Effect Of ARC And IGF-1Against Myocardial Cell Apoptosis In AS Broiler Study

Ascites Syndrome (AS) is a non-communicable diseases. Clinically characterized by significant ascites, right ventricular hypertrophy/failure, pulmonary congestion, edema and liver congestion/swelling. In order to find a good method of control, domestic and foreign scholars made a lot of research on the pathogenesis of AS. The present study about the effect of ARC protein in heart disease mainly focuses on mammalian cardiac cells, and generally agreed that the ARC have good protective effect on mammalian cardiac oxidative damage, infarction and hypertrophy.On the process of Ascites Syndrome, the chicken myocardial cell hypertrophy and associated with serious injury. However, the protective effect of ARC in Broiler chicken on cardiac hypertrophy\injury protection and control mechanism has not been reported. In order to explore the role and molecular mechanisms of ARC in cardiomyocyte hypertrophy/apoptosis with ascitic broiler, and the relationship of ARC with caspases-2, caspases-3, caspases-8, BAX and BCL-2. From molecules, cell and whole respect to clarrify the effect of ARC in cardiomyocyte hypertrophy/apoptosis/ascites formation molecular mechanism. We have carried out related experiments.1. Ascites syndrome in broiler model induces and blood biochemical parameters detectionIt is believed that ascitic broilers die of right heart failure caused by pulmonary hypertension, but the underlying mechanisms of right heart failure are unknown. However, recent studies have shown that reactive oxygen species have the ability to damage heart cells.This study aimed to determine the changes of reactive oxygen species in serum and plasma, and the effect of this variation on myocardial cells during broiler ascites. We used hypoxia and a low-temperature method to induce broiler ascites in the fast-growing group. For controls, we treated a slow-growing group of broilers with70%restricted feeding under the same circumstances as the fast-growing group.The results showed that hypoxia is a more effective and better way to induce broiler ascites than a low-temperature environment and high growth rate. In addition, reactive oxygen species levels were significantly increased in the fast-growing group compared with those in the slow-growing group. This significant increase in reactive oxygen species resulted in myocardial cell apoptosis in the fast-growing group.Our results suggest that cardiomyocyte apoptosis caused by increased reactive oxygen species levels of ascitic broilers is one of the most important reasons for causing heart failure.2. Mouse ARC Gene cloning and expressionAs a template RNA was extracted from mouse heart tissue, using RT-PCR method to amplify mouse ARC full-length cDNA, then amplified product was recombined into the PMD-18T vector, and measured the nucleotide sequence of the gene.Sequence analysis showed that mouse ARC full-length cDNA is663bp long, encoding221amino acids, the same as the sequence in GenBank of NM₀30152.4.PCR and double digested methods were used to recombinant pESATTM-E1-ARC expression vector, and transformed it into host strain BL21(DE3).After inducing by IPTG, mouse ARC fusion protein was successful expression.3. Chick embryo myocardial cells culture, identification and activity determinationHow to get the high activity of myocardial cells has been an important foundation for research broiler cardiovascular disease. After years of accumulation, there are a variety of mature rat cardiac myocytes culture methods, but the chicken myocardial cells rarely reported.In order to acquire chick embryo cardiac cells, tissue block adherent and collagenase digestion method were used to treatment11days AA Avian chick Myocardial tissue. By cell morphological observation and MTT assay to detect cell Purity and grow ability.We found that two methods are available to get sufficient amount of myocardial cells, but the collagenase digestion method is more suitable for basic research.4. The relationship of IGF-1and ARC Insulin-like growth factor-1(IGF-1) and apoptosis repressor with caspase recruitment domain (ARC) play an important role in regulating apoptosis.Although the precise mechanisms of IGF-1and ARC in this process have not been defined, they have similar anti-apoptotic effects in myocardial cells, suggesting that these effects are related. We found that H2O2can induce ARC reduction in H9C2cells, but IGF-1can change this trend. To clarify this trend, using immunofluorescence and immunoblot analysis, we found that LY294002(PI3K inhibitor) blocked IGF-1up-regulation of ARC protein and blocked the protective effect of IGF-1on myocardial cell apoptosis induced by oxidative stress.These results indicate that IGF-1up-regulates ARC protein expression via the PI3K pathway, which protects against myocardial cell apoptosis induced by oxidative stress.5. The effects of ARC protein from murine in the chick embryo myocardial cellsApoptosis repressor with caspase recruitment domain (ARC) is highly involved in apoptosis induced by oxidative stress or ischemia/reperfusion injury. Furthermore, even through the exact mechanism is still unknown; some studies suggested that exogenous ARC also possesses anti-apoptotic ability.In this study, whether mouse-derived ARC acquires anti-apoptotic ability and what pathway of regulation in chick embryo cardiomyocytes were investigated. To evaluate whether mouse-derived ARC can inhibit chick embryo cardiomyocyte apoptosis induced by hydrogen peroxide, recombinant pcDNA3.1/ARC plasmid were acquired and transfected it into chick embryo cardiomyocytes. ARC-related genes (caspases-2,-8,-3, and-9, and cytochrome c, bcl-2, and XIAP) mRNA and protein expression levels were detected by real-time PCR and western blotting, respectively.Hydrogen peroxide induced apoptosis in chick embryo cardiomyocytes and this could be suppressed by mouse-derived ARC expression. Moreover, exogenous ARC was exclusively expressed in the cytoplasm and it down-regulated caspases-2,-8, and-3, and bcl-2, and XIAP gene expression levels. However, only caspase-3protein levels were decreased. In addition, threonine149phosphorylation was required for exogenous ARC to exert an anti-apoptotic effect in chicken embryo cardiomyocytes.These results indicate that mouse-derived ARC plays an important role in protection of chick embryo cardiomyocytes against oxidative stress apoptosis by inhibiting caspase-3mRNA and protein expression levels.