Study On The Mechanism Of Ammonia-Induced Inflammation,Autophagy,and Apoptosis Of Lymphatic Organs In Broilers

Ammonia(NH3),a colorless,water-soluble,and irritant alkaline gas,is produced by the reduction of nitrogenous-containing substances and bacterial decomposition of organic substances.NH3 is one of main environmental pollutants in chicken houses and threatens the health of humans and chickens.In addition,NH3 plays an important role in air pollution.It is a component of atmospheric aerosols and has a negative impact on human beings and other organisms.Birds are highly sensitive to NH3.NH3 in the air can inhibit the immunity of broilers,and even increase the mortality rate of broilers.Broiler lymphatic organs(bursa of fabricius(BF)and thymus)are crucial for immunity.However,biological mechanism of toxic effect of NH3 on chicken lymphoid organs(BF and thymus)is still unclear.Therefore,the aim of the present study was to explore the mechanism of toxicity of NH3 on broiler lymphatic organs(BF and thymus)from multiple perspectives(inflammation,apoptosis,and autophagy).135 experimental chickens(Ross 308)were randomly divided into three groups.The concentration of NH3 in each group was 5.0 mg/m3 NH3 in low NH3 group(control),10.0-15.0 mg/m3 NH3 in middle NH3 group,and 20.0-45.0 mg/m3 NH3 in high NH3 group,respectively.On the 14th,28th,and 42nd days of the experiment,the chickens were euthanized(45 in each group,every time)and the samples of BF and thymus tissues were collected.The harmful effects of NH3 on broiler lymphatic organs BF and thymus tissues were investigated by performed hematoxylin and eosin(HE)staining,transmission electron microscopy(TEM),terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay,immunofluorescent(IF)assay,enzyme-linked immunosorbent assay(ELISA),microplate spectrophotometry,qRT-PCR,and western blot.The following research contents were carried out:Morphological changes of inflammation,apoptosis,and autophagy in BF and thymuses,NO content,iNOS activity,the contents of inflammation-related factors(including interleukin 6(IL-6),IL-1β,and tumor necrosis factor alpha(TNF-α)),mRNA expression of inflammation-related factors((including nuclear factor kappa B(NF-κB),cyclooxygenase-2(Cox-2),toll like receptor 2A(TLR-2A),nitric oxide synthase(iNOS),IL-6,IL10,IL-18,and IL-1β),the contents and activities of oxidative stress-related indexes((including catalase(CAT),glutathione(GSH),glutathione peroxidase(GSH-Px),hydrogen peroxide(H2O2),malondialdehyde(MDA),superoxide dismutase(SOD),gamma glutamyl transferase(GGT),and total antioxidant capacity(T-AOC)),mRNA and protein expression of apoptosis-related factors(including p53,cytochrome C(Cyt-C),B lymphoma-2(Bcl-2),Bcl-2 X protein(Bax),Bcl-2 homologous antagonist killer protein(Bak),Caspase-3,caspase-9,cleaved casapase-3,and cleaved caspase-9),mRNA and protein expression of mitochondrion-related factors(including dynamic protein-related protein 1(Drp-1),mitotic fusion 1/2(Mfnl/2),mitochondrial fission factor(Mff),optic atrophy 1(Opal)),the activities of adenosine triphosphates(ATPases)(including Na+/K-.ATPase,Ca2+ ATPase,Mg2+ATPase,and Ca/Mg2+ ATPase),mRNA and protein expression of autophagy-related factors(including microRNA-99a-3p,rapamycin target(mTOR),microtubule-associated protein light chain light chain 3-Ⅰ(LC3-Ⅰ),LC3-Ⅱ,phosphate and tension protein homologs(PTEN),protein kinase B(AKT),autophagy-related-5(ATG-5),Becline-1 and Dynein),mRNA and protein expression of energy-related factors(including aconitase 2(ACO-2),hexokinase 1(HK-1),hexokinase 2(HK-2),lactate dehydrogenase A(LDHA),lactate dehydrogenase B(LDHB),pyruvate kinase(PK),phosphofructokinase(PFK)and succinate dehydrogenase complex-B(SDB),the activities of serum biochemical parameters(including alkaline phosphatase(AKP),aspartate aminotransferase(AST),Alanine aminotransferase(ALT)and gamma glutamyl transferase(GGT).The results showed that:1.NH3 impaired BF and thymus tissues,including causing inflammation,apoptosis,and autophagy.2.NH3 exposure increase H2O2,GGT,and MDA,and decrease CAT,GSH,GSH-Px,and T-AOC,led to oxidative stress in chicken lymphoid organs.3.Under NH3 exposure,NO content,iNOS activity,and the mRNA expression of inflammation-related factors(NF-κ,TNF-α,TLR-2A,Cox-2,IL-6,IL-1β,iNOS,IL-18 and IL-10)increased,inflammatory reaction occurred.4.NH3 exposure reduced mRNA and protein expression of Bcl-2,mfnl,mfn2 and opA1,and increased the mRNA and proteins expression of Bax,Bak,caspase-3,caspase-9,p-53,Cyt-C,Drp-1,and mff,as well as decreased protein expression of cleaved caspase-3 and cleaved caspase-9,caused apoptosis in chicken lymphoid organs.5.NH3 exposure inhibits mRNA and protein expression of autophagy-related factors(mTOR,LC3-Ⅰ,LC3-Ⅱ,PTEN,AKT,atpg-5,Becline-1 and Dynein),reduced CD8+ B and T lymphocytes,and decreased the activities of ATPase(Na+/K+ATPase,Ca2+ ATPase,Mg2+ATPase),and inhibits the expression of energy metabolism related genes(mRNA and protein)(ACO-2,HK-1,HK-2,LDHA,LDHB,PK,PFK and SDHB),resulted in autophagy in lymphoid organs of chickens.In addition,microRNA-99a-3p was involved in autophagy BF induced by NH3 via negatively regulating its target gene PTEN.(6)NH3 exposure increased the activities of serum biochemical indicators(ALT,AKP,AST),meaning that chickens were negatively affected by NH3.In summary,our findings displayed that,NH3 exposure impaired structural integrity and normal function of broiler lymphatic organs,mitochondrial disfunction and oxidative stress,disturb the process of energy metabolism and ATPase activities,as well as the reduce the CB8+ B and T-lymphocytes through inflammation,apoptosis and,autophagy.Taken together,our findings provide to assess the harmful effects of NH3 exposure on broilers,and clues for human pathophysiology.