蒲公英甾醇对AFB1所致鸡原代肝细胞氧化损伤的保护作用

Protective Effect of Taraxasterol on Oxidative Damage ofChicken Primary Hepatocytes Induced by AFB 1

【目的】探讨蒲公英甾醇对黄曲霉毒素B1(AFB1)诱导的鸡原代肝细胞氧化损伤的保护作用及机制,为应用蒲公英甾醇防治AFB1中毒提供理论依据。【方法】采用组织块酶消化法分离鸡原代肝细胞并进行PAS糖原染色鉴定,通过CCK-8法绘制肝细胞生长曲线;通过MTT法测定AFB1对鸡肝细胞的毒性,结合测定肝细胞上清液中谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,以确定AFB1的体外建模浓度。通过MTT法确定蒲公英甾醇的安全给药浓度后,将试验分为6组:空白对照组、模型组、蒲公英甾醇剂量组(高、中、低剂量组)、阳性对照组。建立AFB1诱导的鸡原代肝细胞损伤模型并给予药物,通过试剂盒测定细胞内活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)以及还原型谷胱甘肽(GSH)水平。通过实时荧光定量PCR法测定Keap1/Nrf2信号通路关键基因血红素加氧酶-1(HO-1)、NADPH醌氧化还原酶1(NQO1)、Kelch样环氧氯丙烷相关蛋白1(Keap1)和核转录因子E2相关因子2(Nrf2)表达量。【结果】试验成功分离鉴定鸡原代肝细胞,在培养24~72 h细胞活力较高;确定0.05μg/mL作为AFB1体外诱导鸡原代肝细胞损伤的建模浓度;确定20、10、5μg/mL作为蒲公英甾醇高、中、低剂量组的给药浓度;与模型组相比,蒲公英甾醇可极显著降低AFB1所致鸡原代肝细胞氧化应激相关指标ROS和MDA含量(P<0.01),极显著提高抗氧化物SOD活性(P<0.01);极显著或显著提高AFB1所致鸡原代肝细胞中HO-1、NQO1和Nrf2基因(除低剂量组外)表达量(P<0.01;P<0.05),降低Keap1基因表达量。【结论】蒲公英甾醇通过调控鸡原代肝细胞Keap1/Nrf2信号通路提高其抗氧化能力,从而对AFB1诱导的鸡原代肝细胞氧化损伤起到保护作用。

【Objective】The purpose of this study was to explore the protective effect and mechanism of taraxasterol on oxidative damage of chicken primary hepatocytes induced by aflatoxin B 1(AFB 1),and provide theoretical basis for the prevention and treatment of AFB 1 poisoning with taraxasterol.【Method】Chicken primary hepatocytes were isolated by tissue block enzyme digestion and identified by PAS glycogen staining.The growth curve of hepatocytes was drawn by CCK-8 method.The toxicity of AFB 1 on hepatocytes was determined by MTT method,and the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the supernatant of hepatocytes were measured to determine the modeling concentration of AFB 1 in vitro.MTT method was used to find out the safe concentration of taraxasterol.The test was divided into 6 groups:Normal group,AFB 1 group,taraxasterol groups(high,medium and low doses)and Sil group.After the establishment of the primary hepatocyte injury model induced by AFB 1 and drug administration,the contents of reactive oxygen species(ROS)and malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in the cells were determined by reagent kits.The expression of heme oxygenase-1(HO-1),NADPH quinone oxidoreductase 1(NQO1),Kelch like ECH associated protein 1(Keap1),and nuclear factor E2 related factor 2(Nrf2)of the key genes in Keap1/Nrf2 signal pathway were determined by Real-time quantitative PCR.【Result】The cells were successfully isolated and identified as chicken primary hepatocytes.The cell viability was higher after 24-72 h culture;0.05μg/mL concentration was used as the modeling concentration of AFB 1 which induced primary hepatocyte injury in vitro;20,10 and 5μg/mL concentrations were used as taraxasterol administration concentrations of the high,medium and low dose groups.Compared with AFB 1 group,taraxasterol could extremely significantly reduce the contents of ROS and MDA(P<0.01),extremely significantly increase the activity of antioxidant SOD in AFB 1-induced chicken primary hepatocytes(P<0.01),and extremely significantly or significantly increase the expression of HO-1,NQO1 and Nrf2(except for 5μg/mL taraxasterol)genes(P<0.01 or P<0.05),and reduce the expression of Keap1 gene in chicken primary hepatocytes induced by AFB 1.【Conclusion】Taraxasterol could improve the antioxidant capacity of chicken primary hepatocytes by regulating the Keap1/Nrf2 signal pathway,thus protecting against the oxidative damage of chicken primary hepatocytes induced by AFB 1.