The Mechanism Of Fumonisin B1 Induced Lysosomal Membrane Permeabilization Through Reactive Oxygen Species

Fumonisins are a class of water-soluble mycotoxins produced by Fusarium,among which the most widely contaminated and most toxic is Fumonisin B1(FB1).With the rapid development of livestock and poultry farming,the supply demand for animal feed and raw materials is also increasing.Crops such as corn and sorghum,which are used as feed raw materials,are prone to contamination by fumonisins due to immature conditions such as temperature and humidity during storage,processing and transportation.Animals fed or accidentally ingesting feed or agricultural products contaminated with toxins can cause great harm to their bodies,greatly affecting the development of the livestock industry.The toxic effects of FB1 vary in different animals.Pigs,as one of the main animals most susceptible to FB1,mainly cause severe pulmonary edema and damage to the liver and kidneys,the reduction of phagocytic ability of alveolar macrophages and the accumulation of substances in the endothelial cells of pulmonary capillaries are unique to pigs or certain cell types.FB1 is structurally similar to sphinganine(SA)and sphingosine(SO),so it may exert its toxic effects by disrupting the biosynthesis and metabolic pathways of myelin lipids,thereby inducing cell apoptosis,autophagy,oxidative stress and mitochondrial damage.Although current research shows that FB1 induces lysosomal membrane imperfection,as a lysosome detergent,it is not clear whether the cytotoxicity of FB1 is similar to the action of SO and SA,what kind of damage it causes to lysosomes.Therefore,this study uses PK15 and 3D4/21 cells as experimental materials to explore the role of lysosomal membrane permeabilization in FB1-induced cytotoxicity and compares with SO and SA,to further explore the mechanism of lysosomal membrane permeabilization,and by screening and analyzing special lysosome groups to further clarify the impact of FB1 on lysosomes.The main research content includes the following two aspects:To explore the toxic effects of FB1,SO and SA on cells and their impact on lysosomes,cell viability and apoptosis-related indicators were examined to comprehensively evaluate cell apoptosis induced by FB1,SO and SA.The state and distribution of lysosomes were detected using confocal microscopy,subcellular distribution of Cathepsin B(CTSB)and Galectin-3(Gal-3)proteins were observed by immunofluorescence,and lysosomes and cytoplasmic components were separated,and the leakage of tissue proteinase was detected by western blot.Changes in mitochondrial membrane potential and mitochondrial morphology were detected to analyze the apoptosis pathway.Subsequently,to explore the mechanism of lysosomal membrane permeabilization induced by FB1,SO and SA,the cysteine protease inhibitor Aloxistatin(E64D)was used to analyze the role of lysosomal membrane permeabilization in cytotoxicity.Inhibitors Bafilomycin A1(Baf A1)and N-acetyl-L-cysteine(NAC)were used to explore the mechanism of lysosomal membrane permeabilization from the internal factors of lysosomal p H,volume and external factors such as reactive oxygen species(ROS).Finally,the mitochondrial division inhibitor Mdivi-1 was used to inhibit the ROS released during mitochondrial division and analyze the relationship between lysosomal membrane permeabilization and mitochondria The results showed that FB1,SO and SA all have cytotoxicity to PK15 and 3D4/21 cells,significantly reduced the expression of the antiapoptotic protein Bcl-2 and increased the cleavage of caspase 3 and caspase 8.Acridine orange results showed lysosomal damage,but the volume and distribution of lysosomes induced by FB1 were different from SO and SA.At 12 h,FB1,SO and SA could induce the leakage of CTSB protein and the punctate aggregation of Gal-3 protein.At the same time,the results of western blot showed that CTSB and CTSD were detected in the cytoplasmic components,indicating that FB1,SO and SA induced the occurrence of lysosomal membrane permeabilization.Mitochondrial morphology showed the destruction of the mitochondrial network and the occurrence of mitochondrial fragmentation,in addition,FB1 also induced the loss of mitochondrial membrane potential and the release of cytochrome C.These results indicate that FB1,SO and SA induce lysosomal membrane permeabilization and induce cell apoptosis through the mitochondrial pathway.E64 D alleviated the cytotoxicity of FB1,SO and SA,but had no effect on lysosomal distribution,and the use of E64 D could alleviate cell apoptosis induced by FB1,indicating that lysosomal membrane permeabilization plays an important role in cell apoptosis induced by FB1.Baf A1 reduced the acidity of lysosomes,increased the volume of lysosomes,and exacerbated the leakage of CTSB and the aggregation of Gal-3 induced by FB1,SO and SA,that is,Baf A1 exacerbated lysosomal membrane permeabilization induced by FB1,SO and SA.Notably,after NAC cleared intracellular ROS,it could alleviate lysosomal membrane permeabilization induced by FB1,but had no effect on lysosomal membrane permeabilization induced by SO and SA.Finally,after inhibiting mitochondrial division,it could also alleviate lysosomal membrane permeabilization induced by FB1.These results indicate that lysosomal membrane permeabilization is the main factor of cell apoptosis induced by FB1,and different from SO and SA,ROS generated by mitochondrial division or damage mediated the lysosomal membrane permeabilization induced by FB1.To explore the relationship between ROS and the distribution of lysosomes,Hyper7 was used to label intracellular hydrogen peroxide(H2O2).To explore the impact of FB1 on lysosomal populations,live-cell imaging and 3D surface analysis were used to analyze lysosomal volume,acidity,and distribution.Four groups were selected based on lysosomal volume and acidity,two groups were selected based on co-localization with mitochondria,and two groups were selected based on distance from the nucleus.After cross-screening,detailed analysis was performed to identify which special lysosomal populations were affected by FB1.The results showed that H2O2 was mainly distributed around the nuclear periphery,and the distribution of lysosomes induced by FB1 was highly consistent with the distribution of H2O2.FB1 generally increased lysosomal volume and acidity,leading to lysosomal accumulation.FB1 did not increase the number of lysosomes in the mitochondrial subcellular region.Lysosomes in the non-mitochondrial subcellular region were more stable than those in the mitochondrial subcellular region.Under the influence of FB1,the volume of lysosomes in the mitochondrial subcellular region fluctuated greatly.Lysosomes in the peripheral region were more stable than those in the perinuclear region.In the perinuclear region,lysosomes in largevolume and high-acidity groups were more susceptible to FB1.In summary,like SO and SA,FB1 has cytotoxicity,induces lysosomal membrane permeabilization,mitochondrial damage,and cell apoptosis.The difference is that lysosomal membrane permeabilization induced by FB1 is mainly caused or mediated by ROS.FB1 induces lysosomal accumulation in the perinuclear region,increases volume,making them more susceptible to ROS-induced lysosomal membrane permeabilization,which is unrelated to lysosomal acidity.In conclusion,this study shows that the toxic effects of FB1 are related to the distribution of various lysosomal populations it induces,and ROS mediates the occurrence of lysosomal membrane permeabilization.These results provide insights into the interaction between organelles and the mechanism of FB1 toxicity,which may have potential significance for future treatment strategies.