The Mechanism Of Rosmarinic Acid Inhibited Acrylamide Induced Oxidative Stress And Endoplasmic Reticulum Stress Alleviated Apoptosis Of BRL-3A Cells

Acrylamide(AA)is a common endogenous contaminant in food,and the liver is the primary target organ for the toxic effects of AA.AA stimulates the body to produce reactive oxygen species(ROS),activates oxidative stress(OS)and endoplasmic reticulum stress(ERS)response to induce apoptosis,which may be one of the mechanisms of AA's toxicity.Rosmarinic acid(Ros A)is a naturally occurring water-soluble polyphenol compound that has good antioxidant activity and can relieve the body's oxidative damage.The purpose of this research is to investigate the mechanism of AA-induced cytotoxicity of BRL-3A in rats.By adding the natural plant-derived antioxidant Ros A to study its protective effect on AA-induced OS and ERS-mediated BRL-3A apoptosis,it provides a theoretical basis for the mechanism of Ros A protecting the body from AA hepatotoxicity.The main research results of this paper are as follows:(1)The scavenging ability of Ros A on free radicals and its effect on the level of oxidative stress induced by AA in BRL-3A cells were studied.Ros A had a good scavenging effect on ABTS,DPPH,FRAP and ORAC free radicals,and the scavenging effect was obviously concentration-dependent.The higher the concentration of Ros A,the higher the free radical scavenging efficiency.Ros A pretreatment reduced the large amount of ROS produced by AA stimulated BRL-3A cells,increased the SOD and GSH activities of BRL-3A cells,and reduced the amount of MDA production,thereby enhancing the antioxidant capacity of cells and reducing the AA-induced oxidative stress of BRL-3A cells damage and lipid peroxidation.(2)The effect of Ros A on the CYP2E1 and MAPK signaling pathways in BRL-3A cells induced by AA was studied.Ros A bound to the active region of CYP2E1 through hydrophobic interaction,and had strong interactions with amino acid residues Ile114,Trp122,Leu368,Phe430,Arg435,Cys437,Ala438 and Gly439.Ros A reduced the protein expression of CYP2E1 in BRL-3A cells and inhibits the activity of CYP2E1 protein.AA increased the expression levels of MAPK pathway proteins p-JNK,p-ERK and p-p38,but Ros A and NAC(ROS inhibitor)pretreatment significantly reduced the expression levels of p-JNK,p-ERK and p-p38 proteins.It showed that Ros A can inhibit the MAPK pathway activated by AA by reducing the production of ROS,thereby protecting cells from AA-induced OS damage.(3)The effect of Ros A on endoplasmic reticulum stress and apoptosis of BRL-3A cells induced by AA was studied.Ros A pretreatment reduced the calcium ions induced by AA and maintains the homeostasis of calcium ions in cells.Ros A reduced the expression levels of IRE1? pathway proteins GRP78,p-IRE1?,TRAF2 and XBP-1s,and reduced the expression of ERS-mediated apoptosis-related proteins P-ASK1,Caspase-12 and CHOP.After NAC pretreatment,the expression of ERS-related proteins IRE1?,GRP78,TRAF2 and CHOP was significantly reduced,indicating that Ros A can inhibit AA-induced ERS response by reducing the production of ROS,thereby protecting cells from AA-induced ERS damage.Ros A pretreatment significantly reduced the apoptotic rate increased by AA,and it reduced the expression of apoptosis-related proteins Caspase-3 and Bax/Bcl-2.NAC and4-PBA(ERS inhibitor)pretreatment reduced the expression levels of apoptosis proteins Caspase-3 and Bax/Bcl-2,indicating that Ros A inhibits cell apoptosis by inhibiting OS and ERS,thereby preventing the hepatotoxicity of AA.In summary,Ros A had good free radical scavenging ability and antioxidant ability,effectively reduced the content of ROS and MDA produced by AA-induced BRL-3A cells,significantly increased the activity of SOD and GSH,and reduced the expression of CYP2E1 protein,thereby improving cell oxidative damage.Ros A inhibited the MAPK pathway activated by AA by reducing the expression of p-JNK,p-ERK and p-p38 proteins,and reduced the OS damage induced by AA.Ros A also reduced ERS damage by maintaining calcium ion homeostasis and inhibiting IRE1?pathway proteins.In addition,Ros A inhibited AA-induced apoptosis by reducing the rate of apoptosis and the expression of apoptosis-related proteins.In short,Ros A slowed down cell apoptosis by inhibiting OS and ERS,thereby preventing hepatotoxicity of AA.This research provided a theoretical basis for the role of Ros A in the field of food protection against AA hazards,and provided a reliable theoretical basis for the application of other natural plant-derived antioxidants in the field of AA protection.