To Explore The Mechanism Of Citrinin On Liver Injury In Mice Based On Endoplasmic Reticulum Stress Signaling Pathway

Background:Citrinin（CTN）is a mycotoxin that is often found in feed and food.It is widely polluted and has hepatotoxicity,nephrotosis and reproductive toxicity.However,the current research on the mechanism of CTN hepatotoxicity is incomplete.Therefore,this experiment took mice as the research object to explore the effect of CTN on liver injury in mice and its mechanism.Objective:The purpose of this study was to investigate the mechanism of endoplasmic reticulum stress（ERs）signaling pathway in CTN-induced liver injury in mice.Methods:Twenty Kunming（KM）male mice were randomly divided into 4 groups（n=5）after one week of pre-bringing,which were control group,low-dose CTN group（1.25 mg/kg CTN）,medium-dose CTN group（5 mg/kg CTN）and high-dose CTN group（20 mg/kg CTN）.After continuous gavage for 14 days,the mice were anesthetized for orbital blood collection,and then the liver tissue was taken out and stored in a-80°C refrigerator,and the following indicators are tested:The body weight,liver weight,liver index,blood aspartate transaminase（AST）,alanine aminotransferase（ALT）and the ratio of AST/ALT;Hematoxylin-Eosinstaining staining（H&E staining）to observe the morphological changes of mouse liver tissue,and fluoroscopy electron microscope to observe the ultramicroscopic structure changes of mouse liver tissue cells;kit to detect liver tissue changes in the activities of superoxide dismutase（SOD）and peroxidase（Catalase,CAT）,malondialdehyde（MDA）,nitric oxide（NO）and glutathione（Changes in Glutathione,GSH）content and total antioxidant capacity（Total antioxidant capacity,T-AOC）;fluorescent probe method to detect the content of reactive oxygen species（ROS）in liver tissue;TUNEL method to detect liver tissue cells Apoptosis changes,the kit detects the changes of Calcium ions(Calcium ions,Ca²⁺)in liver tissue,and WB method detects the marker proteins in the ERs signaling pathway,such as Caspase-12,Glucose Regulated Protein 78（GRP78/BIP）and C/Expression of EBP-homologous protein（CHOP）and key apoptosis proteins such as Bax,Bcl-2 and Caspase-3.On this basis,endoplasmic reticulum stress inhibitor was added to further clarify the liver injury mechanism of citrulin.20 Kunming（KM）male mice were randomly divided into 4groups（n=5）after being pre-bred for one week,namely the control group,the CTN group（5mg/kg CTN）,and the ERs inhibitor group(240 mg/kg 4-phenylbutyric acid（4-Phenylbutyric acid,4-PBA）,ERs inhibitor+CTN group（240 mg/kg 4-PBA+5 mg/kg CTN）.After continuous treatment for 14 days,the mice were anesthetized for orbital blood collection,and then the liver tissue was taken out and stored in a-80°C,and repeat the above indicators.Results:Compared with the control group,different doses of CTN caused a significant dose-dependent decrease in body weight and liver weight of mice（P<0.05）,but there was no significant change in the liver index（P>0.05）.It significantly rises in a dose-dependent manner（P<0.05）.Although the ALT activity and the ratio of AST/ALT increased,there were no significant differences compared with the control group（P>0.05）.The main manifestations are hepatocyte swelling,hepatic cord disorder,nuclear dissolution or fragmentation,etc.Ultramicrosc-opic observation shows that the endoplasmic reticulum of hepatocytes expands under the action of CTN.The above phenomena indicates that CTN can induce liver damage in mice.In addition,different doses of CTN increased the content of ROS（P<0.05）,MDA and NO（P>0.05）by reducing the activities of CAT（P>0.05）and SOD（P<0.01）and the content of GSH（P>0.05）,thereby breaking the homeostasis of the oxidative-antioxidant system in the liver of mice,resulting in a decrease in the level of T-AOC（P>0.05）.Finally,TUNEL assay showed that the apoptosis of mouse liver tissue increased significantly with the increase of CTN dose（P<0.01）,and the Ca²⁺content in liver tissue also increased significantly（P<0.01）;The ratio of key apoptosis proteins Bax/Bcl-2 and cleaved-caspase-3/pro-caspase-3 in liver tissue,the expression of endoparasitic reticulum stress signaling pathway marker proteins GRP78/BIP and CHOP,and the expression of cleaved-caspase-12/pro-caspase-12 ratio increased significantly with the increase of CTN dose（P<0.01）.However,compared with the5 mg/kg CTN group,the protein expressions of GRP78 and CHOP in the liver tissue of 4-PBA pretreated mice,as well as the ratio of cleaved-caspase-12/pro-caspase-12 and cleaved-caspase-3/pro-caspase-3 was significantly decreased（P<0.01）.Correspondingly,the degree of apoptosis in liver tissue was significantly decreased（P<0.01）,and the content of Ca²⁺in hepatocytes also showed a decreasing trend（P>0.05）;further observation found that the dynamic balance of the oxidation-antioxidant system destroyed by CTN and to a certain extent affected the level of T-AOC in the liver tissue increased（P>0.05）;while the hepatocytes in mice were enlarged,nuclear lysis and hepatocyte endoplasmic reticulum expansion,AST and ALT activities in serum and changes in AST/ALT ratio,and changes in mouse body weight,liver weight and liver index were reversed.Conclusion:The results of this experiment indicate that CTN can induce oxidative stress and regulate apoptosis in mouse liver through the Ca²⁺/endoplasmic reticulum signaling pathway.