| 摘要: |
| 【目的】以犬原代肝细胞为模型，研究黄曲霉毒素B1（AFB1）对肝细胞的毒性作用以及N-乙酰-L-半胱氨酸（NAC）对细胞损伤的保护效应。【方法】选取1~2月龄幼犬，取肝细胞培养至对数生长期，分别用0（对照），0.05，0.25，1.25，6.25，31.25 μg/mL AFB1和6.25 μg/mL AFB1+64 mmol/L NAC处理36 h后，观察细胞形态结构，检测肝功能相关指标、氧化与抗氧化功能指标及细胞凋亡相关基因cleaved caspase-3和cleaved-caspase-9 mRNA表达量。【结果】显微观察发现，与对照组相比，0.05~6.25 μg/mL AFB1处理组的细胞数量减少，细胞失去饱满性，细胞凋亡、坏死情况明显；6.25 μg/mL AFB1+64 mmol/L NAC处理组的细胞饱满且活性良好，细胞数目增加，结构完整。与对照组相比，1.25~31.25 μg/mL AFB1处理组的谷丙转氨酶（ALT）和谷草转氨酶（AST）活性均显著上升（P<0.05）；0.05~31.25 μg/mL AFB1处理组的碱性磷酸酶（ALP）活性显著升高（P<0.05）；1.25~31.25 μg/mL AFB1处理组的γ-谷氨酰转移酶（γ-GGT）活性显著降低（P<0.05），0.05~0.25 μg/mL AFB1处理组的γ-GGT活性显著增加(P<0.05)；31.25 μg/mL AFB1处理组的白蛋白（ALB）质量浓度显著降低（P<0.05）。与对照组相比，0.05~0.25 μg/mL AFB1处理组的8 羟基脱氧鸟苷（8-OHdG）和丙二醇（MDA）含量显著增加（P<0.05）；1.25~31.25 μg/mL AFB1处理组的H2O2质量浓度显著增加（P<0.05）。与对照组相比，0.05~31.25 μg/mL AFB1处理组的超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性无显著变化；0.25~31.25 μg/mL AFB1处理组的过氧化氢酶（CAT）活性显著降低（P<0.05）。与6.25 μg/mL AFB1处理组相比，6.25 μg/mL AFB1+64 mmol/L NAC处理组的AST、ALP、r-GGT活性以及ALB、8-OHdG、MDA、H2O2水平显著降低（P<0.05），CAT和GSH-Px活性显著升高（P<0.05）。6.25 μg/mL AFB1处理细胞的凋亡率为12%，极显著高于对照组和6.25 μg/mL AFB1+64 mmol/L NAC处理组。与对照组相比，0.05~6.25 μg/mL AFB1处理组的cleaved-caspase-3和cleaved-caspase-9 mRNA的表达量极显著增加（P<0.01）；与6.25 μg/mL AFB1组相比，6.25 μg/mL AFB1+64 mmol/L NAC处理组的cleaved-caspase-3和cleaved-caspase-9 mRNA表达量均极显著降低（P<0.01）。【结论】AFB1能够抑制犬原代肝细胞活性，引起细胞形态、肝功能指标及细胞氧化与抗氧化功能异常，加快细胞凋亡；NAC能够显著提高抗氧化酶活性，降低AFB1所致肝细胞的损伤和凋亡水平，对肝细胞具有一定的保护性。 |
| 关键词: 黄曲霉毒素B1 N-乙酰-L-半胱氨酸 犬 肝细胞 毒性作用 |
| DOI： |
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| 基金项目:安徽高校协同创新项目（GXXT-2019-013）；安徽农业大学2018年校级大学生创新创业训练计划项目（XJDC2018056） |
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| Toxic effect of aflatoxin B1 on canine hepatocyte and protective effect of N-acetyl-L-cysteine |
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ZHANG Mi,ZHAO Jie,XU Jingru,et al
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| Abstract: |
| 【Objective】The toxicity of aflatoxin B1 (AFB1) on hepatocytes and protective effect of N-acetyl-L-cysteine (NAC) on cell injury were studied using canine primary hepatocytes as model.【Method】Canines at the age of 1-2 months were selected and hepatocytes were cultured to logarithmic growth phase.After being treated with 0 (control),0.05,0.25,1.25,6.25,31.25 μg/mL AFB1 and 6.25 μg/mL AFB1+64 mmol/L NAC for 36 h,cell morphology was observed.Liver function related indicators,oxidation and antioxidant function indicators,and expression levels of apoptosis related genes cleaved-caspase-3 and cleaved-caspase-9 mRNA were detected.【Result】Compared with the control group,0.05-6.25 μg/mL AFB1 treatment reduced number of hepatocytes and caused damage,loss of plumpness,apoptosis and necrosis of cells.Cells in the 6.25 μg/mL AFB1+64 mmol/L NAC treatment were full and well active with increased number and complete structure.Compared with the control group,activities of glutamic-pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST) in 1.25-31.25 μg/mL AFB1 group were significantly increased (P<0.05) and activity of alkaline phosphatase (ALP) in 0.05-31.25 μg/mL AFB1 group was significantly increased (P<0.05).The γ-GGT activity in 1.25-31.25 μg/mL AFB1 group was significantly decreased (P<0.05),while γ-GGT activity in 0.05-0.25 μg/mL AFB1 group was significantly increased (P<0.05).The concentration of ALB in 31.25 μg/mL AFB1 group was significantly decreased (P<0.05).Compared with the control group,concentrations of 8 hydroxydeoxyguanosine (8 OHdG) and propylene glycol (MDA) were significantly increased in 0.05-0.25 μg/mL AFB1 group and concentration of H2O2 in 1.25-31.25 μg/mL AFB1 group increased significantly (P<0.05).The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in 0.05-31.25 μg/mL AFB1 group showed no significant changes while catalase (CAT) activity in 0.25-31.25 μg/mL AFB1 group was significantly decreased compared with the control group (P<0.05).Compared with 6.25 μg/mL AFB1 group,activities of AST,ALP and γ-GGT and levels of ALB,8-OHdG,MDA and H2O2 in the 6.25 μg/mL AFB1+64 mmol/L NAC group were significantly decreased (P<0.05),while CAT and GSH-Px activities were significantly increased (P<0.05).The apoptosis rate of cells treated with 6.25 μg/mL AFB1 was 12%,which was extremely and significantly higher than that of the control group and the 6.25 μg/mL AFB1+64 mmol/L NAC group.Compared with the control group,expression levels of cleaved-caspase-3 and cleaved-caspase-9 mRNA in 0.05-6.25 μg/mL AFB1 group showed extremely significant increases (P<0.01).Compared with the 6.25 μg/mL AFB1 group,expression levels of cleaved-caspase-3 and cleaved-caspase-9 mRNA in 6.25 μg/mL AFB1+64 mmol/L NAC treatment group showed extremely significant reduction (P<0.01).【Conclusion】AFB1 could inhibit activity of canine primary hepatocytes,cause abnormal cell morphology as well as dysfunction of liver function,cell oxidation and anti-oxidation,and accelerate apoptosis of hepatocytes.NAC could protect canine primary hepatocytes by significantly increasing activities of antioxidant enzymes and reducing damage and apoptosis of hepatocytes. |
| Key words: aflatoxin B1 (AFB1) N-acetyl-L-cysteine (NAC) canine hepatocyte toxic effect |
