Based On Autophagy To Explore The Mechanism Of Quercetin Alleviating Acrylamide-induced Liver Injury By Inhibiting Ferroptosis

Acrylamide(ACR)is a hazardous substance formed during the high-temperature processing of carbohydrate-rich foods.ACR has toxic effects on the human body that cannot be ignored,including liver toxicity,neurotoxicity,and carcinogenicity.Quercetin(QCT)is one of the most consumed flavonoids in daily diet and is commonly found in various fruits and vegetables.QCT can resist a variety of chemically induced inflammatory responses or damage,which attributed to its good antioxidant capacity.This article took ACR and QCT as the research object,detected the effect of QCT on ACR-induced liver injury through animal model and cell experiments.Then we predicted its potential mechanism through RNA-seq and further bioinformatics technology.Meanwhile,LC3 dual-fluorescent plasmid transfection was used to detect the effect of QCT treatment on autophagy in ACR-exposed Hep G2 cells.Finally,based on immunofluorescence,Western Blot and protein-ligand docking technology,the molecular mechanism of how QCT inhibited ACR-induced ferroptosis was further studied.The main findings are as follows:1.QCT significantly attenuated ACR-induced hepatotoxicity in vitro and vivo.Through Hematoxylin-Eosin(HE)staining analysis of mouse liver slices,detection of serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT),we found that QCT could significantly alleviate ACR-induced liver injury in vivo.At the same time,the detection of cell viability by CCK-8 showed that ACR significantly inhibited Hep G2 cell viability and was dose-dependent.10 μM QCT effectively alleviated the decrease in cell viability caused by 5 m M ACR.2.QCT alleviated ACR-induced liver injury by inhibiting ferroptosis.RNA-Seq and further bioinformatics analysis showed that QCT downregulated ACR-activated oxidative stress,autophagy,and ferroptosis pathways.At the same time,ACR showed similar signs of toxicity to the ferroptosis inducer Erastin in C57BL/6J mice and Hep G2cells: GSH content decreased while MDA content increased,the level of the ferroptosis marker GPX4 protein decreased and lipid peroxide content increased.In addition,both QCT and the specific inhibitor of ferroptosis Fer-1 were effective in mitigating ferroptosis caused by ACR or Erastin in HepG2 cells.These results suggested that QCT effectively inhibited ACR-induced ferroptosis and thus played a hepatoprotective role.3.QCT alleviated ferroptosis by inhibiting oxidative stress.Using the reagent DHE to detect the reactive oxygen species(ROS)in liver tissues and Hep G2 cells,we found that ACR significantly induced ROS accumulation.Using the ROS scavenger NAC as an inhibitor and using JC-1 reagent to detect the mitochondrial membrane potential(MMP),we further verified that ACR caused oxidative stress by inducing MMP reduction and ROS accumulation.In addition,we found that ACR could significantly induce the expression of the NRF2 protein.Similar to the effect of NAC,QCT significantly reduced the oxidative stress induced by ACR in Hep G2 cells.Interestingly,ACR-induced oxidative stress was attenuated while ferroptosis was inhibited under NAC treatment.In summary,QCT could mitigate ferroptosis by inhibiting ACR-induced oxidative stress.4.QCT alleviated ferroptosis by inhibiting oxidative stress-driven autophagy.We observed an increase in LC3 B protein expression in mouse liver slices.Based on EGFP-RFP-LC3 dual fluorescence transfection and Western Blot,we further found that ACR induced autophagy in Hep G2 cells.The autophagy-specific inhibitor CQ significantly reduced ACR-induced autophagy and inhibited ferroptosis,suggesting that ACR-induced ferroptosis was dependent on autophagy.At the same time,QCT and NAC inhibited ACR-induced autophagy by reducing oxidative stress,indicating that ACR-induced autophagy was driven by oxidative stress.Taken together,QCT inhibited autophagy and alleviated ferroptosis by inhibiting ACR-induced oxidative stress.5.QCT inhibited ferroptosis by reducing autophagy-dependent free iron(Fe2+)accumulation.Ferroptosis occurs by excess lipid peroxides produced by increased free iron.We found that QCT,the autophagy inhibitor CQ and the iron chelator DFO both significantly reduced ACR-induced free iron accumulation and inhibited ferroptosis.Based on Western Blot and protein-ligand docking technology,it was found that QCT might react with the autophagy cargo receptor protein NCOA4,thereby blocking the degradation of the iron heavy chain protein FTH1 to reduce the intracellular iron ion content,and ultimately alleviate ACC-induced ferroptosis.The aim of the present research was to explore the alleviation effect and mechanism of QCT on ACR-induced liver injury.This study implied that QCT recovered the increased ROS,excessive autophagy,and accumulation of free iron induced by ACR.More importantly,QCT reversed the depletion of GSH and significantly decreased the overproduction of Lipid ROS,which could effectively inhibit ferroptosis.Taken together,our results provided the first evidence that QCT established protective effect against ACR via inhibiting autophagy-dependent NCOA4/FTH1 ferroptosis pathway.The findings reported here provided innovative strategies on treating ACR-induced liver injury,especially by utilizing natural antioxidant compounds.These results might provide new ideas for the use of natural plant antioxidants to alleviate ACR-induced liver damage.It might also indicate innovative strategies for the development of dietary supplement products which focused on inhibiting ferroptosis to mitigate the toxicity of food contaminants.