| Ammonia is a harmful gas with irritating odor and higher ammonia concentration was found in intensive poultry houses.Although the toxicity of ammonia is well known,little attention has been given to the mechanism of ammonia poisoning in chicken immune organs(especially immune organs such as spleen).To evaluate the immunotoxicity of ammonia to chickens and investigate ammonia-caused inflammatory damage and autophagy of chicken spleens,in this study,a chicken model for ammonia poisoning was established,and three levels(including mi RNA,m RNA,and protein)were performed using q RT-PCR and western blot.The mechanism of ammonia-induced oxidative stress,energy metabolism disorder,inflammation damage,autophagy and compensatory increase of heat shock protein in chicken spleens was studied by observing the microstructures and ultrastructures,and detecting the levels of oxidative stress and energy metabolism related factors,Th1/Th2 balance related cytokines,inflammatory related factors,autophagy related factors,and heat shock proteins in chicken spleens.135 1-day-old commercial Ross broilers were randomly divided into the group with low ammonia,the group with middle ammonia,and the group with high ammonia,with 45 chickens in each group.Each group was given ad libitum access to drinking water and standard commercial diets,in which ammonia concentration in the group with low ammonia was 5 mg/m3 in 1-6 weeks,ammonia concentration in the group with middle ammonia was 10 mg/m3 in 1-3 weeks and 15 mg/m3 in 4-6 weeks,and ammonia concentration in the group with high ammonia was 20 mg/m3 in 1-3 weeks and 45 mg/m3 in 4-6 weeks.On the 14 th,28th,and 42 nd day of the experiment,15 chickens were randomly sacrificed from each group,and spleen tissue samples were collected.The tests are as follows: microstructures and ultrastructures,oxidative stress related indicators(T-AOC,GSH-Px,CAT,and SOD activity;and GSH,MDA,and H2O2 content),energy metabolism related indicators(Na+-K+ ATPase,Ca2+ ATPase,Mg2+ ATPase,and Ca2+-Mg2+ ATPase activity),mi RNAs(mi R-133 a,mi R-6615,and mi R-202-5p levels)and their target genes(LOC101747543,SMAD7,and PTEN m RNA levels),Th1/Th2 balance related cytokines(IL-1β,IL-2,IL-4,IL-6,and IFN-γ m RNA levels),inflammatory related factors(NO content;i NOS activity;i NOS,HO-1,NLRP3 inflammasome,NF-κB,TNF-α,COX-2,and PTGEs m RNA levels;and i NOS,HO-1,and NF-κB protein levels),autophagy related factors(LC3-I,LC3-II,p62,PTEN,p53,PI3 K,AKT,m TOR,Beclin1,Dynein,ATF3,ATG3,ATG5,ATG7,ATG10,ATG12,ATG14,and ATG16L1 m RNA levels;LC3-I,LC3-II,PI3 K,AKT,and m TOR protein levels)and heat shock protein(HSP27,HSP40,HSP60,HSP70,and HSP90 m RNA levels;HSP60,HSP70,and HSP90 protein levels)in spleen tissues.The experiment results indicate:(1)Ammonia poisoning caused microstructural and ultrastructural changes,and resulted in inflammatory damage and autophagy in chicken spleen tissues.(2)Ammonia poisoning caused the activity of T-AOC,GSH-Px,CAT and SOD,and the content of GSH decreased;and the content of MDA and H2O2 increased,which led to oxidative stress in chicken spleen tissues.(3)Ammonia poisoning caused the activity of Na+-K+ ATPase,Ca2+ ATPase,Mg2+ ATPase,and Ca2+-Mg2+ ATPase activity decreased,which led to energy metabolism disorder in chicken spleen tissues.(4)Ammonia poisoning increased the levels of mi R-133 a,mi R-6615 and mi R-202-5p,and down-regulated the m RNA levels of their target genes LOC101747543,SMAD7 and PTEN,respectively,in chicken spleen tissues.(5)Ammonia poisoning reduced the levels of IL-2 and IFN-γ,and increased the levels of IL-1β,IL-4 and IL-6,resulting in the breakdown of Th1/Th2 balance and drift to Th2 in chicken spleen tissues.(6)Ammonia poisoning reduced anti-inflammatory factor HO-1 m RNA and protein levels;and increased NO content,i NOS activity,i NOS,NLRP3 inflamma some,NF-κB,TNF-α,COX-2 and PTGEs m RNA levels,and i NOS and NF-κB protein levels,which resulted in inflammatory damage in chicken spleen tissues.(7)Ammonia poisoning reduced the levels of PI3 K,AKT,and m TOR,which led to the inhibition of PI3K/AKT/m TOR signaling pathway in chicken spleen s.(8)Ammonia poisoning increased the m RNA levels of LC3-I,LC3-II,p53,Beclin1,ATG14,ATG16L1,ATG12,ATG10,ATG5,ATG3,ATG7 and Dynein,and decreased the protein levels of ATF3 and p62,which resulted in autophagy in chicken spleens.(9)Ammonia poisoning increased the m RNA levels of HSP27,HSP40,HSP60,HSP70 and HSP90,and the protein levels of HSP60,HSP70 and HSP90.The body mobilized HSPs to fight ammonia poisoning.Elevation of HSPs may be a compensatory mechanism in chicken spleens.In summary,excess ammonia gas is immunotoxic to chicken spleen s,and ammonia poisoning leaded to oxidative stress,energy metabolism disorder,Th1/Th2 imbalance,inflammatory damage,autophagy,and HSPs increase in chicken spleens.In the case of ammonia poisoning,the body mobilized protective response of HSPs,and the increase of HSPs may be a compensatory mechanism against ammonia poisoning in chicken spleens. |
