PROTECTIVE EFFECT OF SELENIUM ON THE OXIDATIVE DAMAGE OF LIVER CELLS INDUCED BY SODIUM NITRITE IN GRASS CARP (CTENOPHARYNGODON IDELLA)
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摘要: 以草鱼(Ctenopharyngodon idella)肝细胞(L8824)为研究对象, 设置对照组、亚硝酸钠暴露实验组、亚硒酸钠孵育实验组和亚硒酸钠孵育后亚硝酸钠暴露实验组, 探讨亚硒酸钠对不同浓度亚硝酸钠诱导L8824细胞氧化损伤及凋亡的保护作用。结果显示, 亚硝酸钠暴露能抑制L8824细胞贴壁, 导致细胞凋亡率增加。亚硝酸钠暴露引致L8824细胞的谷胱甘肽过氧化物酶(GPX)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性降低; gpx、sod和cat基因表达下调(P<0.05), DNA损伤诱导转录物3(ddit3)和bcl-2相关X蛋白(bax)基因表达上调(P<0.05)。亚硒酸钠(10 μmol/L)孵育L8824对细胞形态和凋亡率无显著影响, 但GPX、SOD和CAT活性上升, gpx、sod、cat、核因子E2相关因子2(Nrf2)和Kelch样环氧氯丙烷相关蛋白-1(keap1)基因表达上调(P<0.05)。亚硒酸钠孵育后亚硝酸钠暴露实验组, 细胞凋亡率、GPX、SOD和CAT活性较对照组无显著变化, 但B淋巴细胞瘤-2(bcl-2)基因表达显著上调(P<0.05)。研究结果表明, 给草鱼肝细胞补充硒在一定程度上能缓解亚硝酸钠暴露导致的抗氧化系统失衡, 抵抗亚硝酸钠暴露带来的氧化应激, 降低细胞凋亡率, 硒的预孵育作用能上调Nrf2/Keap1通路中的关键基因和酶, 表明硒的保护作用可能是通过介导 Nrf2/Keap1信号通路发挥作用。
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关键词:
- 草鱼肝细胞(L8824) /
- 亚硒酸钠 /
- 亚硝酸钠 /
- 抗氧化损伤 /
- 细胞凋亡 /
- Nrf2/Keap1
Abstract: The protective effects of sodium selenite (Na2SeO3) on oxidative damage and apoptosis of liver cells (L8824) induced by sodium nitrite (NaNO2) were investigated. Cells were pre-incubated by Na2SeO3 for 12h and then exposed to NaNO2 (5, 10 and 25 mg/L) for 24h. The results showed that the apoptotic rate was highly induced by NaNO2, and the activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) as well as the expressions of glutathione peroxidase (gpx), superoxide dismutase (sod) and catalase (cat) genes were reduced (P<0.05). After the cells treated by Na2SeO3 for 24h, the expressions of gpx, sod, cat and the activities of GPX, SOD, CAT increased (P<0.05), the expression of anti-apoptosis gene bcl-2 and the marker gene of Nrf2 pathway Keap1 up-regulated significantly. When the cells were pre-incubated by Na2SeO3 for 12h and then exposed to NaNO2 for 24h, the antioxidant and apoptosis indicators could be maintained at normal levels, as well as the Nrf2 and Keap1. Na2SeO3 might prevent the decrease of GPX, SOD, CAT levels and the increase of apoptotic rate. The results showed that the supplement of selenium in the liver cells of grass carp could prevent the damage of antioxidant system and apoptosis-promoting caused by NaNO2 exposure, and the Nrf2 pathway might play an important role in this process.-
Keywords:
- Liver cells (L8824) of grass carp /
- Sodium selenite /
- Sodium nitrite /
- Antioxidant damage /
- Apoptosis /
- Nrf2/Keap1
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硒(Selenium, Se)是动物的必需微量元素之一。自1957年Schwar等[1]研究发现硒是阻止大鼠(Raltus norvegicus)食饵性肝坏死的第三因子的主要组分, 首次证实动物需要硒元素。目前研究已显示硒元素的重要生物功能包括抗氧化、抗应激、加强机体免疫力和抗肿瘤等[2, 3]。Ferbal等[4]发现在鲤(Cyprinus carpio)饲料中添加适量硒元素, 能有效抵抗铅导致的肝脏和脑部的氧化损伤。Talise等[5]发现, 在饲料中添加亚硒酸钠能有效预防百草枯对斑马鱼(Danio rerio)的氧化损伤。Saffari等[6]证实在鲤幼鱼饲料中补充纳米硒能显著提升鲤生长性能和饲料利用率。而硒缺乏会损伤幼龄草鱼的免疫器官和免疫功能[7]。由于饲料中硒的利用率偏低及各种应激因子的不利影响, 养殖鱼类对硒元素的需求可能会不断提高[8]。
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Keap1-Nrf2介导的信号通路具有抗氧化应激和维持氧化还原平衡的功能, 能够诱导其下游的抗氧化相关基因(如GPX、SOD和CAT等)表达, 提高机体抗氧化能力[17, 18], Fontagne′-Dicharry等[19]发现膳食硒能通过上调Nrf2通路, 提高GPX和CAT等抗氧化酶活性, 来提高虹鳟育苗的抗氧化能力, Noha等[20]在大鼠中也有发现硒能通过上调Nrf2通路来抵抗肝脏砷中毒现象。
草鱼(Ctenopharyngodon idella)属于鲤形目鲤科雅罗鱼亚科草鱼属, 生长速度快, 单位面积产出高, 而且肉质嫩白, 含丰富的营养成分, 具有可观的经济效益和营养价值。研究发现, 给草鱼适当补充硒元素能有效提高肝脏抗氧化性, 维持组织结构完整[21]。肝脏是鱼体重要的代谢和解毒器官, 本研究以草鱼肝细胞为研究对象, 观察和检测硒孵育后肝细胞在不同浓度的亚硝酸钠暴露后, 其氧化应激及细胞凋亡的状况, 以探究硒对亚硝酸钠导致的草鱼肝细胞损伤的干预作用。
1. 材料与方法
1.1 材料
草鱼肝细胞系(L8824)购自中国典型培养物典藏中心; M199培养基、胰蛋白酶、青霉素-链霉素双抗(Penicillin Streptomycin, PS)均购自Gibco公司; 胎牛血清(FBS)购自四季青公司; 磷酸盐缓冲溶液(Phosphate buffered solution, PBS)购自HyClone公司; 细胞增殖-毒性检测试剂盒(Cell Counting Kit-8)购自同仁化学科技有限公司; 总蛋白定量(BCA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)试剂盒均购自南京建成试剂公司; Annexin V-FITC /PI凋亡试剂盒购自上海翊圣生物科技有限公司; 荧光定量试剂盒购自上海翊圣生物科技有限公司; 亚硝酸钠(NaNO2)为分析纯, 购自国药集团; 亚硒酸钠(Na2SeO3)为分析纯, 购自天津市化学试剂研究所。
1.2 草鱼肝细胞L8824的培养
L8824细胞培养于含10%胎牛血清、链霉素(100 μg/mL)和青霉素(100 IU/mL)的M199培养基中, 在28℃和5%CO2饱和湿度培养箱中培养细胞。根据细胞生长状况, 2—3d传代1次, 传代时用含EDTA的0.25%胰蛋白酶消化, 取生长状态良好处于对数生长期的细胞进行实验。
1.3 细胞活性检测
采用CCK-8法检测细胞活性, 取生长良好处于对数生长期的L8824细胞(6—8×105个/mL)接种于96孔板中, 保证每孔数量不低于1000个, 每孔100 μL, 培养12h, 待细胞在孔底铺至70%—80%, 分别加入不同浓度的亚硒酸钠和亚硝酸钠, 使亚硒酸钠的终浓度为0、0.5、1、2、5、10、15、20、25和35 μmol/L, 培养3h、6h、12h、24h、36h和48h, 亚硝酸钠终浓度为0、1、2.5、7.5、10、15、20、25、30和50 mg/L, 培养24h, 空白组为基础培养基, 每组8个复孔, 每孔加入10 μL的CCK-8, 28℃孵育2h, 置于多功能酶标仪上, 检测波长为450 nm的吸光值(Optical density, OD), 细胞存活率(%)=[实验组–空白组]/[对照组–空白组] ×100。
将实验分为4组, 对照组: 加入等量M199培养基; Se组: 10 μmol/L的Na2SeO3作用36h; NaNO2组: 不同浓度的NaNO2暴露, 使NaNO2的终浓度为5、10和25 mg/L, 暴露24h; Se+NaNO2组: 先加入终浓度为10 μmol/L的Na2SeO3预处理12h, 再加入5、10和25 mg/L的NaNO2, 暴露24h。
1.4 细胞形态学观察
取生长状态良好且处于对数生长期的L8824接种于12孔板中, 经对照组、Se组、NaNO2组和Se+NaNO2组的药物处理完毕后, 用PBS洗涤2次, 在倒置显微镜下观察细胞形态变化, 用CCD系统拍照。
1.5 氧化应激指标检测
取生长状态良好且处于对数生长期的L8824接种于12孔板中, 经对照组、Se组、NaNO2组和Se+NaNO2组的药物处理完毕后, 胰蛋白酶消化, 1000 r/min离心6min得到细胞沉淀, 用PBS洗涤2次, PBS悬浮细胞, 分装至1.5 mL离心管中, 在液氮和28℃水浴锅中反复冻融3次, –20℃冻存。BCA法测定样品蛋白浓度, 按照试剂盒说明书进行操作, 用酶标仪在562 nm处测定, 并计算样品蛋白活性; 超氧化物歧化酶(SOD): 按照SOD试剂盒操作, 用酶标仪在550 nm处测定, 并计算SOD的活性; 过氧化氢酶(CAT): 按照CAT试剂盒操作, 用酶标仪在405 nm处测定, 并计算CAT的活性; GSH-PX 活性测定: 按照GSH-PX试剂盒操作, 用酶标仪在 412 nm 处测定, 并计算 GSH-PX 的活性。
1.6 细胞凋亡检测
取生长状态良好且处于对数生长期的L8824接种于12孔板中, 经对照组、Se组、NaNO2组和Se+NaNO2组的药物处理完毕后, 不含EDTA的胰蛋白酶消化, 300×g, 4℃离心5min收集细胞, 用预冷的PBS洗涤细胞2次, 每次均需300×g, 4℃离心5min, 收集1—5×105细胞。吸弃PBS, 加入100 μL 1×Binding Buffer重悬细胞, 再加入5 μL Annexin V-FITC和10 μL PI Staining Solution, 轻轻混匀, 然后避光, 室温反应10—15min, 最后加入400 μL 1×Binding Buffer, 混匀后放置于冰上, 样品在1h内用流式细胞仪检测。
1.7 基因表达检测
取生长状态良好且处于对数生长期的L8824接种于6孔板中, 经对照组、Se组、NaNO2组和Se+NaNO2组的药物处理完毕后, 每孔加入1 mL Trizol, 用Trizol法抽提细胞RNA, 并用1%琼脂糖凝胶电泳和NanoDrop 2000分光光度计检测其浓度和质量。用Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR试剂盒进行反转录, 实际操作根据试剂盒进行。得到cDNA后, 再用Hifair® qPCR SYBR Green Master Mix (Low Rox Plus) 试剂盒进行实时荧光定量分析, β-actin作为内参基因, 设计各个基因的引物(表 1), 反应体系为: Master Mix 10 μL, 上游引物0.4 μL, 下游引物0.4 μL, cDNA1.2 μL, ddH2O 8 μL, 反应条件为: 95℃ 5min, 95℃ 10s, 55—60℃ 20s, 40个循环, 72℃ 20s, 每个样品3个重复。
表 1 qPCR引物Table 1. Primers used for qPCR引物Primer 序列Sequence (5′—3′) 序列号Accession number β-actin AACTGGGACGATATGGAGAAGA
TCACCAGAGTCCATCACGATACDQ983598.1 bcl-2 AGATGGCGTCCCAGGTAGAT
GCTGACCGTACAACTCCACAJQ713862 bax ATCTATGAGCGGGTTCGTCG
CGCAAGACGTTTATGGCTGGKT697992 chop GAATCCGAAACAGCCGAGGA
CCACACCTAGCACACCAGACKX013389 sod AGTTGCCATGTGCACTTTTCT
AGGTGCTAGTCGAGTGTTAGGGU218534.1 cat GTTTCCGTCCTTCATCCACTCT
GACCAGTTTGAAAGTGTGCGATFJ560431.2 gpx CTTTTGTCCTTGAAGTATGTCC
CTTGAGGAAGACGAAGAGAGGGDQ983598.1 Keap-1 TGAGGAGATCGGCTGCACTG
TGGCAATGGGACAAGCTGAAXM_026245355.1 Nrf2 CGCTAACGCAAACCAACACA
GGAGCTGCATGCATTCATCGKX243419.1 1.8 统计分析
实验结果用平均值±标准差(Mean±SD)表示, 数据分析由SPSS 19.0统计软件进行单因素方差分析, 然后通过 Duncan氏法进行多重比较, 显著性水平P<0.05。
2. 结果
2.1 亚硒酸钠和亚硝酸钠对L8824细胞活力的影响
结果显示, 低浓度亚硒酸钠处理后的细胞活力高于对照组, 较对照组有显著性差异(P<0.05), 活力呈先上升后下降趋势, 在10 μmol/L 3—36h阶段活力值最高(图 1), 在实验分组时, 选择10 μmol/L, 12h作为Se+NaNO2组的预处理条件。在亚硝酸钠暴露后, 细胞活力随着浓度的升高而降低(图 2)。
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图 1 不同浓度亚硒酸钠孵育不同时间对L8824细胞活力的影响实验数据以平均值±标准差(Mean±SD)表示, “*”表示与对照组相比差异显著(P<0.05), “**”表示与对照组相比差异极显著(P<0.01); 下同Figure 1. Effects of different concentrations of sodium selenite incubation at different times on the viability of L8824 cellsThe experimental data are expressed as mean±standard deviation (Mean±SD), “*” means a significant difference compared with the control group (P<0.05), and “**” means a very significant difference compared with the control group (P<0.01)![]()
图 2 不同浓度亚硝酸钠暴露24h对L8824细胞活力的影响Figure 2. Effect of sodium nitrite exposure for 24h on the viability of L8824 cells2.2 亚硒酸钠对亚硝酸钠暴露后的L8824细胞形态的影响
结果显示, 对照组L8824细胞呈梭形, 正常贴壁生长, 细胞边界清晰, 圆润, 未出现细胞空泡; Se组细胞正常贴壁生长, 形态清晰, 较对照组无明显差异; 在NaNO2组中, 5 mg/L组细胞贴壁能力开始变弱, 有小范围细胞脱落现象, 脱落细胞周围开始出现空泡; 10 mg/L组细胞脱落范围变大, 漂浮细胞增多, 细胞空泡较明显; 25 mg/L组细胞开始大面积脱落, 皱缩, 毒性作用明显; Se+NaNO2(5、10 和25 mg/L)组细胞贴壁良好, 边界清晰, 无皱缩现象, 有极少数空泡, 经硒孵育后, 在亚硝酸钠暴露下细胞仍生长良好(图 3)。
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图 3 亚硒酸钠和亚硝酸钠暴露后L8824细胞形态观察(10×)a.对照组; b. 亚硒酸钠(10 μmol/L)孵育实验组; c. 亚硝酸钠(5 mg/L)暴露实验组; d. 亚硝酸钠(10 mg/L)暴露实验组; e. 亚硝酸钠(25 mg/L)暴露实验组; f. 亚硒酸钠孵育后亚硝酸钠(5 mg/L)暴露实验组; g. 亚硒酸钠孵育后亚硝酸钠(10 mg/L)暴露实验组; h. 亚硒酸钠孵育后亚硝酸钠(25 mg/L)暴露实验组Figure 3. Morphological observation of L8824 cells after exposure to sodium selenite and sodium nitritea. Control group; b. Sodium selenite (10 μmol/L) incubation experimental group; c. Sodium nitrite (5 mg/L) exposure experimental group; d. Sodium nitrite (10 mg/L) exposure experimental group; e. Sodium nitrite (25 mg/L) exposure experimental group; f. Sodium selenite (5 mg/L) exposure experimental group after sodium selenite incubation; g. Sodium nitrite (10 mg/L) exposure experimental group; h. sodium selenite (25 mg/L) exposure experimental group after incubation2.3 亚硒酸钠对亚硝酸钠诱导L8824细胞凋亡的影响
结果表明, 不同浓度的NaNO2对L8824细胞有不同程度的促凋亡作用, 随着浓度的增高, 早期和晚期凋亡细胞逐步增多, 较对照组有显著性差异(P<0.05), 当NaNO2浓度在25 mg/L时, 细胞凋亡率可达18.69%, 在Se+NaNO2组中, 细胞凋亡水平显著下降, 平均凋亡率为11.34%。结果显示, 硒的预孵育能有效缓解亚硝酸盐暴露导致的细胞凋亡(图 4)。
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图 4 亚硒酸钠预孵育对亚硝酸钠诱导L8824细胞凋亡的影响a. Se+NaNO2(5 mg/L)组对L8824细胞凋亡的影响; b. Se+NaNO2(10 mg/L)组对L8824细胞凋亡的影响; c. Se+NaNO2(25 mg/L)组对L8824细胞凋亡的影响Figure 4. Effects of sodium selenite and sodium nitrite on apoptosis of L8824 cellsa. The effect of Se+NaNO2 (5 mg/L) group on L8824 cell apoptosis; b. The effect of Se+NaNO2 (10 mg/L) group on L8824 cell apoptosis; The effect of c Se+NaNO2 (25 mg/L) group on the apoptosis of L8824 cells2.4 亚硒酸钠对亚硝酸钠诱导L8824细胞氧化损伤的影响
结果表明, 在NaNO2浓度为5 mg/L时, SOD显著上升, GPX显著降低(P<0.05), CAT无明显变化, 在NaNO2浓度为10和25 mg/L时, GPX、SOD和CAT的活性均逐渐降低(P<0.05), 在NaNO2浓度为25 mg/L时, SOD和CAT持续降低(P<0.05), GPX无明显变化, 在亚硒酸钠预孵育12h后, 各组细胞的GPX、SOD和CAT的活性基本能维持在正常水平, 较对照组无显著性差异。结果显示, 硒的预孵育能提高细胞抗氧化能力, 抵抗NaNO2暴露带来的氧化损伤(P>0.05; 图 5)。
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图 5 亚硒酸钠预孵育对亚硝酸钠诱导L8824细胞氧化损伤的影响a. 亚硒酸钠对不同浓度亚硝酸钠诱导氧化损伤的L8824细胞内CAT的影响; b. 亚硒酸钠对不同浓度亚硝酸钠诱导氧化损伤的L8824细胞内GPX的影响; c. 亚硒酸钠对不同浓度亚硝酸钠诱导氧化损伤的L8824细胞内SOD的影响Figure 5. Effect of sodium selenite on oxidative damage of L8824 cells induced by sodium nitritea. The effect of sodium selenite on CAT in L8824 cells with oxidative damage induced by different concentrations of sodium nitrite; b. The effect of sodium selenite on GPX in L8824 cells with oxidative damage induced by different concentrations of sodium nitrite; c. The effect of sodium selenite on SOD in L8824 cells with different concentrations of sodium nitrite induced oxidative damage2.5 亚硒酸钠对亚硝酸钠暴露后的L8824细胞抗氧化及凋亡相关基因的影响
结果显示, 与对照组细胞相比, Se组细胞中的chop、bax、bcl-2和keap1 mRNA表达量差异不显著(P>0.05), Nrf2、gpx、sod和cat mRNA表达量显著上升(P<0.05), NaNO2组细胞中, chop和bax mRNA表达量显著上升(P<0.05), 当NaNO2浓度为5 mg/L时, sod mRNA表达量显著上升(P<0.05), 浓度为10和25 mg/L时, sod、gpx和cat mRNA表达量显著下降(P<0.05), 在Se+NaNO2组中, chop、bax、gpx、sod和cat mRNA表达量差异不显著(P>0.05), 其中NaNO2浓度为5和10 mg/L时, bcl-2 mRNA表达量显著上升(P<0.05)。结果表明, 细胞抗氧化能力的增强可能是通过Nrf2-Keap1通路来实现的(图 6)。
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图 6 亚硒酸钠预孵育对L8824细胞酶活及凋亡相关基因的影响Figure 6. Effect of sodium selenite preconditioning on enzyme activity and expression of apoptosis-related genes in L8824 cells3. 讨论
3.1 硒对草鱼肝细胞抗氧化功能的影响
已有研究表明, 硒可通过增强机体的抗氧化功能, 清除过氧化物, 保护生物膜和生物大分子免受氧化损伤, 降低水产动物的发病率和死亡率[22－25]。在草鱼日粮中添加0.6—1.2 mg/kg的硒(Na2SeO3)能增强草鱼机体抗氧化功能[21], 在鲤和虹鳟幼鱼日粮中分别添加0.7和0.5—1.7 mg/kg的硒元素, 也发现了同样的现象[6,19], 与本实验中硒元素浓度基本一致。在本实验中, 10 μmol/L 亚硒酸钠的预孵育能有效提高肝细胞SOD、CAT和GPX的活性(P<0.05)。GPX的活性中心是硒半胱氨酸, 是衡量机体硒水平的一项重要生化指标[19], 硒孵育后能显著提高GPX的活性, 表明10 μmol/L 的亚硒酸钠作用12h后, 硒元素已进入胞内并发挥作用。
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而在硒预孵育12h后再用亚硝酸钠暴露, 肝细胞GPX、SOD和CAT均能基本维持在正常水平(P>0.05)。可能是细胞经硒预孵育后, 胞内抗氧化酶活性升高, 当亚硝酸盐暴露产生大量自由基后, 这些抗氧化酶能及时有效的清除自由基, 使细胞维持正常的生理机制, 表明此时细胞抗氧化能力增强, 并能抵抗一定浓度亚硝酸盐暴露导致的氧化损伤。
3.2 硒对抗氧化基因和Nrf2/Keap1通路基因表达的影响
Nrf2/Keap1介导的信号通路具有抗氧化应激和维持氧化还原平衡的功能, 非应激状态时, Keap1在细胞质和Nrf2相结合, 促进Nrf2蛋白泛素化和蛋白酶体降解, 维持Nrf2在低水平状态, 应激状态时, Nrf2的泛素化和蛋白酶体降解被阻断, Nrf2转移至细胞核内, 诱导细胞保护相关基因的表达[31, 32]。本实验结果显示, 在补硒组中, keap1、sod、cat和gpx mRNA表达量显著上升(P<0.05), 在硒预孵育后, 再经亚硝酸盐暴露, 细胞中gpx、sod和cat mRNA表达量也能维持在正常水平, 较正常组无显著性差异(P>0.05)。Nrf2/Keap1通路能够诱导与ROS清除相关的基因表达, 如SOD和CAT等, 减少细胞内ROS含量[33]。在鸡的肝细胞中也发现硒能通过激活Nrf2/Keap1通路来抵抗重金属镉诱导的细胞凋亡[34]。在本实验中细胞可能通过上调Nrf2/Keap1通路, 诱导gpx、sod和cat基因表达, 提高抗氧化酶活性, 来增强细胞抗氧化能力, 并能在一定程度上抵抗亚硝酸盐暴露带来的抗氧化酶活性降低, 维持细胞抗氧化系统平衡。
3.3 硒对草鱼肝细胞凋亡的作用
细胞凋亡是指为了维持内环境的稳定, 由凋亡相关基因调控的细胞自主和有序的死亡, 又称程序性细胞死亡, 是细胞为了更好的适应环境而采取的一种主动的方式[35]。长时间或高浓度的亚硝酸盐暴露能诱导细胞凋亡[36]。本实验从细胞形态上看, 随着亚硝酸盐的浓度升高, 细胞贴壁能力下降, 成片脱落, 而硒预孵育12h后的细胞生长良好, 形态正常, 基本接近正常细胞组, 表明硒的预孵育有利于细胞在亚硝酸盐的暴露中维持正常生长形态, 维持贴壁能力。在FITC和PI双荧光染色后, 观察细胞凋亡特点, 由亚硝酸盐引起的细胞凋亡具有浓度依赖性, 且主要变化集中在凋亡晚期, 较正常组及补硒组凋亡细胞显著增加(P<0.05), 相反的是, 硒的预孵育能显著降低L8824的凋亡率, 并使其恢复至正常水平。
硒能有效降低细胞中bax/bcl-2比值[37], 而长时间高浓度亚硝酸盐的暴露会诱导细胞凋亡[38], 在基因表达的结果中, 亚硝酸盐暴露能上调L8824细胞中chop和bax的表达, bax/bcl-2比值显著上升(P<0.05), 而在硒预孵育12h后的细胞中, bcl-2上调, bax/bcl-2比值显著下降(P<0.05), 但在25 mg/L时, bcl-2下降到无显著性差异(P>0.05), 在亚硝酸盐5 mg/L组下调了chop和bax的表达。从流式和凋亡基因结果分析, 随着亚硝酸盐暴露浓度升高, 硒的保护作用可能会逐渐失效。亚硝酸盐能启动凋亡信号通路, 引发了L8824的凋亡, 而亚硒酸钠的预孵育能上调抗凋亡基因表达, 维持促凋亡基因表达在正常水平, 能有效缓解亚硝酸盐带来的细胞损伤和凋亡。
综上所述, 给草鱼肝细胞适当补充硒元素能在一定程度和范围内有效缓解亚硝酸盐暴露带来的抗氧化系统失衡, 其中Keap1-Nrf2介导的信号通路可能发挥了重要作用, 同时可以降低细胞凋亡率, 维持细胞正常生长, 但随着亚硝酸盐浓度的增高, 硒的保护作用可能会逐渐失效。
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图 1 不同浓度亚硒酸钠孵育不同时间对L8824细胞活力的影响
实验数据以平均值±标准差(Mean±SD)表示, “*”表示与对照组相比差异显著(P<0.05), “**”表示与对照组相比差异极显著(P<0.01); 下同
Figure 1. Effects of different concentrations of sodium selenite incubation at different times on the viability of L8824 cells
The experimental data are expressed as mean±standard deviation (Mean±SD), “*” means a significant difference compared with the control group (P<0.05), and “**” means a very significant difference compared with the control group (P<0.01)
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图 2 不同浓度亚硝酸钠暴露24h对L8824细胞活力的影响
Figure 2. Effect of sodium nitrite exposure for 24h on the viability of L8824 cells
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图 3 亚硒酸钠和亚硝酸钠暴露后L8824细胞形态观察(10×)
a.对照组; b. 亚硒酸钠(10 μmol/L)孵育实验组; c. 亚硝酸钠(5 mg/L)暴露实验组; d. 亚硝酸钠(10 mg/L)暴露实验组; e. 亚硝酸钠(25 mg/L)暴露实验组; f. 亚硒酸钠孵育后亚硝酸钠(5 mg/L)暴露实验组; g. 亚硒酸钠孵育后亚硝酸钠(10 mg/L)暴露实验组; h. 亚硒酸钠孵育后亚硝酸钠(25 mg/L)暴露实验组
Figure 3. Morphological observation of L8824 cells after exposure to sodium selenite and sodium nitrite
a. Control group; b. Sodium selenite (10 μmol/L) incubation experimental group; c. Sodium nitrite (5 mg/L) exposure experimental group; d. Sodium nitrite (10 mg/L) exposure experimental group; e. Sodium nitrite (25 mg/L) exposure experimental group; f. Sodium selenite (5 mg/L) exposure experimental group after sodium selenite incubation; g. Sodium nitrite (10 mg/L) exposure experimental group; h. sodium selenite (25 mg/L) exposure experimental group after incubation
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图 4 亚硒酸钠预孵育对亚硝酸钠诱导L8824细胞凋亡的影响
a. Se+NaNO2(5 mg/L)组对L8824细胞凋亡的影响; b. Se+NaNO2(10 mg/L)组对L8824细胞凋亡的影响; c. Se+NaNO2(25 mg/L)组对L8824细胞凋亡的影响
Figure 4. Effects of sodium selenite and sodium nitrite on apoptosis of L8824 cells
a. The effect of Se+NaNO2 (5 mg/L) group on L8824 cell apoptosis; b. The effect of Se+NaNO2 (10 mg/L) group on L8824 cell apoptosis; The effect of c Se+NaNO2 (25 mg/L) group on the apoptosis of L8824 cells
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图 5 亚硒酸钠预孵育对亚硝酸钠诱导L8824细胞氧化损伤的影响
a. 亚硒酸钠对不同浓度亚硝酸钠诱导氧化损伤的L8824细胞内CAT的影响; b. 亚硒酸钠对不同浓度亚硝酸钠诱导氧化损伤的L8824细胞内GPX的影响; c. 亚硒酸钠对不同浓度亚硝酸钠诱导氧化损伤的L8824细胞内SOD的影响
Figure 5. Effect of sodium selenite on oxidative damage of L8824 cells induced by sodium nitrite
a. The effect of sodium selenite on CAT in L8824 cells with oxidative damage induced by different concentrations of sodium nitrite; b. The effect of sodium selenite on GPX in L8824 cells with oxidative damage induced by different concentrations of sodium nitrite; c. The effect of sodium selenite on SOD in L8824 cells with different concentrations of sodium nitrite induced oxidative damage
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图 6 亚硒酸钠预孵育对L8824细胞酶活及凋亡相关基因的影响
Figure 6. Effect of sodium selenite preconditioning on enzyme activity and expression of apoptosis-related genes in L8824 cells
表 1 qPCR引物
Table 1 Primers used for qPCR
引物Primer 序列Sequence (5′—3′) 序列号Accession number β-actin AACTGGGACGATATGGAGAAGA
TCACCAGAGTCCATCACGATACDQ983598.1 bcl-2 AGATGGCGTCCCAGGTAGAT
GCTGACCGTACAACTCCACAJQ713862 bax ATCTATGAGCGGGTTCGTCG
CGCAAGACGTTTATGGCTGGKT697992 chop GAATCCGAAACAGCCGAGGA
CCACACCTAGCACACCAGACKX013389 sod AGTTGCCATGTGCACTTTTCT
AGGTGCTAGTCGAGTGTTAGGGU218534.1 cat GTTTCCGTCCTTCATCCACTCT
GACCAGTTTGAAAGTGTGCGATFJ560431.2 gpx CTTTTGTCCTTGAAGTATGTCC
CTTGAGGAAGACGAAGAGAGGGDQ983598.1 Keap-1 TGAGGAGATCGGCTGCACTG
TGGCAATGGGACAAGCTGAAXM_026245355.1 Nrf2 CGCTAACGCAAACCAACACA
GGAGCTGCATGCATTCATCGKX243419.1 
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[1] Combs G F, Gombs S B. The role of selenium in nutrition [J]. The Quarterly Review of Biology, 1988, 63(1): 15-40.
[2] Das B, Ghosh T, Gangopadhyay S. Assessment of ergonomic and occupational health-related problems among female prawn seed collectors of Sunderbans, West Bengal, India [J]. International Journal of Occupational Safety and Ergonomics, 2012, 18(4): 531-540. doi: 10.1080/10803548.2012.11076949
[3] Zeng H, Cao J J, Combs G F Jr. Selenium in bone health: roles in antioxidant protection and cell proliferation [J]. Nutrients, 2013, 5(1): 97. doi: 10.3390/nu5010097
[4] Özkan-Yılmaz F, Özlüer-Hunt A, Gül Gündüz S, et al. Effects of dietary selenium of organic form against lead toxicity on the antioxidant system in Cyprinus carpio [J]. Fish Physiology and Biochemistry, 2014, 40(2): 355-363. doi: 10.1007/s10695-013-9848-9
[5] Müller T E, Nunes M E, Menezes C C, et al. Sodium selenite prevents paraquat-induced neurotoxicity in zebrafish [J]. Molecular Neurobiology, 2018, 55(5): 1928-1941.
[6] Saffari S, Keyvanshokooh S, Zakeri M, et al. Effects of dietary organic, inorganic, and nanoparticulate selenium sources on growth, hemato-immunological, and serum biochemical parameters of common carp (Cyprinus carpio) [J]. Fish Physiology and Biochemistry, 2018, 44(4): 1087-1097. doi: 10.1007/s10695-018-0496-y
[7] Zheng L, Feng L, Jiang W D, et al. Selenium deficiency impaired immune function of the immune organs in young grass carp (Ctenopharyngodon idella) [J]. Fish & Shellfish Immunology, 2018(77): 53-70.
[8] Ling Q, Hong F. Antioxidative role of cerium against the toxicity of lead in the liver of silver crucian carp [J]. Fish Physiology and Biochemistry, 2010, 36(3): 367-376. doi: 10.1007/s10695-008-9301-7
[9] Larsen E H, Willumsen N J, Christoffffersen B C. Role of proton pump of mitochondria-rich cells for active transport of chloride ions in toad skin epithelium [J]. The Journal of Physiology, 1992, 450(1): 203-216. doi: 10.1113/jphysiol.1992.sp019124
[10] Onken H, Putzenlechner M. AV-ATPase drives active, electrogenic and Na+ independent Cl– absorption across the gills of Eriocheir sinensis [J]. Journal of Experimental Biology, 1995, 198(3): 767-774. doi: 10.1242/jeb.198.3.767
[11] Jensen L, Willumsen N, Larsen E. Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater [J]. Journal of Comparative Physiology, 2002, 172(6): 503-511. doi: 10.1007/s00360-002-0276-x
[12] 高明辉, 马立保, 葛立安, 等. 亚硝酸盐在水生动物体内的吸收机制及蓄积的影响因素 [J]. 南方水产, 2008, 4(4): 73-80. Gao M H, Ma L B, Ge L A, et al. Nitrite uptake mechanism and the influencing factors of accumulation in aquatic animals [J]. South China Fisheries Science, 2008, 4(4): 73-80.
[13] Lin Y, Miao L H, Pan W J, et al. Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis [J]. Fish & Shellfish Immunology, 2018(76): 126-132.
[14] Park I S, Lee J, Hur J W, et al. Acute toxicity and sublethal effects of nitrite on selected hematological parameters and tissues in dark-banded rockfish, Sebastes inermis [J]. World Aquaculture, 2007, 38(2): 188-199. doi: 10.1111/j.1749-7345.2007.00088.x
[15] Sun S M, Ge X P, Zhu J, et al. Identification and mRNA expression of antioxidant enzyme genes associated with the oxidative stress response in the Wuchang bream (Megalobrama amblycephala Yih) in response to acute nitrite exposure [J]. Comparative Biochemistry and Physiology, Part C, 2014(159): 69-77.
[16] Liu M, Sun T. Effects of D-galactose, sodium nitrite and alchlor on cognitive competence in mice [J]. Chinese Journal of Integrative Medicine on Cardio-/Cerebrovascular Disease, 2017, 5(3): 34-35.
[17] Zhao C Y, Wang X L, Peng Y K. Role of Nrf2 in neurodegenerative diseases and recent progress of its activators [J]. Acta Pharmaceutica Sinica, 2015, 50(4): 375-384.
[18] Bell J G, Pirie B J, Adron J W, et al. Some effects of selenium deficiency on glutathione peroxidase (EC 1.11. 1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri) [J]. British Journal of Nutrition, 1986, 55(2): 305-311. doi: 10.1079/BJN19860038
[19] Fontagne′-Dicharry S, Godin S, Liu H, et al. Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry [J]. British Journal of Nutrition, 2015, 113(12): 1876-1887. doi: 10.1017/S0007114515001300
[20] Shafik N M, El Batsh M M. Protective effects of combined selenium and Punica granatum treatment on some inflammatory and oxidative stress markers in arsenic-induced hepatotoxicity in rats [J]. Biological Trace Element Research, 2016, 169(1): 121-128. doi: 10.1007/s12011-015-0397-1
[21] 苏传福, 罗莉, 李芹, 等. 硒对草鱼抗氧化功能及组织结构的影响 [J]. 西南师范大学学报: 自然科学版, 2008, 33(5): 69-75. Su C F, Luo L, Li Q, et al. Effects of dietary selenium on the anti-oxidation capability and histological structure of grass carp, Cenopharyngodon idellus [J]. Journal of Southwest China Normal University (Natural Science Edition)
, 2008, 33(5): 69-75. [22] 李杨, 陈永生, 吉红, 等. 硒在水产养殖中的应用研究进展 [J]. 安康学院学报, 2014, 26(3): 82-85. doi: 10.3969/j.issn.1674-0092.2014.03.022 Li Y, Chen Y S, Ji H, et al. Status and perspective of selenium in the application study of aquaculture [J]. Journal of Ankang Teachers College, 2014, 26(3): 82-85. doi: 10.3969/j.issn.1674-0092.2014.03.022
[23] 金明昌, 汪开毓. 不同硒水平对幼鲤生产性能和免疫功能的影响 [J]. 营养饲料, 2008, 44(5): 32-36. Jin M C, Wang K Y. Effect of Selenium on performance and immune function of juvenile carp [J]. Nutrition and Feed Stuffs, 2008, 44(5): 32-36.
[24] 崔恒阳, 张电光, 凌仕诚, 等. 饲料硒含量对黄颡鱼肠系膜脂肪组织中脂类代谢和miRNAs表达水平的影响 [J]. 水生生物学报, 2020, 44(4): 685-692. Cui H Y, Zhang D G, Ling S C, et al. Effects of dietary selenium on lipid metabolism and miRNAs expression in mesenteric adipose tissue of yellow catfish Pelteobagrus fulvidraco [J]. Acta Hydrobiologica Sinica, 2020, 44(4): 685-692.
[25] Behne D, Alber D, Kyriakopoulos A. Long-term selenium supplementation of humans: selenium status and relationships between selenium concentrations in skeletal muscle and indicator materials [J]. Journal of Trace Elements in Medicine and Biology, 2010, 24(2): 99-105. doi: 10.1016/j.jtemb.2009.12.001
[26] Steinbrenner H, Sies H. Selenium homeostasis and antioxidant selenoproteins in brain: implications for disorders in the central nervous system [J]. Archives of Biochemistry and Biophysics, 2013, 536(2): 152-157. doi: 10.1016/j.abb.2013.02.021
[27] Huertas M, Gisbert E, Rodríguez A, et al. Acute exposure of Siberian sturgeon (Acipenser baeri, Brandt) yearlings to nitrite: median-lethal concentration (LC(50)) determination, haematological changes and nitrite accumulation in selected tissues [J]. Aquatic Toxicology, 2002, 57(4): 257-266. doi: 10.1016/S0166-445X(01)00207-7
[28] Sun S, Ge X, Zhu J, et al. Identification and mRNA expression of antioxidant enzyme genes associated with the oxidative stress response in the Wuchang bream (Megalobrama amblycephala Yih) in response to acute nitrite exposure [J]. Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology, 2014(159): 69-77.
[29] Cao L A, Huang W, Liu J H, et al. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure [J]. Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology, 2010, 151(3): 386-392.
[30] Krych-Madej J, Gebicka L. Interactions of nitrite with catalase: Enzyme activity and reaction kinetics studies [J]. Journal of Inorganic Biochemistry, 2017(171): 10-17.
[31] Dinkova-Kostova A T, Kostov R V, Canning P Keap1. the cysteine-based mammalian intracellular sensor for electrophiles and oxidants [J]. Archives of Biochemistry and Biophysics, 2017(617): 84-93.
[32] Stępkowski T M, Kruszewski M K. Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis [J]. Free Radical Biology and Medicine, 2011, 50(9): 1186-1195. doi: 10.1016/j.freeradbiomed.2011.01.033
[33] Li X H, Tang N F, Li Y Q, et al. Cytoprotective effect of p62/Nrf2 signaling pathway [J]. Acta Pharmaceutica Sinica, 2018(9): 57-59.
[34] Zhang C, Lin J, Ge J, et al. Selenium triggers Nrf2-mediated protection against cadmium-induced chicken hepatocyte autophagy and apoptosis [J]. Toxicology in Vitro, 2017(44): 349-356.
[35] Susan Elmore. Apoptosis: a review of programmed cell death [J]. Toxicology and Pathology, 2007, 35(4): 495-516. doi: 10.1080/01926230701320337
[36] Jia R, Han C, Lei J L, et al. Effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus) [J]. Aquatic Toxicology, 2015(169): 1-9.
[37] 王苏苏, 胡昕迪, 顾璇, 等. 基于Bax/Bcl-2比值探讨硒对镉致睾丸间质细胞凋亡的保护作用 [J]. 营养学报, 2018, 40(6): 616-618. doi: 10.3969/j.issn.0512-7955.2018.06.019 Wang S S, Hu X D, Gu X, et al. Protection of selenium on TM3 cell apoptosis induced by cadmium based on the Bax/Bcl-2 ratio [J]. Acta Nutrimenta Sinica, 2018, 40(6): 616-618. doi: 10.3969/j.issn.0512-7955.2018.06.019
[38] Elsherbiny N M, Maysarah N M, El-Sherbiny M, et al. Renal protective effects of thymoquinone against sodium nitrite-induced chronic toxicity in rats: Impact on inflammation and apoptosis [J]. Life Sciences, 2017(180): 1-8.
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