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小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用

邹翠云 陈小晶 邬颖欣 黄锦雄 谭小红 胡心月 甘松永 吴锦辉

邹翠云, 陈小晶, 邬颖欣, 黄锦雄, 谭小红, 胡心月, 甘松永, 吴锦辉. 小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用[J]. 南方水产科学. doi: 10.12131/20210362
引用本文: 邹翠云, 陈小晶, 邬颖欣, 黄锦雄, 谭小红, 胡心月, 甘松永, 吴锦辉. 小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用[J]. 南方水产科学. doi: 10.12131/20210362
ZOU Cuiyun, CHEN Xiaojing, WU Yingxin, HUANG Jinxiong, TAN Xiaohong, HU Xinyue, GAN Songyong, WU Jinhui. Protective effect of Xiaochaihu Decoction on D-GalN/ LPS-induced hepatocyte injury in hybrid grouper (Epinephelus lanceolatusy♂× E. fuscoguttatus♀)[J]. South China Fisheries Science. doi: 10.12131/20210362
Citation: ZOU Cuiyun, CHEN Xiaojing, WU Yingxin, HUANG Jinxiong, TAN Xiaohong, HU Xinyue, GAN Songyong, WU Jinhui. Protective effect of Xiaochaihu Decoction on D-GalN/ LPS-induced hepatocyte injury in hybrid grouper (Epinephelus lanceolatusy♂× E. fuscoguttatus♀)[J]. South China Fisheries Science. doi: 10.12131/20210362

小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用

doi: 10.12131/20210362
基金项目: 大学生创新创业训练计划项目 (S202011347016);广东省现代海水种业科技示范园项目;广东省科技创新战略专项资金 (攀登计划专项资金) 项目 (pdjh2020b0292)
详细信息
    作者简介:

    邹翠云 (1982—),女,副教授,博士,从事水产动物营养与免疫研究。E-mail: zoucuiyun@zhku.edu.cn

    通讯作者:

    吴锦辉 (1979—),男,工程师,硕士,从事工厂化循环水养殖研究。E-mail: wjhin@sina.com

  • 中图分类号: S 917.4

Protective effect of Xiaochaihu Decoction on D-GalN/ LPS-induced hepatocyte injury in hybrid grouper (Epinephelus lanceolatusy♂× E. fuscoguttatus♀)

  • 摘要: 为探讨小柴胡汤对虎龙杂交石斑鱼 (Epinephelus lanceolatus♂×E. fuscoguttatus♀) 化学性肝细胞损伤的免疫功能影响,用D-氨基半乳糖 (D-GalN, 20 mmol·L–1) 和脂多糖 (LPS, 1 μg·mL–1) 作用肝细胞24 h构建虎龙杂交石斑鱼肝细胞损伤模型,用不同质量浓度的小柴胡汤 (100、200和400 μg·mL–1) 对肝细胞进行前处理 [D-氨基半乳糖联合脂多糖 (D-GalN/LPS) 损伤前加药],通过CCK-8法检测肝细胞活力,HE染色观察肝细胞形态,收集细胞上清液检测炎症因子INF-γ、COX-2和PGE2的含量,以及细胞沉淀中凋亡和免疫相关基因的表达。结果表明,小柴胡汤可以提高肝细胞的成活率,不同程度地抑制D-GalN/LPS损伤引起的肝细胞培养上清液中INF-γ活性以及COX-2和PGE2含量的升高,降低细胞内乳酸脱氢酶 (LDH)、谷草转氨酶 (AST) 和谷丙转氨酶 (ALT) 活性以及细胞凋亡率,对肝细胞结构具有明显的保护作用;同时显著抑制凋亡相关基因caspase-3、caspase-9和P53的上调,促进免疫相关基因TLR3的上调。数据统计分析显示,添加200 μg·mL–1小柴胡汤组的抑制效果最明显。综合以上结果,小柴胡汤对虎龙杂交石斑鱼化学性肝损伤有一定的保护作用,可预防肝脏疾病的发生和发展,为鱼类保肝药物研究提供了新的见解。
  • 图  1  小柴胡汤改善D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的成活率

    注:不同小写字母表示差异显著 (P<0.05);图 3、图 4、图 5-f、图 6 同此。

    Figure  1.  Hepatocyte viability of D-GalN/LPS-induced hepatocyte injury in hybrid grouper by Xiaochaihu Decoction

    Note: Different small letters indicate significant difference (P<0.05); the same case in Fig. 3, Fig. 4, Fig. 5-f and Fig. 6.

    图  2  小柴胡汤对D-GalN/LPS诱导的肝细胞形态的影响

    注:a. 对照组;b. 模型组;c. 100-XD;d. 200-XD;e. 400-XD;图5-a—5-e同此。

    Figure  2.  Effect of Xiaochaihu Decoction on morphology of D-GalN/LPS-treated hepatocytes

    Note: a. Control; b. Model; c. 100-XD; d. 200-XD; e. 400-XD; the same case in Fig. 5-a−5-e.

    图  3  小柴胡汤对D-GalN/LPS诱导的上清液中细胞因子的炎症反应

    Figure  3.  Effect of Xiaochaihu Decoction on D-GalN/LPS-induced inflammatory responses in hepatocytes

    图  4  小柴胡汤对D-GalN/LPS诱导的细胞内肝功能指标的影响

    Figure  4.  Effect of Xiaochaihu Decoction on D-GalN/LPS-induced liver function index in hepatocytes

    图  5  小柴胡汤降低D-GalN/LPS诱导的细胞凋亡

    注:f. 细胞凋亡率 (UR+LR):将肝细胞进行 Annexin V-APC/7AAD 双染后,用流式细胞仪检测细胞凋亡率。

    Figure  5.  D-GalN/LPS-induced apoptosis reduced by Xiaochaihu Decoction

    Note: f. Apoptotic cell ratio (Percentage of cells in quadrants UR and LR): The cells were double stained with Annexin V-APC/7AAD, and the apoptosis rate was determined using fow cytometry.

    图  6  小柴胡汤对D-GalN/LPS诱导的肝细胞免疫和凋亡相关基因的影响

    Figure  6.  Effect of Xiaochaihu Decoction on D-GalN/LPS-induced hepatocytes mRNA expression of immune and apoptosis genes

    表  1  本研究所用相关的引物序列

    Table  1.   Primer sequence of related genes in this study

    基因
    Gene
    引物序列 (5'—3')
    Primer sequence (5'–3')
    半胱氨酸天冬氨酸蛋白酶-3
    Caspase-3
    F: CGCAAAGAGTAGCGACGGA
    R: CGATGCTGGGGAAATTCAGAC
    53抑癌基因 P53 F: GGCACCAAACAAACCAAAAAAC
    R: GTCAAGCAACTCCAGACCATCA
    半胱氨酸天冬氨酸蛋白酶-9
    Caspase-9
    F: TTTTCCTGGTTATGTTTCGTGG
    R: TTGCTTGTAGAGCCCTTTTGC
    抑制蛋白κBα IKKα F: ACACCGACACAACGGCTCAT
    R: CCAGACGGCACAGTTTCACAG
    谷胱甘肽过氧化物酶 GPx F: TACCCTACCAAGTCCTCCAACC
    R: AACAAACACCCGACACCCA
    Toll样受体3 TLR3 F: TCTCCATTCCGTCACCTTCC
    R: TCATCCAGCCCGTTACTATCC
    内参 β-Actin
     
    F: TACGAGCTGCCTGACGGACA
    R: GGCTGTGATCTCCTTCTGC
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-12-01
  • 修回日期:  2022-01-31
  • 录用日期:  2022-03-15
  • 网络出版日期:  2022-03-30

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