蒲公英甾醇对AFB_(1)诱导鸡原代肝细胞凋亡和自噬的影响

Effect of Taraxasterol on Apoptosis and Autophagy of Chicken Primary Hepatocytes Induced by AFB_(1)

【目的】探究蒲公英甾醇对黄曲霉毒素B 1(AFB_(1))诱导鸡原代肝细胞凋亡、自噬的影响及其作用机制。【方法】采用MTT法测定蒲公英甾醇对鸡原代肝细胞的毒性,确定蒲公英甾醇安全作用浓度。将鸡原代肝细胞分为空白组(Normal)、模型组(AFB_(1),0.05μg/mL)、水飞蓟宾阳性组(Sil,2μg/mL)以及蒲公英甾醇低(L-dose,5μg/mL)、中(M-dose,10μg/mL)、高(H-dose,20μg/mL)剂量组。各试验组经相应浓度药物处理后收集细胞,利用流式细胞术和CYTO-ID法检测鸡原代肝细胞凋亡和自噬情况。采用实时荧光定量PCR检测鸡原代肝细胞中细胞色素酶、凋亡和自噬相关基因mRNA表达量。【结果】蒲公英甾醇浓度为5~25μg/mL时对鸡原代肝细胞无明显毒性,因此后续选用蒲公英甾醇低、中、高剂量组给药浓度分别为5、10、20μg/mL。流式细胞术和CYTO-ID检测结果显示,与空白组相比,模型组鸡原代肝细胞中绿色荧光标记的自噬滤泡数量明显增多。与模型组相比,水飞蓟宾组、蒲公英甾醇各剂量组鸡原代肝细胞中绿色荧光标记的自噬滤泡数量明显减少。实时荧光定量PCR结果显示,与空白组相比,模型组鸡原代肝细胞中细胞色素酶P4501A5(CYP1A5)、CYP 3 A 37 A、半胱氨酸蛋白水解酶3(Caspase-3)、Caspase-9、苄氯素1(Beclin-1)、自噬蛋白5(ATG-5)、微管相关蛋白轻链3Ⅰ(LC3-Ⅰ)和LC 3-Ⅱ基因mRNA表达量均极显著升高(P<0.01)。与模型组相比,水飞蓟宾和蒲公英甾醇各剂量组鸡原代肝细胞中CYP 1 A 5、CYP 3 A 37、Caspase-3、Caspase-9、Beclin-1、ATG-5、LC 3-Ⅰ、LC 3-Ⅱ基因mRNA表达量均极显著减低(P<0.01)。【结论】蒲公英甾醇通过影响细胞色素酶、凋亡和自噬相关基因的表达,进而对AFB_(1)所致鸡原代肝细胞损伤起到保护作用。

【Objective】This experiment was aimed to explore the effect and mechanism of taraxasterol on apoptosis and autophagy of chicken primary hepatocytes induced by aflatoxin B 1(AFB_(1)).【Method】The toxicity of taraxasterol to chicken primary hepatocytes was determined by MTT assay and the safe concentration of taraxosterol was determined.Chicken primary hepatocytes were divided into normal,model(AFB_(1),0.05μg/mL),silybinin-positive groups(Sil,2μg/mL)and low(5μg/mL),medium(10μg/mL)and high(20μg/mL)dose of taraxasterol groups.Cells of each test group were collected after treatment with the corresponding concentration of drug.The apoptosis and autophagy were measured by flow cytometry and CYTO-ID.The mRNA expression of cytochrome enzyme,apoptosis and autophagy related genes in chicken primary hepatocytes were determined by Real-time quantitative PCR.【Result】Taraxosterol had no obvious toxicity to chicken primary hepatocytes when the concentration of taraxosterol was 5-25μg/mL.Therefore,the concentrations of taraxosterol in low,medium and high dose groups were set as 5,10 and 20μg/mL,respectively.Flow cytometry and CYTO-ID detection results showed that compared with normal group,the number of green fluorescent labeled autophagy follicles in the chicken primary hepatocytes of AFB_(1)group was significantly increased.Compared with AFB_(1)group,the number of green fluorescentially labeled autophagy follicles in chicken primary hepatocytes in silybin and taraxosterol different dose groups was significantly reduced.Real-time quantitative PCR results showed that compared with normal group,the mRNA expressions of cytochrome enzyme P4501A5(CYP1A5),CYP 3 A 37 A,cysteine proteolytic enzyme 3(Caspase-3),Caspase-9,Beclin-1,autophagy protein 5(ATG-5),microtubule-associated protein light chain 3Ⅰ(LC3-Ⅰ)and LC 3-Ⅱgenes of chicken primary hepatocytes in AFB_(1)group were extremely significantly increased(P<0.01).Compared with AFB_(1)group,mRNA expression levels of CYP 1 A 5,CYP 3 A 37,Caspase-3,Caspase-9,Beclin-1,ATG-5,LC 3-Ⅰand LC 3-Ⅱgenes of chicken primary hepatocytes in silybin and taraxosterol different dose groups were extremely significantly decreased(P<0.01).【Conclusion】Taraxosterol could protect chicken primary hepatocytes induced by AFB_(1)by influencing the expression of cytochrome enzyme,apoptosis and autophagy related genes.