Spatiotemporal Association Of Oxidative Stress,Ferroptosis And Inflammatory In Pelteobagrus Fulvidraco Induced By Ammonia Nitrogen

In the process of intensive aquaculture,about 75%of the input nitrogen can’t be used by the aquaculture animals.It accumulates in the aquaculture system in the form of feed residues and aquatic animal excreta,resulting in the normalization of excessive ammonia nitrogen in the aquaculture water body.Ammonia mainly exists in ionic state（NH₄⁺）and non-ionic state（NH₃）.Compared with NH₄⁺,NH₃ is easier to cross the cell membrane and cause harmful effects.Ammonia nitrogen is highly toxic to fish and can cause a large number of deaths.Based on the existing studies,the toxicity of ammonia nitrogen is mainly related to oxidative stress and excessive inflammatory response in fish,but its inducing mechanism is not clear.Ferroptosis is a newly discovered non apoptotic cell death mode involving iron,which is driven by lipid peroxidation,and the imbalance of cell redox homeostasis leads to oxidative damage.Our previous studies confirmed that ammonia nitrogen could induce ferroptosis in the head kidney macrophages of Pelteobagrus fulvidraco.In mammals,it has been proved that ferroptosis is a key regulatory signal pathway in the process of oxidative stress and inflammatory response,but there are few studies in aquatic animals.Liver and brain are the most important metabolic organs and detoxification organs of aquatic animals,and are also the main target organs for the toxic effects of ammonia nitrogen.It is still unclear how the time correlation between oxidative stress,inflammatory response and ferroptosis in fish under ammonia nitrogen stress,and whether there is tissue difference in the time expression of the three.This experiment took P.fulvidraco as the research object to explore the time correlation of the expression of oxidative stress,inflammatory reaction and ferroptosis in fish liver and brain under acute ammonia nitrogen stress,so as to provide theoretical reference for further explaining the mechanism of fish ammonia poisoning.1.Effects of ammonia nitrogen stress on the expression of oxidative stress,ferroptosis and inflammatory response genes in the liver of P.fulvidracoThrough 96 h acute ammonia nitrogen stress test,this experiment analyzed the expression of biochemical indicators and key regulatory genes related to oxidative stress,inflammatory response and ferroptosis in the liver of P.fulvidraco under different concentrations of ammonia nitrogen and different stress times,so as to provide theoretical reference for further explaining the mechanism of fish ammonia poisoning.Three kinds of ammonia nitrogen concentrations(0 mg L^-1,5.7 mg L^-1,28.5 mg L^-1)were set in the experiment for 96 h.This study showed that high concentration of ammonia nitrogen significantly reduced the survival rate of P.fulvidraco,increased the content of hydroxyl radical（·OH）in the liver of P.fulvidraco,and significantly increased the content of MDA and free Fe.The content of GSH was significantly reduced and had nothing to do with stress concentration.At the early stage of stress,the activities of antioxidant enzymes T-SOD and CAT were significantly increased,and at the later stage,the activities of antioxidant enzymes T-SOD and CAT were inhibited.In gene expression,the m RNA expression of antioxidant genes SOD and CAT was significantly up-regulated.The expression of ferroptosis gene TFR1 m RNA was significantly up-regulated,and the expression of GPX4 and System x_c^-m RNA was significantly inhibited.The m RNA expression levels of four inflammatory genes were significantly up-regulated.This shows that,on the one hand,ammonia nitrogen stress leads to intracellular free Fe overload,triggers Fenton reaction,increases the content of hydroxyl radical（·OH）and MDA,and forms lipid peroxidation,thus inducing oxidative stress.On the other hand,ammonia nitrogen significantly reduced the content of GSH,resulting in decreased expression of System x_c^-and GPX4,resulting in arachidonic acid（AA）oxidation and NF-κB pathway is activated to cause inflammatory reaction.2.Effects of ammonia nitrogen stress on the expression of oxidative stress,ferroptosis and inflammatory response genes in the brain of P.fulvidraco.Through 96 h acute ammonia nitrogen stress test,the study analyzed the expression of biochemical indicators and key regulatory genes related to oxidative stress,inflammatory response and ferroptosis in the brain of P.fulvidraco under three ammonia nitrogen concentrations and different stress times.The results showed that ammonia nitrogen significantly increased the content of hydroxyl radical（·OH）and free Fe in the brain of P.fulvidraco.In the early stage,T-SOD and CAT activities were up-regulated,and in the late stage,T-SOD and cat activities were inhibited.High concentration of ammonia nitrogen significantly increased the content of GSH in the brain of P.fulvidraco at 12 h.In terms of gene expression level,ammonia nitrogen significantly increased the expression of SOD and CAT m RNA,and started quickly,which was significantly higher than that of CON group at 1 h.Ammonia nitrogen significantly increased the expression of brain ferroptosis related genes GPX4 m RNA and System x_c^-m RNA,which also started at 1 h.However,the key gene TFR1 m RNA in the Fe transport pathway started later than GPX4 and s System x_c^-（started at 3 h）.Ammonia nitrogen significantly up regulated the key inflammatory factor NF-κB p65,TNF,COX-2 and LOX-15b were expressed,but the m RNA activation of LOX-15b gene was later than that of the other three genes.This indicates that ammonia nitrogen can significantly increase the content of free iron,generate free radicals through Fenton reaction to participate in lipid peroxidation,or activate COX-2,LOX-15b and other factors to participate in lipid peroxidation,leading to inflammatory reaction.When the body is stimulated by ammonia nitrogen,it will trigger cellular immunity and produce hydroxyl free radicals（·OH）and MDA,resulting in the imbalance of antioxidation in the body and the occurrence of oxidative stress.3.Spatiotemporal correlation of oxidative stress,ferroptosis and inflammatory response in P.fulvidraco under ammonia nitrogen stressThe results showed that the liver and brain of P.fulvidraco had tissue specificity at 1 h,3 h,48 h and 96 h.In terms of temporal and spatial expression,the earliest ferroptosis in the liver was initiated first（1 h）,followed by inflammatory reaction（3h）,and oxidative stress（6 h）.In the 12 h-24 h period,inflammatory reaction was dominant,and in the 48 h-72 h period,ferroptosis occurred violently.This shows that ferroptosis first occurs in the liver of P.fulvidraco,and then the inflammatory reaction begins to occur.The body starts the antioxidant system to deal with oxidative stress.When the antioxidant system of P.fulvidraco is not enough to deal with strong oxidative stress,it will cause irreversible damage to the antioxidant system.The sequence of temporal and spatial expression in the brain was that inflammation and ferroptosis occurred simultaneously（1 h）,and oxidative stress was subsequently initiated（3 h）.This shows that under ammonia nitrogen stress,inflammation and ferroptosis first occur in the brain,and then the body starts the antioxidant system.When the antioxidant system of P.fulvidraco is insufficient to cope with strong oxidative stress,then persistent inflammatory reaction occurs.