Effect of Taraxasterol on Apoptosis and Autophagy of Chicken Primary Hepatocytes Induced by AFB1
[Objective]This experiment was aimed to explore the effect and mechanism of taraxasterol on apoptosis and autophagy of chicken primary hepatocytes induced by aflatoxin B1(AFB1).[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(AFB1,0.05 μg/mL),silybinin-positive groups(Sil,2 μg/mL)and low(5 μg/mL),medium(10 μg/mL)and high(20 μg/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 5-25 μg/mL.Therefore,the concentrations of taraxosterol in low,medium and high dose groups were set as 5,10 and 20 μg/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 AFB1 group was significantly increased.Compared with AFB1 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 P450 1A5(CYP1A5),CYP3A37 A,cysteine proteolytic enzyme 3(Caspase-3),Caspase-9,Beclin-1,autophagy protein 5(ATG-5),microtubule-associated protein light chain 3 Ⅰ(LC3-Ⅰ)and LC3-Ⅱ genes of chicken primary hepatocytes in AFB1 group were extremely significantly increased(P＜0.01).Compared with AFB1 group,mRNA expression levels of CYP1A5,CYP3A37,Caspase-3,Caspase-9,Beclin-1,ATG-5,LC3-Ⅰ and LC3-Ⅱ genes 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 AFB1 by influencing the expression of cytochrome enzyme,apoptosis and autophagy related genes.
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