Expression Of Ferroptosis Related Genes In Head Kidney Macrophages Of Pelteobagrus Fulvidraco Induced By Ammonia Nitrogen Stress

Water environment pollution is an important cause of fish diseases,which has a serious impact on aquaculture industry.It needs timely treatment and control to remedy.As an important pollutant in aquaculture water environment,ammonia nitrogen causes oxidative stress,tissue damage,inflammatory reaction and physiological metabolism disorder in fish.Ferroptosis is an iron dependent cell death mode characterized by the accumulation of reactive oxygen species and lipid peroxides,which plays an important role in diseases.Studies have shown that macrophages are activated when ferroptosis occurs,which promotes the release of inflammatory factors and the accumulation of reactive oxygen species and lipid peroxides.Macrophages also accumulate when ammonia nitrogen stress occurs.Pelteobagrus fulvidraco is one of the most important economical fishes in our province and even in our country.The high-density intensive culture mode has become an important reason for its ammonia nitrogen pollution of water environment.Quercetin,as a natural flavonoid,has the effect of inhibiting or slowing down iron death.So far,ferroptosis in fish diseases has not been elucidated,but related studies have shown that some important genes,metabolites and signal networks of iron related death in fish are similar to those in vertebrates.In view of this,aiming at the treatment strategy of vertebrates,this study used ammonia nitrogen as the stress source and quercetin as the inhibitor to preliminarily explore the effect of ammonia nitrogen on ferroptosis related genes of P.fulvidraco macrophages and the effect of quercetin on them,which can provide reference for the prevention and treatment of fish-related diseases.1.Effects of ammonia nitrogen on the expression of ferroptosis genes in head kidney macrophages of P.fulvidraco（1）Determine the half-inhibitory concentration of ammonia nitrogen on macrophages in the head kidney of P.fulvidraco(24h IC₅₀):There were 9 concentration gradients in the experiment,namely the control group（complete medium,0 mg/L）and the experimental group（0.01mg/L,0.02mg/L,0.04mg/L,0.08mg/L,0.16mg/L,0.32 mg/L,0.64mg/L and 1.28mg/L）,the macrophages of the head kidney of the P.fulvidraco treated with different concentrations of ammonia nitrogen were cultured in a 28℃,5%CO2 incubator for 24h.The MTT method was used to determine the effect of ammonia nitrogen on the cell viability of P.fulvidraco head kidney macrophages,and then the Graph Pad 5 software was used to perform nonlinear regression analysis of the ammonia nitrogen concentration with Log logarithm,and the concentration-effect curve was fitted to obtain the half inhibitory concentration(24h IC₅₀).The result shows:24h IC₅₀=0.23mg/L.（2）The effect of ammonia nitrogen on the expression of ferroptosis genes in the head kidney macrophages of P.fulvidraco:According to the results of 24 h IC₅₀determination,four concentration gradients were set,namely control group（complete medium,0 mg/L）,0.12 mg/L,0.23 mg/L and 0.46 mg/L.the stress treatment time was 24h.MTT assay was used to detect macrophage activity,DCFH-DA fluorescent probe was used to detect ROS,GSH detection kit was used to detect GSH content,and q PCR was used to detect ferroptosis gene expression（System Xc,GPX4,15-LOXb,TFR1,FTL and NCOA4）.The results showed that With the increase of ammonia nitrogen concentration,the cell viability decreased significantly（P<0.05）;With the increase of the concentration of ammonia nitrogen,the level of ROS increased significantly（P<0.05）,and reached the highest level at 0.23mg/l.Compared with the control group,different concentrations of ammonia nitrogen could significantly reduce the GSH content of head kidney macrophages of P.fulvidraco（P<0.05）.The System Xc m RNA level was significantly decreased with the increase of ammonia concentration（P<0.05）;GPX4 m RNA level was significantly increased（P<0.05）,and the highest expression was at 0.23mg/l.The m RNA level began to decrease significantly at 0.46mg/l（P<0.05）.15-LOXb m RNA level was significantly up-regulated at 0.12mg/l of ammonia nitrogen（P<0.05）,TFR1 and NCOA4m RNA levels were significantly up-regulated at 0.12mg/l of ammonia nitrogen（P<0.05）,and down regulated at 0.23mg/l of ammonia nitrogen（P<0.05）,the expression of FTL m RNA was down regulated by different concentrations of ammonia nitrogen（P<0.05）.Conclusion:Different concentrations of ammonia nitrogen can promote the occurrence of ferroptosis by activating the metabolic pathways of ferroptosis.When treated with low concentration,15-LOXb m RNA level in lipid metabolism signaling pathway was increased to catalyze lipid peroxidation,the TFR1 m RNA level of iron metabolism signaling pathway was increased,FTL and NCOA4 m RNA levels were decreased,fenton reaction between excessive iron ionons and hydroxyl radicals was promoted,and ROS production was increased,then,in high concentration group,System Xc m RNA level was decreased,GSH synthesis was limited,GPX4 m RNA level was decreased,antioxidant system activity was decreased,and ferroptosis was promoted.2、Effects of quercetin on ferroptosis gene expression levels in head kidney macrophages induced by ammonia nitrogen stress of P.fulvidracoIn the first chapter,the half inhibitory concentration(IC₅₀)of ammonia nitrogen on head kidney macrophages of P.fulvidraco was determined to be 0.23mg/l in 24h.The ammonia nitrogen(IC₅₀)treatment group was divided into control group（0 mg/L）、ammonia nitrogen(IC₅₀)group、quercetin（50μmol/L）、quercetin（50μmol/L）plus ammonia nitrogen group.They were treated with 5%CO2 and 28℃for 6h,12h,18h and 24h.The intracellular ROS was detected by DCFH-DA fluorescent probe,the GSH content was detected by GSH detection kit,and the m RNA expression of ferroptosis related genes（System Xc,GPX4,15-LOXb,TFR1and FTL）was detected by q PCR.Results:（1）There was no significant difference in reactive oxygen species（ROS）among the groups at 6h（P>0.05）.The level of ROS in the ammonia nitrogen group increased gradually from 12h to 18h,which was significantly higher than that in the other three groups（P<0.05）.There was no significant difference in reactive oxygen species（ROS）among the groups at 24h（P>0.05）.（2）GSH content in 6h,12h,18h and 24h ammonia nitrogen groups decreased significantly（P<0.05）,and showed a decreasing trend with the action time.GSH content in quercetin group and quercetin plus ammonia nitrogen group increased significantly（P<0.05）,and showed a decreasing trend with the action time.（3）Compared with the control group,the m RNA level of System Xc in each group was significantly increased at 6h（P<0.05）,the m RNA level of System Xc in ammonia nitrogen group was significantly increased at 12h（P<0.05）,and the expression level of System Xc in quercetin plus ammonia nitrogen group was significantly increased at 6h and 24h（P<0.05）.The GPX4 m RNA level in ammonia nitrogen group was significantly lower than that in other groups at 18h（P<0.05）,and the GPX4 m RNA level was significantly increased after quercetin intervention（P<0.05）.Compared with the control group,15-LOXb m RNA level in the ammonia nitrogen group increased from 6h to 12h,decreased significantly from 18h to 24h,and then increased.In the quercetin group,15-LOXb m RNA level increased significantly from 6h（P<0.05）and then decreased,which were higher than those in the ammonia nitrogen group and the control group.In the quercetin plus ammonia nitrogen group,15-LOXb m RNA level decreased significantly at12h（P<0.05）,the expression level was significantly increased（P<0.05）.Compared with the control group,TFR1 m RNA level increased at 6h and 24h in the ammonia nitrogen group,and increased significantly at 6h（P<0.05）.The expression trend was that TFR1 m RNA level decreased at 6h and then increased at 24h;In the quercetin plus ammonia nitrogen group,TFR1m RNA level decreased significantly at 6 h（P<0.05）and increased significantly at 24h（P<0.05）.Compared with the control group,FTL m RNA level increased from 12h to 24h in the ammonia nitrogen group,and decreased from 6h to 12h in the quercetin plus ammonia nitrogen group（P<0.05）.Conclusion:Quercetin can control intracellular iron overload and ROS overproduction by regulating the m RNA level of ferroptosis signaling pathway factor,that is,inhibiting the over expression of TFR1 m RNA and up regulating FTL m RNA;System Xc m RNA level in amino acid signaling pathway was increased,GSH synthesis was promoted,and GPX4 m RNA level was up-regulated;In addition,quercetin could also up regulate the 15-LOXb m RNA level in lipid metabolism pathway,thereby enhancing the activity of antioxidant system and improving the ferroptosis induced by ammonia nitrogen in head kidney macrophages of P.fulvidraco.