Patulin Induces Autophagy-Dependent Apoptosis In HepG2 Cells

Objective: Patulin(PAT)is a polyketide lactone(4-hydroxy-4H-furo[3,2-c]Pyran-2(6H)-one)produced by the fungi of Penicillium,Aspergillus and Fusariums.It is widespread in apple and apple-derived products,posed a health hazard to humans and living organisms.PAT can enter the body through inhalation,ingestion and skin contact,resulting in genotoxicity,embryo toxicity,immunotoxicity,and teratogenicity in some microorganisms and animals.It has been shown that the kidney,liver,gastrointestinal tract and immune system are the main target organs for mammalian PAT toxicity.PAT induces hepatotoxicity in mice and cells,however the molecular mechanism of hepatic toxicity with PAT remains unclear.Recently,It has been focused on the link between autophagy and apoptosis.These processes involve the transformation and degradation of intracellular proteins and organelles,and there is ample evidence that there is a close correlation between the two processes.It has been shown that autophagy precedes apoptosis,and autophagy can directly induce apoptosis.Therefore,we attempted to detect possible associations between autophagy and apoptosis in HepG2 cells with the aim of providing a comprehensive view of the role of PAT-whether PAT can induce lysosomal membrane permeabilization(LMP),the effect of LMP in PAT-induced apoptosis,and the molecular mechanism between autophagy and apoptosis.Methods: HepG2 cells were treated with 2 ?M PAT at different time points(0,3,6,12,24 h),and Western blotting was used to detect LC3-II,cathepsin B,cytochrome c and caspase 3 protein expression level in HepG2 cells.The expression levels of various related proteins were changed;the effect of PAT on the stability of lysosomal membrane in HepG2 cells was detected by AO staining relocation method and fluorescence microscopy(Olympus BX-63);using JC-1 staining and fluorescence microscopy to detect PAT on HepG2 cells Effects of mitochondrial transmembrane potential;Hoechst staining and flow cytometry to detect whether PAT induces apoptosis in HepG2 cells;detection of PAT-treated HepG2 cells after RNA interference by knockdown of Atg5 gene or autophagy inhibitor 1 mM 3MA.The relationship between autophagy and LMP,Mitochondrial outer membrane permeabilization(MOMP)and apoptosis;specificity of cathepsin B after treated with inhibitor CA-074 Me(10 ?M),the effect of cathepsin B on PAT-induced MOMP and apoptosis was detected by fluorescence microscopy and Western blot.Results: 2 ?M PAT treated HepG2 cells at 0,3,6,12,and 24 h at various time points.From the early 6 h of treatment,the expression of LC3-II protein was increased,and then the cells were treated with chloroquine.It was found that chloroquine promoted the accumulation of LC3-II and was enhanced 6 hours after PAT treatment.After treated with PAT for 12 h,the level of LMP increased significantly.At the same time,the release amount and expression level of cathepsin B increased.The addition of3 MA pretreatment revealed that the increase of LMP and the expression level of cathepsin B were significantly relieved.Subsequently,it was detected that the mitochondrial transmembrane potential was significantly decreased after 12 hours of PAT treatment,and the release amount and expression level of cytochrome c also increased.Similarly,to the above treatment,the specific inhibitors 3MA and CA-074 Me were added for pretreatment,Inner mitochondrial membrane potential(??m)decreased were found.When the level is increased,the release level of cytochrome c was significantly alleviated.Hoechst staining and flow cytometry revealed that HepG2 cells began to apoptosis after 12 hours of PAT treatment.Western blotting also detected a significant increase in caspase 3 protein expression at 12 h after PAT treatment.3MA,siRNA Atg5 and CA-074 Me were pretreated.It was found that the apoptosis was relieved.Conclusion: PAT induces autophagy-dependent apoptosis in HepG2 cells.PAT can activate autophagy early,which leads to an increase in LMP and a decrease in ??m.The lysosome-mitochondrial axis ultimately leads to apoptosis of HepG2 cells.