Ammonia Inhuced Apoptosis Of Porcine Intestinal Epithelial Cells IPEC-J2 By Promoting ROS Production

Ammonia is one of the main metabolic toxic products in animal intestines.Ammonia can cause dysfunction of many organs and different types of tissues and cells,and cause related diseases.The intestine is one of the most important organs in the animal body.The integrity of the structure of the intestine is closely related to animal immunity,metabolism and development.Different endogenous or exogenous stimuli can cause intestinal damage and cause related intestinal diseases.Therefore,studying the effect of ammonia on the structure and function of intestinal epithelial cells,and analyzing the mechanism of its possible impact,is of great significance for the treatment of intestinal diseases caused by excessive ammonia.In this study,porcine intestinal epithelial cells（IPEC-J2）were used as an in vitro research model to study the relationship between ammonia,free radical metabolism,intestinal epithelial cell apoptosis and intestinal barrier function,and to preliminarily explore the possible damage of ammonia to porcine intestinal epithelial cells The mechanism provides more reference for the prevention and treatment of intestinal diseases in pigs caused by ammonia.The main findings of this study are as follows:1.Ammonia reduces the antioxidant capacity and apoptosis of porcine intestinalepithelial cells（IPEC-J2）In this study,the change of cell viability and morphology after ammonia treatment was the first choice to determine the ammonia concentration of 40 m M as the optimal concentration of ammonia to induce cell damage.And further studied the effects of ammonia treatment on cell antioxidant capacity,channel proteins,apoptotic proteins,inflammatory factors and cell barrier function.The test results showed:（1）The ammonia-induced porcine intestinal epithelial cell ROS,MDA content significantly increased,SOD,GPx activity significantly decreased;mitochondrial membrane potential was significantly reduced,COX-2 expression was significantly up-regulated,intracellular calcium(Ca²⁺)The concentration increases significantly,and the rate of cell apoptosis increases;（2）The m RNA expression of ammonia transporter RHCG and aquaporin AQP8 is up-regulated,and ammonia enters the cell;apoptosis-related molecules Caspase-3,Caspase-8,Caspase-9 and Bax The m RNA and protein expression levels of Bcl-2 were significantly increased,and the m RNA and protein expression levels of the anti-apoptotic molecule Bcl-2 were significantly decreased;（3）The m RNA and protein levels of inflammatory factors IL-1β,IL-6 and IL-8 were significantly increased,Claudin-1 and ZO-1 expression levels were significantly reduced.The above results indicate that ammonia enters the cell,induces oxidative stress in porcine intestinal epithelial cells,reduces the cell’s antioxidant capacity,leads to a decrease in mitochondrial membrane potential（MMP）,an increase in Ca²⁺concentration,and activation of cell mitochondrial pathways and death receptor pathways.The two types of apoptosis destroy the integrity of the tight junction structure of intestinal epithelial cells,and at the same time promote the inflammatory response of the cells.2.The antioxidant NAC alleviates ammonia-induced oxidative damage and apoptosis of porcine intestinal epithelial cells（IPEC-J2）To further explore the mechanism of ammonia-induced oxidative damage and apoptosis in porcine intestinal epithelial cells,we used a powerful antioxidant—NAC（N-acetylcysteine）to treat the cells.As the precursor of GSH,NAC can effectively eliminate ROS.In this study,the optimal concentration of NAC was first screened out,and the cells were treated with this concentration and 40m M NH₄Cl to detect changes in cell antioxidant capacity,mitochondrial membrane potential,apoptosis-related factors,and structural proteins.（1）After ammonia stimulates the cells,adding NAC reduces the content of ROS and MDA in the cells,increases the activity of SOD and GPx,increases the level of mitochondrial membrane potential,significantly down-regulates the expression of COX-2,and intracellular calcium ions.The concentration of(Ca²⁺)is significantly reduced,and the rate of cell apoptosis is reduced;（2）The m RNA and protein expression levels of apoptosis-related molecules Caspase-3,Caspase-8,Caspase-9,and Bax are significantly reduced,and the apoptosis-inhibiting molecule Bcl-2 The expression levels of m RNA and protein of the protein increased significantly;（3）The expression levels of tight junction proteins Claudin-1 and ZO-1 increased significantly.The above results indicate that the antioxidant NAC helps to eliminate ROS in cells and greatly reduces the oxidative damage of ammonia to intestinal epithelial cells.In summary,ammonia can reduce the viability of porcine intestinal epithelial cells IPEC-J2,inhibit cell proliferation,and have serious toxic effects on cells.Ammonia reduces the antioxidant capacity of cells by inducing excessive production of ROS,reduces MMP,breaks Ca²⁺homeostasis,causes Ca²⁺accumulation,damages mitochondria,leads to apoptosis,and destroys the integrity of tight junctions between cells,causing intestines.Damage to epithelial cells.Ammonia-induced apoptosis of porcine intestinal epithelial cells may be the result of simultaneous activation of mitochondrial pathway and death receptor pathway.In addition,the antioxidant NAC can reduce the damage to porcine intestinal epithelial cells by eliminating ROS in cells.