Study On The Role Of Calpains In The Apoptosis Of Myocardial Cells Of Broilers And The Regulation Mechanism Of Taurine

The heart and blood circulatory system cannot meet the need of oxygen for chicken's weight fast growing is the main reasons for broiler ascites syndrome?AS?,right ventricular hypertropy and then failure is the important cause for AS and chicken deaths.The study found that the addition of taurine can reduce the incidence of ascites syndrome and reduce the activity of calpains in the right ventricle.Objective:Hypoxia induced apoptosis in chicken's primary cardiomyocytes,with the treat of taurine and PD150606?a specific calpains inhibitor?separate or together,to compare and discuss whether taurine can inhibit myocardial cell apoptosis through affecting the activity of Calpains.The study will provide the theoretical basis for the prevention and treatment of broiler AS.Methods:The primary cardiomyocytes were separated from 12 days chicken embryos,authenticated by cell immunochemical method,the MTS method was used to monitor myocardial cell vitality.There were eight groups:blank control group?C?,taurine control group?CT?,PD150606 control group?CP?,taurine and PD150606 control group?CTP?,hypoxia model group?M?,taurine model group?MT?,PD150606 model group?MP?,taurine and PD150606 model group?MTP?.The addition concentration of taurine and PD150606 was10 mmol/L and 10 umol/L separately,the pretreatment time was 12 h and 0.5 h,and the time of hypoxia was 12 h.Cell apoptosis in each group was checked by AO/EB.Fluorescence probe method was used to detect intracellular Ca²⁺concentration.The relative mRNA expression levels of Calpain-1,Calpain-2 and Caspase-3 were detected by RT-PCR.The relative protein expression levels of Calpain-1,Bcl-2,Cyt-C,Caspase-9,and Caspase-3 were detected by WB.The results are as follows.1)Separating and authenticate of myocardial cells in chicken embryo:the methods we use to separate myocardial cells,can get myocardial cells with 95%purity and 97%activity in 5 days,all the cultural cells can meet the test requirements.2)The effects of taurine on apoptosis rate in hypoxic chicken myocardial cells:hypoxia with 12 h significantly induced the myocardial cell apoptosis rate increase?P<0.01?,the Tau and PD150606 pretreatment could significantly reduce myocardial cell apoptosis rate?P<0.05?under anoxic condition,the effect of Tau and PD150606 treated together was better than that treated separately?P<0.01?.3)The effects of taurine on the damage index and antioxidant index in hypoxic chicken myocardial cells:the hypoxia treatment could improve the MDA content in myocardial cell culture supernatant and intracellular?P>0.05,P<0.05?,the Tau could reduce MDA content.The SOD activity of the myocardial cell culture supernatant was significantly increased compared with the control group?P<0.05?under hypoxia condition,Tau could increase the SOD activity significantly compared with M group?P<0.05,P>0.05?.PD150606 had no significant impact to the MDA content and SOD activity,PD150606 could increase the MDA content compared with C group under normal condition?P<0.05?.Hypoxia and Tau also had no significant effect on LDH activity in the myocardial cells,PD150606 could make the LDH activity in supernatant slightly increase under hypoxia?P>0.05?and make the LDH activity in cells decrease?P>0.05?.4)The effects of taurine on the mRNA expression level related with cell apoptosis in hypoxic chicken myocardial:hypoxia treatment induced the significantly increase in Calpain-1,Calpain-2,Caspase-9 and Caspasse-3 mRNA expression level?P<0.01?.Tau could inhibit the mRNA level of Calpain-1 and Calpain-2 in chicken myocardial cells under hypoxia?P>0.05,P<0.01?,significant inhibit Caspase-9 and Caspasse-3 mRNA expression?P<0.05,P<0.01?.PD150606 had no effect of Calpains mRNA expression,but inhibited Caspase-9mRNA expression?P<0.01?and increased the mRNA expression of Caspase-3?P<0.01?.5)The effects of taurine on the protein expression level related with cell apoptosis in hypoxic chicken myocardial cells:the hypoxia made Calpain-1 protein expression level significantly increased?P<0.01?,the Tau and PD150606 could inhibit the increase significantly?P<0.01?,but they add together had no statistical effect?P>0.05?.The hypoxia led the Bcl-2 protein expression to decrease significantly?P<0.01?,the Tau and PD150606could significantly maintain the Bcl-2 protein levels?P<0.01?.Hypoxia made myocardial cell Cyt-C protein increased significantly?P<0.01?,the Tau can significantly reduce the Cyt-C protein content under hypoxia?P<0.01?,PD150606 can reduce the Cyt-C protein content?P<0.05?.The Caspase-9 protein expression in M group was significantly higher than other groups?P<0.01?.Tau and PD150606 could reduce the Caspase-9 protein expression level significantly?P<0.01?,the effect of Tau was significant higher than that of PD150606?P<0.05?.Hypoxia reduced the pre-caspase-3 protein expression in M group significantly?P<0.05?,the Tau and PD150606 could significantly reverse the reduction?P<0.01?,the together effect was the best;this results indicated that hypoxia could cause Caspase-3 protein activation,while the Tau and PD150606 could significantly reduce the activation.Conclusion:When myocardial cells in chicken suffer hypoxic injury,the preventive application of taurine has the ability to improve the antioxidant capacity of myocardial cells and can inhibition of genes and proteins expression in the Cyt-C-mediated apoptosis pathway by regulating the expression of Calpains,which effectively inhibited the occurrence of myocardial cells apoptosis.Which suggested that Calpains can promote the myocardial cell apoptosis induced by hypoxia through the Cyt-C mediated apoptosis;taurine could protect the myocardial cell partly through the calpains mediated apoptosis.The above results provide a theoretical basis for the clinical application of taurine in the prevention and treatment of ascites syndrome in broiler chickens.