| Sudden death syndrome(SDS)in broilers is characterized by rapid death after sudden and transient flapping of the wings in well-nourished and seemingly healthy broilers,and the occurrence of the disease is not significantly seasonal.SDS is a common disease associated with cardiac dysfunction in broilers and one of the leading non-infectious causes of broiler death,with an incidence of 0.5 – 5%,causing significant economic losses to the poultry breeding industry.Due to the acute onset of SDS and no obvious clinical symptoms,it is difficult to obtain fresh clinical cases.Based on literature reports and previous proteomics sequencing results of the research group,it is found that fast large broilers are more likely to have acid-base balance disorders under greater metabolic pressure.Compared with healthy broilers,broilers that died of sudden death syndrome had significantly higher blood lactate content.As the main product of glycolysis,lactate will aggravate lactate accumulation and then change the body p H under metabolic stress and stress,resulting in acidosis and cell damage in myocardial cells.Therefore,in this study,we used an acidotic cardiomyocyte injury model to investigate the mechanism of myocardial injury when SDS occurs.The research group previously found that SDS broilers had disorganized and broken myocardial fibers,kinetic disturbances and structural damage in mitochondria.In order to deeply explore the causes and mechanisms of SDS,it is planned to study from the following aspects.1.Effect of lactic acid on injury of primary myocardial cells of chicken embryoChicken embryo primary cardiomyocytes were cultured,cell isolation and culture conditions were optimized and a cell model of lactic acidosis was established.Cardiomyocytes were treated with 10 m M,20 m M,and 40 m M lactic acid,and the cells were collected for subsequent assays.(1)optical microscopy was used to observe the changes in cardiomyocyte morphology and cardiomyocyte contraction frequency after 24 hours of lactate treatment;(2)flow cytometry and Western blot(WB)were used to detect the apoptosis of cardiomyocytes after lactate treatment;(3)transmission electron microscopy was used to observe the changes in the number of mitochondria in cells;(4)flow cytometry was used to detect the changes in cardiomyocyte mitochondrial membrane potential levels after lactate treatment;(5)WB was used to detect the expression levels of cardiomyocyte mitochondrial kinetic-related proteins Drp1,Fis1,Opa1,and Mfn2 proteins;(6)ROS detection kit was used to detect the effect of lactate on ROS levels in cardiomyocytes;and(7)transmission electron microscopy and WB were used to detect the effect of lactate on cardiomyocyte mitophagy.The results showed that after broiler primary cardiomyocytes were treated with different concentrations of lactic acid,(1)compared with the control group,with the increase of lactic acid concentration,the stereoscopic sensation of broiler primary cardiomyocytes in the lactic acid-treated group gradually disappeared,the refraction gradually decreased,and the frequency of cardiomyocyte contraction significantly decreased(P < 0.01);(2)after 24 hours of lactic acid treatment of cardiomyocytes,the DNA damage repair enzyme PARP-1 in cardiomyocytes was significantly down-regulated,and the expression of cleaved Caspase-3,a key protein of apoptosis,was significantly down-regulated(P < 0.01),and the flow cytometry results showed that the apoptosis rate of cardiomyocytes significantly increased with the increase of lactic acid concentration(P < 0.01);(3)ultrastructural results showed that lactic acid could change the mitochondrial morphology of chicken embryo primary cardiomyocytes and significantly reduce the number of mitochondria(P < 0.01);(4)lactic acid could lead to a significant decrease in mitochondrial membrane potential(P < 0.01);(5)WB results showed that lactic acid treatment of cardiomyocytes could up-regulate the expression levels of mitochondrial split proteins Drp1 and Fis1(P <0.01),And it was time-dependent;however,the expression levels of the fusion proteins Mfn2 and Opa1 were significantly down-regulated with time(P < 0.01);(6)After lactate treatment of cells for 24 h,the ROS level increased,which was significantly higher than that of the control group at 40 m M(P < 0.01);(7)Compared with the control group,the number of intracellular autophages was significantly increased in the lactate treatment group,and the p62 expression was significantly down-regulated and the LC3 expression was significantly up-regulated(P < 0.01).2.Protective effect of inhibition of mitochondrial division on myocardial damage induced by lactic acidTo further investigate the effect of mitochondrial division and fusion on cardiomyocytes,cardiomyocytes were co-treated with 5 μMMdivi-1(mitochondrialdivisioninhibitor1,an inhibitor of mitochondrial division-related protein Drp1)with different lactate concentrations to study the effect of Drp1 on cardiomyocyte injury.(1)CCK-8 was used to determine the effect of inhibiting mitochondrial division on cardiomyocyte survival;(2)immunofluorescence was used to observe the state of mitochondrial networking after inhibiting mitochondrial division;(3)WB was used to detect changes in cleavedcaspase-3 expression;and(4)changes in cardiomyocyte ROS levels were detected after inhibiting mitochondrial division.The results showed that after inhibition of cardiomyocyte mitochondrial division using Mdivi-1,(1)Mdivi-1 could reduce cardiomyocyte mortality induced by lactate;(2)co-treatment of cardiomyocytes with 20 m M lactate and Mdivi-1 could effectively restore the network state between intracellular mitochondria;(3)the expression level of cleavedcaspase-3 was relatively low and did not change significantly in the untreated Mdivi-1 alone treatment group;the expression level of cleavedcaspase-3 was significantly increased after treatment with lactate alone(P < 0.01),and the expression of cleavedcaspase-3 was significantly decreased after co-treatment with Mdivi-1 and lactate compared with the lactate alone treatment group(P < 0.05);(4)Mdivi-1 could significantly reduce ROS in cardiomyocytes treated with lactate compared with the lactate alone treatment group.3.The model of sudden death syndrome of broilers was establishedAccording to the results of the acidosis in vitro model,an in vivo model of sudden death in broilers was preliminarily established using ammonium chloride gavage.(Fig.1)to detect arterial blood p H,serum bicarbonate concentration and carbon dioxide arterial partial pressure in broilers;2)to observe the clinical symptoms of broilers in the control and model groups;(3)to dissect the cardiac lesions of broilers;and(4)to examine the pathological changes of broiler hearts by histopathology.The results showed that the results of in vivo model of sudden death in broilers showed that:(1)compared with the control group,the blood p H,serum bicarbonate concentration and carbon dioxide partial pressure of sudden death model in broilers were significantly decreased(P < 0.05);(2)the broilers in the control group had healthy appearance,good mental status and normal feed intake;while the broilers in the model group mostly showed lack of energy and sudden death occurred,and the broilers with sudden death showed sudden loss of balance,inciting wings and screaming;(3)the anatomy of sudden death broilers showed that the heart volume was significantly dilated,and there was a large amount of pericardial effusion;the lungs were congested and swollen,dark red;(4)the results of histopathological examination showed that the myocardial cells of sudden death broilers had granular degeneration,muscle fiber ruptureThese results indicate that lactic acid can reduce the contraction frequency,change the morphology,increase the apoptosis of primary cardiomyocytes,damage the mitochondrial structure,decrease the membrane potential,abnormal dynamics,increase the level of oxidative stress,and increase the autophagy of mitochondria.After the addition of mitochondrial division inhibitors,the survival rate of primary cardiomyocytes after lactic acid treatment increased,ROS level decreased,and apoptosis decreased,which could effectively protect the structure and function of cardiomyocytes and mitochondria.Ammonium chloride can be used to successfully establish the model of sudden death in broilers,and the clinical case characteristics of SDS in broilers can be preliminarily replicated.The successful establishment of sudden death models for broilers in vitro and in vivo laid a foundation for the prevention and control of SDS,and provided ideas for the mechanism research of sudden death diseases in humans from the perspective of comparative medicine. |
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