Ammonia Exposure Exacerbates LPS-induced Myocardial Necroptosis In Chicken

With the rapid development of modern intensive aquaculture,the growth and health of livestock and poultry are extremely susceptible to the influence of the barn environment.Raising animals in poor conditions reduces the ratio of survival and speed of growth,and increases susceptibility to disease.For the broiler chickens,the humidity,the temperature,as well as air quality are the important factors that constitute the influence of the barn environment on the disease resistance of livestock and poultry,among which ammonia gas(NH3)is one of the most important factors.As a colorless toxic gas,NH3 has a strong pungent odor,which is mainly decomposited by feces and feed residue in poultry houses Living in the environment of high concentration NH3 too long,animals’ appetite would decrease,the speed of growing up slowed down,and may also induced respiratory diseases.Lipopolysaccharide(LPS)can cause body fever,changes in the number of white blood cells,shock and nervous system damage and so on.To investigate the effect of environmental NH3 exposure on LPS-induced programmed myocardial necrosis in chickens,eighty 1-day-old broilers were randomly divided into two groups,group C and group N,with 40 chickens in each group.Both groups were raised in the environmental control chamber(chamber C and chamber N,respectively).NH3 was continuously metered into chamber N,and the concentration of NH3 was controlled at 19.5-20.5 mg/m3 during0-3 weeks.The concentration of NH3 was controlled at 44.5-45.5 mg/m3 during 4-6 weeks.No additional NH3 was added to chamber A,and the concentration of NH3 was not exceeding 5 mg/m3.At 42 days of age,group A and group B were randomly divided into two groups,with 20 chickens in each group.Group C was devided into group C and group P,group N is divided into group N and group NP.200 mg/kg body weight LPS were intraperitoneal injected into Chickens in gourp P and group NP,and group C and group N were treated with normal saline by the same amount and method.After 5 h injection,the myocardial tissue was taken from the euthanasia chickens.HE method,real-time fluorescence quantitative PCR method and western blot method were used to detect myocardial tissue morphology,oxidative stress index(T-AOC,NO,T-NOS,i NOS,CAT,GPx,SOD and MDA),energy metabolism related index(PKR,PKC-α,NOX1,p47,ERK1,STAT3,PPAR-γ),inflammatory pathways related index(IKKβ,IκB-α,NF-κB,TNF-α,NLRP3)and parameters related to Th1/Th2 balance(IL-1β,IL-4,IL-5,IL-6,IL-12,IL-13,IFN-γ),parameters related to necroptosis pathways(RIPK1,RIPK3,Caspase1,Caspase8,JNK,NOD1),and the results were as follows:(1)Morphological observation results showed that there were myocardial cell necrosisaccompanied by neutrophil infiltration in group P,myocardial fiber rupture accompanied by cell necrosis in group N,and a large number of myocardial cell necrosis in group NP,which was significantly higher than that in group N and group P.NH3 can aggravate the inflammatory injury and necrosis of chicken myocardial tissue induced by LPS.(2)Compared with group C,T-AOC of group P and group N was significantly lower(P<0.05),CAT,GPx,SOD activity decreased(P<0.05),NO,MDA,T-NOS as well as i NOS increased significantly(P<0.05).The activity of T-AOC,GPx CAT,SOD in NP group was significantly lower than that in group P and group N(P<0.05),and the activity of MDA,T-NOS,NO,i NOS was significantly higher than those in group N and group P(P>0.05).The results showed that NH3 stimulation can aggravate LPS-induced the oxidative stress of heart organ in chickens.(3)LPS or NH3 stimulation can down-regulate the expression of PPAR-γ gene,and up-regulate the expression of PKR,PKC-α,NOX1,STAT3 and ERK1 genes(P<0.05),but have no effect on the expression of p47 gene(P>0.05).NH3 exposure caused further decrease of LPS-induced PPAR-γ gene expression(P<0.05),further increase of PKR,PKC-α,NOX1,STAT3,ERK1 genes expression than group C(P<0.05).The expression level of p47 gene than in group N is significant higher than group C(P <0.05).NH3 can aggravate the disorder of LPS-induced energy metabolism in chicken myocardium.(4)The expression levels of IL-1β,IL-4,IL-5,IL-6 and IL-13 were significantly increased by NH3 or LPS stimulation(P<0.05),and the expression levels of IL-12 and IFN-γ were significantly decreased(P<0.05).NH3 further significantly increased the expression levels of IL-1β,IL-4,IL-5,IL-6 and IL-13 genes induced by LPS(P 0.05),while the m RNA levels of IFN-γ,IL-12 were significantly lower than which were stimulated by NH3 or LPS(P<0.05).These elucidated that NH3 can aggravate LPS-induced imbalance of Th1/Th2.(5)The expression levels of IKKβ,NF-κB,TNF-α,NLRP3 were significantly increased by NH3 or LPS stimulation(P<0.05),and the expression level of IκB-α were significantly decreased(P<0.05).The expression levels of IKKβ,NF-κB,TNF-α,NLRP3 and IκB-α were further increased by the stimulation of NH3 and LPS collectively(P<0.05).The protein levels of IKKβ,IκB-α,NF-κB,TNF-α,NLRP3 were similar to the result of m RNA expression.These results suggest that NH3 can exacerbate the LPS-induced myocardial inflammatory response via NF-κB pathway.(6)The gene expression levels of Caspase1,JNK,RIPK1,RIPK3 and NOD1 were significantly increased by NH3 or LPS stimulation(P<0.05),while the gene expression levels of Caspase8 were significantly decreased(P<0.05).NH3 further significantly increased LPS-induced expression levels of Caspase1,JNK,RIPK1,RIPK3,and NOD1(P<0.05),while m RNA levels of Caspase8 were significantly lower than LPS or NH3(P<0.05).The protein levels of Caspase1,JNK,RIPK3 were similar to the result of m RNA expression.This suggests that NH3 exposure can aggravate programmed myocardial necrosis induced by LPS.In summary,NH3 exposure can cause oxidative stress and energy metabolism disorder in myocardium,activate inflammatory pathways and induce programmed necrosis of myocardium cells,and can aggravate the programmed necrosis of myocardium cells induced by LPS through the above pathways.This result enriched the toxicological data of NH3 and provided scientific basis for the prevention and treatment of NH3 poisoning.