Effects Of Excess Iron On Oxidation-antioxidant Function Of Chicken Embryo Primary Hepatocytes And Liver

As an indispensable trace element in the process of life activities,iron has a variety of important physiological functions.The body mainly carries oxygen through the combination of iron divalent ion(Fe²⁺)and hemoglobin.Excess iron will destroy iron homeostasis and cause pathological damage to tissues and organs.Iron is mainly stored in the liver,playing a crucial role in iron metabolism regulated by a variety of iron-related proteins.This experiment was conducted to investigate the effects of iron excess on liver oxidation and antioxidant function of chicken embryo primary hepatocytes and10-day-old SPF chicks from both in vitro and in vivo.In vitro experiments,chicken embryo primary hepatocytes were prepared under aseptic conditions,and iron excess model of chicken embryo primary hepatocytes was established by CCK-8 and Calcein AM.The optimal Fe²⁺concentration and action time were determined.Chemical colorimetry was used to determine the contents of NO,MDA and H₂O₂and the activities of CAT,SOD,GPx,i NOS and T-AOC.The m RNA expression levels of CAT,GPx4,i NOS,p53,SLC7A11,Tf R1 and Nrf2 were detected by q PCR.The contents of GPx4 and i NOS were detected by ELISA.Western Blot was used to detect CAT protein expression.Thirty 1-day-old SPF chicks were randomly divided into two groups:control group and iron excess group.Chicks in iron excess group were fed 300 mg/m L normal saline with Fe SO₄·7H₂O for three consecutive days at 10days of age.At 7,14 and 21 days after iron excess,5 chicken were randomly selected to calculate the organ index and observe the liver pathological changes,which were observed by paraffin section,Prussian blue staining and HE staining.MDA,NO,GPx,CAT and T-AOC in liver were detected by chemical colorimetry.Main results of the experiment are as follows:（1）The iron excess model of chicken embryo primary hepatocytes was successfully established in this experiment.The cells reached their logarithmic growth phase after 72 h of culture and were given a certain concentration of Fe²⁺.Cell viability and LIP relative level decreased after iron excess.According to the cell viability and LIP,the concentration of Fe²⁺was determined as 40μmol/L and the reaction time was 24 h,36 h and 48 h.（2）In vitro studies showed that the contents of MDA,NO,H₂O₂and i NOS in chicken embryo primary hepatocytes increased after iron excess,levels of T-AOC,GPx,CAT and SOD decreased,and them RNA and protein expressions of i NOS,GPx4 and CAT also decreased,which indicated that oxidants increased,the contents of antioxidants decreased in chicken embryo primary hepatocytes after ironexcess.Oxidative stress occurred and induced ferroptosis.（3）In vivo studies showed that after iron excess,the contents of MDA and NO in liver of chicks increased,the activities of CAT and GPx and T-AOC in liver decreased,and the pathological changes such as inflammatory cell infiltration,connective tissue hyperplasia and hemosiderin deposition occurred in liver.It indicated that excess iron caused pathological damage to liver,imbalanced the oxidation-antioxidant reaction.