Corynoline Attenuates Zearalenone-induced Liver Injury By Inhibiting Ferroptosis Through Regulating Nrf2/GPX4 Signaling Pathway

Zearalenone（ZEA）is a toxin produced by Fusarium fungi,widely present in foods such as corn,wheat,and peanuts,and is one of the most common pollutants.It can be absorbed by the human body with food,and has reproductive toxicity,liver toxicity,and teratogenic effects,causing significant harm to human health.Corynoline is an isoquinoline alkaloid isolated from the traditional Chinese medicine Corydalis.It has biological characteristics such as anti-inflammatory,antibacterial,and antioxidant properties,and can protect against liver damage caused by carbon tetrachloride,thioacetamide,and acetaminophen in mice.It is currently confirmed that zearalenone can induce liver damage through oxidative stress,apoptosis,autophagy,etc.However,it is unclear whether ferroptosis plays a role in zearalenone induced liver damage and whether corynoline can alleviate zearalenone induced liver damage.This study uses mouse hepatocytes and liver tissue as research models to reveal the mechanism of zearalenone-induced liver injury,explore the relationship between ferroptosis and liver injury,and clarify the effect of corynoline on zearalenone-induced liver injury in mice.protective effect.1.Study on the mechanism of zearalenone induced ferroptosis in mouse liver tissueConstruction of zearalenone-stimulated mouse liver injury model.The pathological changes of liver tissue,mouse transaminase（ALT and AST）levels,inflammatory factor（TNF-αand IL-1β）content,ferroptosis-related factors(GSH,GPX4,x CT,ATP,MDA and Fe²⁺)were detected,and The nuclear factor erythroid2-related factor（Nrf2）signaling pathway was analyzed.The results showed that compared with the control group,with the increase of concentration of zearalenone（10,20,40 mg/kg）,dose-dependent pathological damage to liver tissue occurred,with an increase in serum transaminase content and inflammatory factor content in liver tissue,indicating that zearalenone can cause liver tissue damage in mouse.Ferroptosis-related factors in mouse liver tissue were detected,and the results showed that the GSH,GPX4,x CT,and ATP contents of liver tissue decreased,and the MDA and Fe²⁺contents increased under the action of zearalenone.Analysis of ferroptosis-related signaling pathways revealed that zearalenone can reduce the expression levels of Nrf2 and heme oxygenase-1（HO-1）in liver tissue.In Nrf2-deficient mice,it can also lead to more severe liver tissue damage and ferroptosis.It shows that zearalenone can induce liver tissue damage and ferroptosis by inhibiting the Nrf2/GPX4 pathway.After adding the ferroptosis inhibitor Fer-1,the related indicators of mouse liver tissue were detected,and it was found that Fer-1could significantly inhibit the pathological changes,inflammatory response and expression of ferroptosis-related factors in liver tissue induced by zearalenone,indicating that the inhibition of ferroptosis reduces zearalenone-induced liver tissue damage.2.Study on the protective effect of corynoline on zearalenone-induced liver cell damage in mice.Using the mouse liver cell line AML-12 as the model and corynoline as the research object,the ultrastructure was observed,cell viability,inflammatory factors,ferroptosis related factors,ROS,mitochondrial membrane potential,apoptosis and autophagy related proteins were detected,and the Nrf2 signaling pathway was analyzed to observe its protective effect and mechanism against zearalenone induced liver cell damage.The results showed that corynoline（2,4,8μM）could reverse the inhibitory effect of zearalenone（40μM）on mouse liver cell activity in a dose-dependent manner and downregulate the levels of inflammatory factors,suggesting that corynoline can protect zearalenone-induced hepatocellular injury.Under the action of zearalenone,the levels of caspase 3 and p53 proteins related to apoptosis,as well as Beclin1 and p62 proteins related to autophagy,increased in mouse liver cells.However,after the addition of corynoline,these proteins did not change,indicating that corynoline had no effect on zearalenone induced cell apoptosis and autophagy.Mitochondrial electron microscopy observed that corynoline can alleviate the reduction in mitochondrial size caused by zearalenone.The results of detecting mitochondrial membrane potential showed that corynoline can alleviate the decrease in mitochondrial membrane potential caused by zearalenone.At the same time,corynoline can inhibit the production of MDA and ROS induced by zearalenone,and upregulate the expression of GPX4 and x CT,indicating that corynoline can alleviate ferroptosis induced by zearalenone.corynoline can also upregulate the protein expression of Nrf2 and HO-1.After knocking down the Nrf2 gene in liver cells with si RNA,the protective effect of corynoline on zearalenone-induced ferroptosis of hepatocytes is weakened,further indicating that corynoline can inhibit ferroptosis and protect zearalenone-induced liver cell damage by activating the Nrf2 signaling pathway.3.Protective effect of corynoline on liver tissue damage induced by zearalenone in miceUsing a mouse liver tissue injury model induced by zearalenone,analyze the protective effect of corynoline.Detected liver tissue pathological changes,transaminase levels,inflammatory factor content,ferroptosis related factors,and analyzed Nrf2 and NF-κB signal pathway.The results showed that corynoline（15,30,60 mg/kg）could dose-dependently alleviate liver tissue pathological damage induced by zearalenone in mice,reduce serum transaminase content and liver tissue inflammatory factor content,inhibit liver tissue ferroptosis induced by zearalenone,activate Nrf2 pathway,and inhibit NF-κB pathway.But after intervention with the SIRT1 inhibitor EX527（SIRT1 is an upstream molecule of the Nrf2 signaling pathway）,the effect of corynoline on liver tissue pathological changes,ferroptosis related factors,and inflammatory factors is weakened.Using the Nrf2 deficient mouse model,the above indicators were tested,and the results showed that the protective effect of corynoline on zearalenone induced ferroptosis and liver injury in liver tissue was weakened.Further in vivo experiments have confirmed that corynoline can protect liver tissue damage induced by zearalenone by activating the Nrf2 signaling pathway and inhibiting ferroptosis.In summary,zearalenone induces ferroptosis in hepatocytes and liver tissue by inhibiting the Nrf2/GPX4 pathway,causing liver damage.Corynoline can activate the Nrf2 pathway,inhibit zearalenone-induced ferroptosis through the system XC-/GPX4 pathway,lipid metabolism pathway,and ferroptosis pathway,and play a role in protecting liver damage.The results of this study provide certain theoretical basis and experimental data for the prevention and treatment of zearalenone-induced liver injury.