Patulin Induced ROS-dependent Autophagic Cell Death In Human Hepatoma G2 Cells

Objective:Patulin(PAT)is a kind of secondary metabolite which is produced by certain species of Penicillium,Byssochlarnys and Aspergillus.It is widespread in fruits,vegetables and their products.Patulin contamination in apple products has been a worldwide problem without a satisfactory solution yet.At present,there are many research methods for the inactivation and attenuation of patulin.However,due to the widespread distribution of PAT,it can only control the content proportion in food but not completely eliminate or effectively antagonize its toxicity.PAT has many toxic effects such as carcinogenesis,teratogenicity and mutagenicity.The liver is one of its main target organs,but the possible molecular mechanism of PAT's hepatotoxicity still needs to be further studied and clarified.Studies have shown that the liver DNA toxicity induced by PAT is closely related to the intracellular ROS level.This study focused on exploring whether PAT can induce autophagy in HepG2 cells,whether the induced autophagy is related to the oxidative stress pathway,and the possible molecular mechanism involvedMethods:HepG2 cells were treated with PAT at different concentrations(0-80 uM),and the changes in cell survival rate were detected by MTT assay(tetramethazole salt microenzyme reaction colorimetry).Four concentration groups(0 ?M,0.5?M,1 uM,2 u,1M)were set as PAT to treat HepG2 cells for 24 h according to MTT results.1 mM 3-ma was used to pretreat HepG2 cells and PAT was added for 2 hours to observe the effect of inhibition of autophagy on cell survival.The HepG2 cells were pretreated with 50 nM rapamycin for 1 h,and PAT was added to observe the effect of promoting autophagy on cell survival.5 mM NAC was used to pretreat HepG2 cells for 1 h,and PAT was added to observe the effect of inhibiting ROS level on cell survival.Dcfh-da was added to detect the ROS production level in HepG2 cells after PAT treatment by fluorescence microscopy.Western blot was used to detect the changes of LC3,P62,AKT,mTOR and other protein expressions in HepG2 cells,and the relationship between the protein expression and PAT dose was observed.The protein expression was pretreated with NAC for 1 h,and then treated with PAT at 2 ?M concentration for 24 h Application of dyeing,JC-1 with fluorescent microscopy,PAT HepG2 cells mitochondrial transmembrane potential(??m)of the changes.All the cells used in the experiment were in the logarithmic growth phase,and the concentration,time and sequence of drug treatment were consistentResults:MTT results showed that PAT caused dose-dependent death of HepG2 cells.Autophagy inhibitor 3-MA reduced the toxic effect of PAT on HepG2 cells,while autophagy promoter rapamycin further enhanced the cytotoxicity of PAT on HepG2 cells.Thus it is speculated that the toxic effects of PAT on HepG2 cells may be related to autophagy.After treatment with HepG2 cells by PAT,the expression of LC3-? in the cells increased,and the expression of P62 decreased,and it showed a concentration-dependent manner.This indicates that PAT can induce the occurrence of autophagy in HepG2 cells.At the same time,the expression levels of p-Aktl and p-mTOR in cells decreased significantly after PAT treatment.In addition,after PAT treatment,the ROS level of the cells increased significantly,and ??m decreased significantly.After pretreatment with NAC,PAT-induced cytotoxicity was attenuated,LC3-? protein expression was reduced,and levels of p-Aktl and p-mTOR were up-regulated.This indicates that PAT-induced autophagy in HepG2 cells is ROS-dependentConclusion:The reason that PAT can induce autophagic cell death in HepG2 cells is related to its promotion of ROS production and inhibition of aktl-mtor pathway.