Protective Effects Of Quercetin To Aflatoxin B₁-Induced Cytotoxicity In BRL-3A Cells

Aflatoxin B₁?AFB₁?is the most toxic secondary metabolite produced by Aspergillus flavus under specific conditions and is widely present in corn,peanut and other agricultural products.Quercetin?QUE?is a flavonoid compound with anti-oxidant,anticancer and other physiological functions.This article intends to study the protective effect of quercetin on rat hepatocytes?BRL-3A?induced by AFB₁ and its related mechanism.The cell viability of BRL-3A measured by CCK-8 assay showed that the IC₅₀0 of AFB₁on BRL-3A cells was 23?mol/L,while QUE below 15?mol/L did not cause adverse impact on BRL-3A cells.After pretreatment of the cells with QUE for 24 h,AFB₁ was applied to the cells for another 24 h.The experiment results of cell proliferation,LDH,CYP1A2 activity,reactive oxygen species?ROS?,lipid peroxide?MDA?,and antioxidant enzymes?GST,SOD,CAT,GSH/GSSG ratio?,demonstrated that QUE enhanced the activity of the antioxidative denfense system,reduced the release of LDH and CYP1A2activity,Thus,QUE pretreatment decreased the oxidative damage and cytotoxicity induced by AFB₁ and maintained the cell viability.The study on the effect of AFB₁ on BRL-3A apoptosis showed that AFB₁ significantly decreased the cellular ATP level and mitochondrial membrane potential.The irregular fragmentation and dense staining of nucleus were also observed.However,QUE pretreatment significantly increased the mitochondrial membrane potential and intracellular ATP levels,lessened the fragmentation and dense staining of the nucleus,alleviated the degree of DNA damage and inhibited the occurrence of apoptosis.RT-qPCR experiments indicated that AFB₁ can induce the expression of pro-apoptotic gene of Bax while QUE treatment notablly increased the expression of Bcl-2 genes.Thus,QUE also protects the BRL-3A cells from a mitochondria-mediated intrinsic apoptosis.Western blotting experiments revealed that AFB₁ inhibited the phosphorylation of AKt and nuclear transfer of Nrf2.However,QUE pretreatment significantly increased AKt phosphorylation and Nrf2 level in the nucleus.As it is known the function of AKt/Nrf2signaling pathway,the inhibition of the AKt/Nrf2 signaling pathway might be an important toxicity target.The AKt/Nrf2 signaling pathway increased antioxidant capability and reduced cell apoptosis after QUE pretreatment indicating the AKt/Nrf2 signaling pathway might be a main mechanism for its protective effect on cells after exposure to AFB₁.In summary,QUE can increase AKt phosphorylation and stimulate Nrf2 nuclear translocation to activate the AKt/Nrf2 signaling pathway and to regulate expression of its target genes including HO-1.The increased activity of phase II enzymes,and decreased activity of phase I enzymes may result in a reduction of the degree of cytotoxicity and oxidative damage caused by AFB₁ and enhance the stress resistance of the BRL-3A cells.At the same time,it can also maintain the normal physiological functions of mitochondria and reasonably regulate the expression of apoptosis-related genes.Thus,QUE has the anti-apoptosis and anti-oxidation functions to protect cells from AFB₁-induced cytotoxicity.