| Patulin(PAT)is a mycotoxin produced by the genus Penicillium,Aspergillus,and Silkworm,commonly found in moldy fruits and their products.The target organs for PAT toxicity are mainly kidneys.The results of the study indicate that PAT induces cytotoxicity through oxidative damage and mitochondrial damage pathway,which can cause rise of ROS,decrease of mitochondrial membrane potential,ATP content,antioxidant enzyme activity and oxidative damage.In this study,the mechanism of ROS production and ROS clearance in PAT-induced HEK293 cells apoptosis were studied in detail based on the previous studies.(1)NADPH oxidase is a membrane-bound protein located in the plasma membrane and is the main enzyme that produces ROS.The expression of NADPH oxidase regulatory subunit p47 phox,catalytic subunit NOX2 gene and protein showed that the expression of p47 phox and NOX2 gene and protein up-regulated with the increase of PAT concentration,and the highest gene expression increased by 121.9% and 1387.7%.The highest protein expression was increased by 121.9% and 155.3%,respectively.Co-treatment with NADPH oxidase inhibitor DPI greatly attenuated PAT-induced renal cell toxicity.The experimental results showed that the cell viability of the DPI-PAT co-treatment group was increased by 24.4%,LDH release was decreased by 8.8%,the apoptosis rate was decreased by 17.5%,the mitochondrial membrane potential was increased by 17.4%,and the ATP content was increased by 25.1% compared with the PAT alone group.While intracellular ROS content decreased by 62%.PAT can up-regulate the production of ROS by regulating the expression of NADPH oxidase subunits.(2)Mitochondria are both targets of ROS and ROS generation sites.The regulation of gene expression of complex I and II by PAT increased first and then decreased with the increase of PAT concentration.The expression of complex IV increased with the increase of PAT concentration.The gene expression of complex III and V(ATP6、8)decreased with the increase of PAT concentration,and the protein expression of the complex was basically consistent with gene expression.This suggests that ROS produced by the plasma membrane may act on mitochondria,induce disordered expression of mitochondrial respiratory chain complexes,and stimulate product more ROS.(3)In vivo,ROS are in a dynamic balance of production and clearance under normal conditions.The ROS clearance system includes antioxidant enzymes and antioxidants.The experimental results showed that PAT significantly inhibited the activity of antioxidant enzymes SOD,CAT,GR,and GPx;PAT significantly reduced intracellular GSH content and increased GSSG content;except for SOD,exogenous ROS scavenger NAC significantly inhibited PAT induced antioxidant enzyme activity and antioxidant content decrease;MTT,LDH,Hoechst 33342 apoptotic staining,cell apoptosis rate results show that NAC can alleviate apoptosis induced by PAT in HEK293 cells;NAC clears excess intracellular ROS and regulates mitochondrial respiratory chain Complex gene and protein expression maintain normal mitochondrial function;caspase activity and gene expression,NAC can inhibit the increase of PAT-induced caspase activity and gene expression.The results showed that NAC can alleviate PAT-mediated apoptosis in HEK293 cells. |
