AFB_(1)对犬肝细胞的毒性作用及NAC的保护效应

Toxic effect of aflatoxin B_(1) on canine hepatocyte and protective effect of N-acetyl-L-cysteine

[目的]以犬原代肝细胞为模型,研究黄曲霉毒素B_(1)(AFB_(1))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),0.05,0.25,1.25,6.25,31.25μg/mL AFB_(1)和6.25μg/mL AFB_(1)+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,0.05~6.25μg/mL AFB_(1)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;6.25μg/mL AFB_(1)+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,1.25~31.25μg/mL AFB_(1)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);0.05~31.25μg/mL AFB_(1)处理组的碱性磷酸酶(ALP)活性显著升高(P<0.05);1.25~31.25μg/mL AFB_(1)处理组的γ-谷氨酰转移酶(γ-GGT)活性显著降低(P<0.05),0.05~0.25μg/mL AFB_(1)处理组的γ-GGT活性显著增加(P<0.05);31.25μg/mL AFB_(1)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,0.05~0.25μg/mL AFB_(1)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);1.25~31.25μg/mL AFB_(1)处理组的H2O2质量浓度显著增加(P<0.05).与对照组相比,0.05~31.25μg/mL AFB_(1)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;0.25~31.25μg/mL AFB_(1)处理组的过氧化氢酶(CAT)活性显著降低(P<0.05).与6.25μg/mL AFB_(1)处理组相比,6.25μg/mL AFB_(1)+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).6.25μg/mL AFB_(1)处理细胞的凋亡率为12%,极显著高于对照组和6.25μg/mL AFB_(1)+64 mmol/L NAC处理组.与对照组相比,0.05~6.25μg/mL AFB_(1)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6.25μg/mL AFB_(1)组相比,6.25μg/mL AFB_(1)+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]AFB_(1)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AFB_(1)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.

【Objective】The toxicity of aflatoxin B_(1)(AFB_(1))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),0.05,0.25,1.25,6.25,31.25μg/mL AFB_(1) and 6.25μg/mL AFB_(1)+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,0.05-6.25μg/mL AFB_(1) treatment reduced number of hepatocytes and caused damage,loss of plumpness,apoptosis and necrosis of cells.Cells in the 6.25μg/mL AFB_(1)+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 1.25-31.25μg/mL AFB_(1) group were significantly increased(P<0.05)and activity of alkaline phosphatase(ALP)in 0.05-31.25μg/mL AFB_(1) group was significantly increased(P<0.05).Theγ-GGT activity in 1.25-31.25μg/mL AFB_(1) group was significantly decreased(P<0.05),whileγ-GGT activity in 0.05-0.25μg/mL AFB_(1) group was significantly increased(P<0.05).The concentration of ALB in 31.25μg/mL AFB_(1) 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 0.05-0.25μg/mL AFB_(1) group and concentration of H 2O 2 in 1.25-31.25μg/mL AFB_(1) group increased significantly(P<0.05).The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in 0.05-31.25μg/mL AFB_(1) group showed no significant changes while catalase(CAT)activity in 0.25-31.25μg/mL AFB_(1) group was significantly decreased compared with the control group(P<0.05).Compared with 6.25μg/mL AFB_(1) group,activities of AST,ALP andγ-GGT and levels of ALB,8-OHdG,MDA and H 2O 2 in the 6.25μg/mL AFB_(1)+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 6.25μg/mL AFB_(1) was 12%,which was extremely and significantly higher than that of the control group and the 6.25μg/mL AFB_(1)+64 mmol/L NAC group.Compared with the control group,expression levels of cleaved-caspase-3 and cleaved-caspase-9 mRNA in 0.05-6.25μg/mL AFB_(1) group showed extremely significant increases(P<0.01).Compared with the 6.25μg/mL AFB_(1) group,expression levels of cleaved-caspase-3 and cleaved-caspase-9 mRNA in 6.25μg/mL AFB_(1)+64 mmol/L NAC treatment group showed extremely significant reduction(P<0.01).【Conclusion】AFB_(1) 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.