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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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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