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郁浓, 石瑶瑶, 叶元土. 大豆和豆粕水提物致草鱼原代肝细胞损伤作用的研究[J]. 水生生物学报, 2021, 45(4): 709-721. DOI: 10.7541/2021.2020.025
引用本文: 郁浓, 石瑶瑶, 叶元土. 大豆和豆粕水提物致草鱼原代肝细胞损伤作用的研究[J]. 水生生物学报, 2021, 45(4): 709-721. DOI: 10.7541/2021.2020.025
YU Nong, SHI Yao-Yao, YE Yuan-Tu. OXIDATIVE DAMAGE OF SOYBEAN AQUEOUS EXTRACT AND SOYBEAN MEAL AQUEOUS EXTRACT TO GRASS CARP (CTENOPHARYNGODON IDELLUS) PRIMARY HEPATOCYTES[J]. ACTA HYDROBIOLOGICA SINICA, 2021, 45(4): 709-721. DOI: 10.7541/2021.2020.025
Citation: YU Nong, SHI Yao-Yao, YE Yuan-Tu. OXIDATIVE DAMAGE OF SOYBEAN AQUEOUS EXTRACT AND SOYBEAN MEAL AQUEOUS EXTRACT TO GRASS CARP (CTENOPHARYNGODON IDELLUS) PRIMARY HEPATOCYTES[J]. ACTA HYDROBIOLOGICA SINICA, 2021, 45(4): 709-721. DOI: 10.7541/2021.2020.025

大豆和豆粕水提物致草鱼原代肝细胞损伤作用的研究

OXIDATIVE DAMAGE OF SOYBEAN AQUEOUS EXTRACT AND SOYBEAN MEAL AQUEOUS EXTRACT TO GRASS CARP (CTENOPHARYNGODON IDELLUS) PRIMARY HEPATOCYTES

  • 摘要: 为了探讨大豆和豆粕对肝细胞是否具有损伤作用及其作用机制, 以大豆水提物(Soybean aqueous extract, SAE)和豆粕水提物(Soybean meal aqueous extract, SMAE)为实验材料, 以草鱼(Ctenopharyngodon idella)离体培养的原代肝细胞为试验对象, SAE和SMAE分别以终浓度为0、0.5、5.0和10.0 mg/mL加入到细胞培养液中。采用CCK-8法检测细胞活力, 电子显微镜观察细胞超微结构, Hoechst 33285染色观察细胞核形态, 检测细胞培养液中乳酸脱氢酶(LDH)和丙二醛(MDA)的含量, 检测细胞内超氧化物歧化酶(SOD)和还原性谷胱甘肽(GSH)的活性, 流式细胞仪检测细胞凋亡率、线粒体膜电位(MMP)及活性氧(ROS)。利用转录组技术分析对照组、SAE组(5.0 mg/mL)和SMAE组(5.0 mg/mL)肝细胞差异表达基因(Differentially expressed genes, DEGs)及其富集的相关通路。结果表明, 随着水提物浓度的升高, 肝细胞活力逐渐下降, 有明显的剂量效应关系(P<0.05)。在相同浓度下, SAE组细胞相对活力小于SMAE组。肝细胞超微结构可见染色质沿核膜的环状凝结、稀疏的光密度、线粒体肿胀和数量的减少、脂滴堆积等。与对照组相比, 细胞培养液中LDH和MDA含量显著升高, 细胞内抗氧化酶活性具有极显著差异(P<0.01)。SAE和SMAE能够降低MMP, 增加细胞内ROS, 并诱导肝细胞凋亡。转录组结果显示, 肝细胞DEGs主要有氧化应激、免疫应答、甘油三酯代谢、氨基酸代谢、信号转导通路及转录因子调控等。SAE和SMAE通过线粒体依赖途径诱导草鱼原代肝细胞损伤和凋亡, 且SAE与SMAE表现出相同的损伤作用结果。

     

    Abstract: In order to investigate whether soybean meal and soybean have a damaging effect on hepatocytes and its mechanism, Soybean aqueous extract (SAE) and Soybean meal aqueous extract (SMAE) were used as the experimental material for culturing primary hepatocytes of grass carp for 24 hours. The final concentrations of SMAE and SAE were 0, 0.5, 5.0 and 10.0 mg/mL. The results showed that with the increase of the concentration of aqueous extract, the activity of hepatocytes decreased gradually, with a significant dose-response relationship (P<0.05). No significant difference in relative cell activity was observed among the low SAE and SMAE concentration (0.5 and 2.5 mg/mL) groups and the control group. In the 5.0 and 10.0 mg/mL SAE dose groups, the relative cell viability was negatively correlated with the concentration. The cell viability was (78.10±8.57)% and (65.97±7.35)% respectively, which was significantly different from the control group (P<0.01). The relative cell viability in the SMAE groups (5.0 and 10.0 mg/mL) was (86.35±7.17)% and (80.26±7.08)%, respectively, which was significantly lower than that of the control group (P<0.01). At the same concentration, the relative activity of cells in SAE group was lower than that in SMAE group. The ultrastructure of hepatocytes showed that the annular condensation of chromatin along the nuclear membrane, sparse light density, swelling of mitochondria, decrease of number and accumulation of lipid droplets. Hoechst 33285 staining showed that the fluorescence intensity of SAE and SMAE groups (10.0 mg/mL) was significantly weakened, the staining was uneven (chromatin agglutination), the nucleus was broken, and some apoptotic bodies appeared. Compared with the control group, the contents of Lactate dehydrogenase (LDH) and Malondialdehyde (MDA) in the cell culture medium were significantly increased, and the activities of Superoxide dismutase (SOD) and Glutathione (GSH) were significantly different (P<0.05). SAE and SMAE decreased MMP and increased ROS. With the increase of water extract concentration, the early apoptosis rate of cells increased. There was no significant difference in the apoptotic ratio between the low dose group (0.5, 2.5 mg/mL) and the control group. The apoptotic ratio of the SAE group with 5.0 and 10.0 mg/mL concentrations was (22.55±4.35)%, (55.03±2.76)%, and the SMAE group was (32.67±5.79)%, (37.11±8.57)%, respectively, which was significantly different from the control group (P<0.01). Transcriptome results showed that the enriched pathways were mostly related to inflammation, protein metabolism, amino acid metabolism and lipid metabolism. The above results show that the degree of damage to hepatocytes by SAE and SMAE is positively correlated with the amount of addition, and SAE and SMAE have the same damage effect. SAE and SMAE can cause cell ultrastructure changes, increase ROS levels, abnormal cell antioxidant systems and abnormal mitochondrial structure and function, which ultimately affect cell viability and trigger cell death pathways.

     

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