Mechanism Of AflatoxinB1 Blocking Autophagy Flow And Inducing Apoptosis In HepG2 Cells

Mycotoxins are highly toxic metabolites secreted by fungi.They are widely distributed in the soil and are highly contaminated.Among the various types of aflatoxins,aflatoxin B1(AFB1)is the most commonly in natural foods and has the widest pollution,so it has received extensive attention.Researches have shown that as a target organ of aflatoxin,it can lead to a serious damage to liver function under the action of low dose of AFB1.At present,research on AFB1-induced cell autophagy mainly focuses on immune cells and liver cells of broilers.However,there is no correlation between aflatoxin-induced autophagy in human hepatocytes.This article intends to use human hepatocellular carcinoma cell line HepG2 to study the changes of cell autophagy level and the whole process of autophagy flow after AFB1 treatment.In the course of the study,it was found that AFB1 caused an increase in autophagosomes in HepG2 cells,and was blocked in the late stage of autophagy flow.Lysosomes play an important role in the degradation of autophagosomes.In order to explore the reasons for the blockage of autophagy flow caused by AFB1,we deeply studied the damage on lysosomes,and then revealed the specific mechanism of AFB1 blocking the autophagy flow of HepG2 cells.Finally we measured the apoptosis induced by AFB1,and analyze the relationship between AFB1-induced apoptosis and lysosomal damage.We are aimed to provide new ideas for studying AFB1-induced liver cell damage.At same time,it has more important value on doing hazard assessment and Danger defense on AFB1.The main research contents and results are as follows:(1)Effects of AFB1 exposure on autophagy in HepG2 cells.First,the MTT experiment was used to screen out the experimental conditions that AFB1 had no toxic effect on HepG2 cells.Cells were treated by different time and concentration of AFB1,the expression of autophagy-labeled protein LC3-? was evaluated by Western Blot,and finally 20?M AFB1 treatment was selected for 6h as the treatment condition for subsequent autophagy research.Through MDC staining experiments,it was found that fluorescence staining of autophagosomes in HepG2 cells increased significantly after AFB1 treatment.Furthermore,increased autophagosomes was fund in HepG2 cells treated with AFB1 by transmission electron microscope.We proved that AFB1 caused the increase of autophagosomes in HepG2 cells from both molecular and cellular morphology.We introduced two autophagy inhibitors,CQ and 3-MA,to investigate whether AFB1 affects the integrity of autophagy flow.We evaluate the expression of two autophagy marker proteins LC3-? and P62 by Western Blot.We fund that AFB1 can inhibition the autophagy flow in HepG2 cells by blocking autophagosome degradation.Confocal microscope observation of mRFP-GFP-LC3 fluorescence expression can also confirm that AFB1 caused the blocking of autophagy flow.(2)AFB1 induced lysosomal damage.In order to explore the mechanism of AFB1-induced autophagy blockade in HepG2 cells,we focused on the lysosomal function that plays an important role in the autophagy phase.Through the colocalization of LAMP1 and LC3,it was found that the fusion process of lysosome and autophagosome was not significantly affected by AFB1.Lysotracker Red and AO staining revealed that AFB1 caused the rise of lysosome internal pH and induced lysosome alkalization.Western Blot showed that cathepsin leaked from lysosomal cavity,which indicated that lysosomal membrane permeabilization occurred.The protein TFEB that associated with lysosome and autophagosome formation was activated.The protein TFEB which related to lysosome and autophagosome synthesis was activated by nuclear ectopic,indicating that the lysosomal biosynthesis process was activated.It shows that AFB1 induced autophagy flow block in HepG2 cells was mainly caused by lysosomal damage.(3)AFB1 can cause apoptosis in HepG2 cells.The MTT experiment,Annexin V-FITC/PI double staining flow cytometry and DAPI staining experiment proved that different concentrations of AFB1 treated HepG2 cells can induce apoptosis after 48 h.Detected apoptosis-related proteins: Bax / Bcl-2,Caspase-3,and Caspase-9 by Western Blot.The results showed that after AFB1 treatment,the apoptosis rate increased,intracellular chromatin agglutination,and the expression of each apoptotic protein increased.From the molecular and morphological point of view,it is proved that AFB1 induces apoptosis,and it is found that autophagy occurs before apoptosis.Under the experimental conditions of AFB1-induced apoptosis,we detected the expression of CTSD in lysosomes once again.We fund that CTSD leaked from lysosomal into cytoplasm after treated by AFB1.The trend of cathepins leakage in HepG2 cells is the same as that of apoptosis.We speculate that the apoptosis induced by AFB1 may be related to lysosomal damage.