidative stress damage induced by hydrogen peroxide[J].Journal of Cellular Physiology, 2018, 233(8):6262-6272.
[5] DIESEN D L, KUO P C.Nitric oxide and redox regulation in the liver:Part Ⅰ.General considerations and redox biology in hepatitis[J].The Journal of Surgical Research, 2010, 162(1):95-109.
[6] LAI K P, CHEUNG A H Y, TSE W K F.Deubiquitinase Usp18 prevents cellular apoptosis from oxidative stress in liver cells[J].Cell Biology International, 2017, 41(8):914-921.
[7] KIM S H, YOON D, LEE Y H, et al.Transformation of liver cells by 3-methylcholanthrene potentiates oxidative stress via the downregulation of glutathione synthesis[J].International Journal of Molecular Medicine, 2017, 40(6):2011-2017.
[8] LIN S, LIN J M, ZHANG L, et al.Shexiang tongxin dropping pill protects against Na₂S₂O₄-induced hypoxia-reoxygenation injury in H9C2 cells[J].Chinese Journal of Integrative Medicine, 2019, 25(6):439-445.
[9] ABUDARA V, JIANG R G, EYZAGUIRRE C.Behavior of junction channels between rat glomus cells during normoxia and hypoxia[J].Journal of Neurophysiology, 2002, 88(2):639-649.
[10] LUO L, LV L, LU Y, et al.Effects of hypoxia on progranulin expression in HT22 mouse hippocampal cells[J].Molecular Medicine Reports, 2014, 9(5):1675-1680.
[11] YU H N, MA X L, YANG J G, et al.Comparison of effects of epigallocatechin-3-gallate on hypoxia injury to human umbilical vein, RF/6A, and ECV304 cells induced by Na₂S₂O₄[J].Journal of Endothelial Cell Research, 2007, 14(4-5):227-231.
[12] 姚瑞芹, 王兴启, 刘轩, 等.新生大鼠少突胶质前体细胞低糖缺氧损伤模型的建立[J].神经解剖学杂志, 2011, 27(2):135-139. YAO R Q, WANG X Q, LIU X, et al.The establishment of oligodendrocyte precursor cells low glucose and oxygen deprivation model in neonatal rat[J].Chinese Journal of Neuroanatomy, 2011, 27(2):135-139.(in Chinese)
[13] CHENG M L, NAKIB D, PERCIANI C T, et al.The immune niche of the liver[J].Clinical Science, 2021, 135(20):2445-2466.
[14] 刘飞祥, 林子璇, 张怀亮, 等.人参抗疲劳的作用机制和潜在靶点研究[J].中国中药杂志, 2019, 44(24):5479-5487. LIU F X, LIN Z X, ZHANG H L, et al.Analysis of anti-fatigue mechanism and potential targets of ginseng[J].China Journal of Chinese Materia Medica, 2019, 44(24):5479-5487.(in Chinese)
[15] MARJOT T, RAY D W, WILLIAMS F R, et al.Sleep and liver disease:A bidirectional relationship[J].The Lancet Gastroenterology & Hepatology, 2021, 6(10):850-863.
[16] SEEN S.Chronic liver disease and oxidative stress-A narrative review[J].Expert Review of Gastroenterology & Hepatology, 2021, 15(9):1021-1035.
[17] ABDUL K S M, FAIZ N, JOVANOVIĆ A, et al.Isosteviol protects H9C2 cells against hypoxia-reoxygenation by activating ERK1/2[J].Cardiovascular & Hematological Disorders Drug Targets, 2021, 21(1):73-77.
[18] LIU X J, LV Y F, CUI W Z, et al.Icariin inhibits hypoxia/reoxygenation-induced ferroptosis of cardiomyocytes via regulation of the Nrf2/HO-1 signaling pathway[J].FEBS Open Bio, 2021, 11(11):2966-2976.
[19] ZHENG H, YU Z, WANG H, et al.miR-125b-5p ameliorates hypoxia/reoxygenation-induced endothelial cell dysfunction and attenuates reduced uterine perfusion pressure-induced hypertension in pregnant rats via targeting BMF[J].Hypertension in Pregnancy, 2022, 41(2):79-88.
[20] WU Z, WANG H, FANG S, et al.Roles of endoplasmic reticulum stress and autophagy on H₂O₂ induced oxidative stress injury in HepG2 cells[J].Molecular Medicine Reports, 2018, 18(5):4163-4174.
[21] LI L, LIN M, ZHANG L, et al.Cyclic helix B peptide protects HK-2 cells from oxidative stress by inhibiting ER stress and activating Nrf2 signalling and autophagy[J].Molecular Medicine Reports, 2017, 16(6):8055-8061.
[22] ZHAO H, WANG R, YE M, et al.Genipin protects against H₂O₂-induced oxidative damage in retinal pigment epithelial cells by promoting Nrf2 signaling[J].International Journal of Molecular Medicine, 2019, 43(2):936-944.
[23] WANG C, LIAO Y, WANG S, et al.Cytoprotective effects of diosmetin against hydrogen peroxide-induced L02 cell oxidative damage via activation of the Nrf2-ARE signaling pathway[J].Molecular Medicine Reports, 2018, 17(5):7331-7338.
[24] XIE L, WU Y, FAN Z, et al.Astragalus polysaccharide protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injury:The role of PI3K/Akt, Bax/Bcl-2 and caspase-3[J].Molecular Medicine Reports, 2016, 14(1):904-910.
[25] NAN B, YANG C, LI L, et al.Allicin alleviated acrylamide-induced NLRP3 inflammasome activation via oxidative stress and endoplasmic reticulum stress in Kupffer cells and SD rats liver[J].Food and Chemical Toxicology, 2021, 148:111937.
[26] ZHAO X C, ZHANG L, YU H X, et al.Curcumin protects mouse neuroblastoma Neuro-2A cells against hydrogen-peroxide-induced oxidative stress[J].Food Chemistry, 2011, 129(2):387-394.
[27] LI L, SHA Z, WANG Y, et al.Pre-treatment with a combination of Shenmai and Danshen injection protects cardiomyocytes against hypoxia/reoxygenation- and H₂O₂-induced injury by inhibiting mitochondrial permeability transition pore opening[J].Experimental and Therapeutic Medicine, 2019, 17(6):4643-4652.
[28] CESARATTO L, VASCOTTO C, CALLIGARIS S, et al.The importance of redox state in liver damage[J].Annals of Hepatology, 2004, 3(3):86-92.
[29] HAN J Y, CHO S S, YANG J H, et al.The chalcone compound isosalipurposide (ISPP) exerts a cytoprotective effect against oxidative injury via Nrf2 activation[J].Toxicology and Applied Pharmacology, 2015, 287(1):77-85.
[30] DAS S, MARAS J S, HUSSAIN M S, et al.Hyperoxidized albumin modulates neutrophils to induce oxidative stress and inflammation in severe alcoholic hepatitis[J].Hepatology, 2017, 65(2):631-646.
[31] MOSTAFA D G, KHALEEL E F, BADI R M, et al.Rutin hydrate inhibits apoptosis in the brains of cadmium chloride-treated rats via preserving the mitochondrial integrity and inhibiting endoplasmic reticulum stress[J].Neurological Research, 2019, 41(7):594-608.
[32] ZHANG Z K, ZHOU Y, CAO J, et al.Rosmarinic acid ameliorates septic-associated mortality and lung injury in mice via GRP78/IRE1α/JNK pathway[J].Journal of Pharmacy and Pharmacology, 2021, 73(7):916-921.
[33] PAN X, YAN D, WANG D, et al.Mitochondrion-mediated apoptosis induced by acrylamide is regulated by a balance between Nrf2 antioxidant and MAPK signaling pathways in PC12 cells[J].Molecular Neurobiology, 2017, 54(6):4781-4794.