Citreoviridin Induces Autophagy-dependent Apoptosis Through Lysosomal-mitochondrial Axis In Human Liver HepG2 Cells

Objective: Citreoviridin(CIT)is a mycotoxin derived from fungal species(Penicillium Citreoviridin mainly)in mouldy cereals.It was reported that Penicillium Citreoviridin started to grow when the water conent in grains reached 14.6%.Therefore,if the storage conditions is moist and time is too long,the fungus grow rapidly and the pollution of the fungus and its toxin aggravates.Although,the technology of food production,processing and storage becomes more scientific,its health standards formulating and monitoring mechanisms to implement more strict and continuous improvement now,the phenomenon of the stored grain encountered fungal contamination is still serious.Resently,the search of the toxicity of CIT focus on its cardiotoxicity,genotoxicity and neurotoxicity.Liver is the important organ of metabolism and detoxication,however,CIT hepatotoxicity has been less investigated,so the research of its mechanism is not clear and this is the concerntion of this testing of citreoviridin toxicity mechanism.With the in-depth research of autophagy,people found persistent stress may lead to an intensification of autophagy that lead to cell death-called type II programmed cell death,which is different from apoptosis(type I programmed cell death).Although there are significant difference between autophagic cell death and apoptosis in the metabolic and morphologic changes,it is revealed that there are extensive crosstalk between autophagic cell death and apoptosis.In our previous study,we reported that CIT stimulated autophagosome formation in human liver HepG2 cells.In this study,we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells.This study may provide experimental data for the research of the relationship between autophagy and apoptosis,and take a deep insight in understanding the molecular mechanisms of CIT hepatotoxicity.Methods: In our previous study,we found that the IC50 of CIT was 7.2mM in HepG2 cells and the cell viability(%)could reach to 80% when the concentration of CIT was 5 mM.So in this study,we used 5 mM CIT to treat the HepG2 cells at different time.We used Westernt blot to measure the expression of LC3(microtubule-associated protein light chain 3)and cathepsin D in HepG2 cells.The lysosomal membrane stability of HepG2 cells was measured by AO staining under a fluorescence microscope.The mitochondrial transmembrane potential(??m)was evaluated by JC-1 staining under a fluorescence microscope.Caspase-3 activity was determined using colorimetric method.Cell apoptosis was observed by TUNEL staining under a fluorescence microscope.After pretreatment with the autophagosome formation inhibitor 3-MA,or si RNA against Atg5,we investigated the relationship of autophagy with lysosomal membrane stability and apoptosis in CIT-treated cells.After pretreatment with cathepsin D inhibitor pepstain A,we investigated the role of cathepsin D in CIT-induced apoptosis.Results: In this study,we found that the expression of LC3-II was significantly increased after treatment with 5 mM CIT for 6 h,12 h and 24 h.But after treatment with 5 mM CIT for 3 h,the LC3-II levels did not change significantly as compared with the control.Lysosomal membrane permeabilization and the release of cathepsin D were induced after treatment with CIT for 12 h.Inhibition of autophagosome formation with 3-MA and si RNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization and the release of cathepsin D.The mitochondrial transmembrane potential(??m)collapsed after treatment with 5 mM CIT for 12 h and it was attenuated by 3-MA and si RNA against Atg5 by the same.The activity of caspase-3 was activated after treatment with 5 mM CIT for 24 h,and it was relieved by 3-MA,pepstain A and si RNA against Atg5.Futhermore,after treatment with 5 mM CIT for 24 h,apoptosis was induced in HepG2 cells as shown in TUNEL assay.CIT-induced apoptosis was relieved by pretreatment with 3-MA and si RNA against Atg5,while apoptosis was not induced after treatment with 5 mM CIT for 6 h and 12 h.Conclusion: We demonstrated that CIT would induce apoptosis in HepG2 cells.CIT-induced apoptosis was autophagy-independent.CIT-activated autophagic flux might be an upstream event that triggered apoptosis through the lysosomalmitochondrial axis.CIT-activated autophagy caused lysosomal membrane permeabilization,the lysosomal release of cathepsin D,collapse of mitochondrial trasmembrane potential and apoptosis in HepG2 cells eventually.