Mechanism Of L-Selenomethionine Alleviating Intestinal Injury In Pigs Caused By Ammonia Exposure

Ammonia is recognized as the main harmful gas in livestock production.The intestine,as the largest digestive and absorption organ for nutrients,is also one of the target organs for ammonia poisoning.However,the potential toxicity of ammonia poisoning to the intestine remains unclear.L-selenomethionine is an important source of organic selenium,which has the advantage of being highly bioavailable,safe and efficient.Although environmental ammonia exposure has been associated with changes in intestinal media,the effect of Lselenomethionine on the regulatory mechanisms of ammonia-induced physiological responses in intestinal cells has not been reported.In order to comprehensively analyze the toxic effects of ammonia and its mechanisms,and to screen for effective antidotes,a multidimensional ammonia toxicity model was established in this study,in which L-selenomethionine was used to intervene in ammonia toxicity from two dimensions: in vivo and in vitro.In the in vivo experiment,twentty-four 125-day-old fattening pigs were selected and randomly divided into four groups: control group,selenium group,ammonia group and ammonia + selenium group.In the in vitro experiments,IPEC-J2 cell line culture was performed and divided into two models with six groups each.Model I-programmed necroptosis model: namely control group,selenium group,ammonium chloride group,ammonium chloride + selenium group,ammonium chloride + NAC group and ammonium chloride + Nec-1 group;Model II-ferroptosis model:namely control group,selenium group,ammonium chloride group,ammonium chloride +selenium group,ammonium chloride + ML334 group,ammonium chloride + Fer-1 group.On the basis of this grouping,a preliminary pathological assessment of ammonia-intoxicated small intestinal tissues and IPEC-J2 cells was firstly performed,followed by the use of various cutting-edge experimental techniques to explore and validate the molecular mechanisms of action of ammonia exposure on intestinal injury and the protective mechanisms of Lselenomethionine from the perspectives of oxidative stress,necroptosis,ferroptosis,Nrf2 pathway,Th1/Th2 imbalance and inflammation.This study not only revealed a new mechanism of ammonia toxicity and enriched the toxicological theory of ammonia,but also pointed out the direction to elucidate the molecular mechanism of L-selenomethionine to reduce ammoniainduced enterotoxicity.Meanwhile,this study also improved the immunity,production performance and economic efficiency of livestock,provided a guarantee for healthy and green breeding,and provided a direction for feed development.The results indicated that:(1)Ammonia exposure increased the levels of MDA and ROS in pig small intestinal tissues and IPEC-J2 cells,decreased the activities of antioxidant enzymes(SOD,GSH-Px)and the content of GSH,and significantly altered the expression of Nrf2 signaling pathway(Keap1,Nrf2,HO-1),necroptosis(RIPK1,RIPK3,MLKL,and Caspase-8),inflammatory factors(IFN-γ,IκBα,IL-2,TNF-α,IL-4,IL-6,IL-1β,NF-κB p65,IKKβ,i NOS,and COX-2),intestinal tight junctions(Claudin-1,Occludin,and ZO-1)and other related genes,suggesting that ammonia exposure inhibited Nrf2/Keap1/HO-1 signaling pathway and induced necroptosis by causing oxidative stress,thus mediating intestinal inflammation and destroying intestinal barrier.(2)Ammonia exposure also upregulated the levels of iron,ROS,and LPO in pig small intestinal tissues and IPEC-J2 cells,inhibited the Nrf2 pathway,and significantly changed the expression of ferroptosis(TFR-1,FPN-1,FTH1,SLC7A11,GPX4,ACSL4)and intestinal tight junctions(Claudin-1,Occludin,ZO-1)related genes,indicating that ammonia exposure could lead to intracellular iron overload,lipid peroxidation and ROS accumulation in pig intestinal cells,induce cellular ferroptosis by regulating the Nrf2 pathway,and eventually damage the intestinal barrier.(3)L-selenomethionine down-regulated the levels of MDA,ROS and iron in pig small intestine tissue and IPEC-J2 cells,increased the activities of antioxidant enzymes(SOD,GSHPx)and the content of GSH,and partially restored the expression of genes related to Nrf2 signaling pathway,necroptosis,ferroptosis,inflammatory factors,and intestinal tight junctions,indicating that the addition of L-selenomethionine could effectively attenuate ammoniainduced intestinal oxidative stress,and antagonized cellular necroptosis and ferroptosis by regulating ROS level and Nrf2/Keap1/HO-1 pathway,thus reducing ammonia exposureinduced intestinal inflammation and intestinal barrier damage in pigs.In conclusion,intestinal tract is one of the target organs of ammonia toxicity.Ammonia exposure led to oxidative stress,lipid peroxidation,excessive accumulation of ROS and inhibited of the Nrf2 pathway in pig small intestine tissue and IPEC-J2 cells,which induced necroptosis and ferroptosis and targeted the inflammatory response leading to disruption of intestinal junctions,thereby damaging the intestinal barrier.L-selenomethionine could effectively reduce the intestinal damage caused by ammonia exposure by regulating oxidative stress/necroptosis/ferroptosis/inflammatory axis,thus antagonizing the intestinal toxicity of ammonia.This study not only revealed a new mechanism of ammonia toxicity,but also enriched the toxicological theory of ammonia,and provided a theoretical basis for elucidating the molecular mechanism of L-selenomethionine alleviating ammonia-induced enterotoxicity.