Effects Of Oxidative Stress-mediated Energy Metabolism,Apoptosis,and Autophagy On Ammonia-induced Liver Injury In Chickens

Ammonia?NH₃?is the most harmful gas in livestock and poultry houses.Excess NH₃ reduces animal welfare,has adverse effects on animal health,and seriously affects the development of breeding industry.Chickens are sensitive to NH₃.Chickens grow in the environment of a high-concentration NH₃ for a long time,which can lead to a variety of diseases.Liver is the largest gland in the body,and plays an important role in metabolism,detoxification,and immunity.However,there are few studies on the toxic effects of NH3 on chicken livers.Thus,in this study,the NH3poisoning model was successfully established with chicken as the research object.Toxic effects of NH₃ on broiler livers were evaluated via investigating the effects and molecular mechanisms of NH₃exposure on oxidative stress,energy metabolism,apoptosis and autophagy.One hundred and eight healthy 1-day-old Ross 308 broilers were randomly divided into the low NH₃ group?the control group?,the middle NH₃ group,and the high NH₃ group.All chickens were kept in three different environmental control cabins.The NH3 concentration in the low NH3 group was kept below 5 mg/m³all time.The NH₃ concentrations in the middle NH₃ group:10±0.5 mg/m³ during 1-21 days old;20±0.5 mg/m³ during 22-42 days old.The NH₃ concentrations in the high NH₃ group:15±0.5mg/m³ during 1-21 days old;45±0.5 mg/m³ during 22-42 days old.Temperature and humidity in three environmental control cabins are the same.On the 14^th,28^th,and 42^nd days,chickens were euthanized and liver samples were collected quickly.Morphological changes of liver tissue were observed using light microscope and electron microscope;apoptotic cells were detected through TUNEL analysis;oxidative stress indexes and ATPase activity were detected with kits;mi RNA expression,m RNA and protein expression of energy metabolism,apoptosis and autophagy related genes were detected using quantitative Real-time PCR and Western blot.The main results are as follows:?1?Histopathological changes showed that excess NH₃ exposure caused apoptosis and autophagy of livers,leading to liver damage in broilers.?2?TUNEL analysis showed that excess NH₃ exposure increased the number of apoptotic cells and apoptotic rate in a dose-dependent manner.?3?Excess NH₃ caused significant changes in oxidative stress indexes?MDA,CAT,SOD,T-AOC,and GSH-Px?.Moreover,the oxidative stress indexes showed dose-and time-dependent changes with the increase of NH₃ concentration and exposure time,indicating that excess NH₃induced oxidative stress in chicken livers.?4?The results of energy metabolism indexes showed that NH₃ exposure significantly inhibited ATPase activities and the expression of energy metabolism-related genes in livers,and activated AMPK pathway,indicating that NH₃ exposure caused energy metabolism disorder and compensatively activated AMPK to meet cellular energy requirements.?5?The results of apoptosis indexes further confirmed from molecular level that NH₃ exposure triggered the mitochondrial apoptosis signaling pathway and induced apoptosis in chicken livers.Moreover,mi R-187-5p was first revealed to be involved in oxidative stress-mediated mitochondrial pathway apoptosis by targeting apaf-1.?6?The results of autophagy indexes from m RNA and protein levels further confirmed that NH₃exposure activated APMK/m TOR/ULK1 signaling pathway and induced autophagy in chicken liver tissues.In conclusion,excess NH₃ induced oxidative stress through disrupting the balance between oxidation and antioxidation,energy metabolism disorder,apoptosis via mitochondrial pathway,and autophagy through triggering AMPK/m TOR/ULK1 signaling pathway in chicken livers.NH₃exposure exerted toxic effects on livers through oxidative stress-mediated apoptosis and autophagy.