The Role And Mechanism Of Thioredoxin(Trx) In Chicken Cardiomyocyte Injury

Thioredoxin is a small protein that ubiquitous in prokaryotic and eukaryotic organisms,its function of redox had been demonstrated.our former study found that selenium deficiency can cause cardiomyocytes damage,and also suppress the expression of Trx,thus,we speculate that there may be some relationship between Trx and the damage to the cardiomyocytes in chicken,but its mechanisms are still unclear.Therefore,we established the model of Trx knock down in cardiomyocytes and use RNA interference,western blot,real-time PCR to detected the gene expression of selenoproteins,energy metabolism level,Akt phosphorylation level,heat shock proteins,ER stress,calcium concentration and calcium channel,autophagy,apoptosis,inflammation and cytokine.To explore the role and mechanism of Trx in chicken cardiomyocyte injury,to provide important evidence for better and more comprehensive understanding of the function and function of trx,and to provide reference for comparative medicine.The results showed that:.?1?Trx knock down can significantly decreased the expression of IGF1,IGF2,IGFBP2,IGFBP4,IR,IRS1,IRS2,GLUT1,GLUT3,GLUT8,PI3K,AKT,P-PI3K in mRNA and protein levels,and significantly increased the expression of IGFBP3,FOX,P-FOX,JNK in mRNA and protein levels.It indicates that Trx suppression can cause dysfunction of insulin-modulated cardiac energy metabolism and increase insulin resistance through PI3K-AKT pathway inhibition?2?Trx knock down can significantly increase the calcium content in cardiomyocytes,its significantly decreased the expression of NCX,PMCA,CALM in mRNA level,and significantly increased the expression of SERCA,STIM1,CACNA1S,TRPC1,TRPC3,Orail,TRP1,TRP2,TRP3,RyR1,RyR2,RyR3 in mRNA level.Meanwhile,significantly increased the expression of SERCA in protein level,and significantly decreased the expression of NCX,PMCA,CALM in protein level.It indicates that Trx knockout can induce calcium overload in chicken cardiomyocytes.?3?Observed by electron microscope,obvious autophagy bodies were found in cardiac myocytes of Trx knocdown group,suggesting That trx knockdown can induce autophagy in chicken cardiomyocytes and induce myocardial cell damage.At the same time,by detecting the kurtosis of genes associated with autophagy and apoptosis,we found that Trx knockdown significantly decreased the expression of mTOR in mRNA level,and significantly increased the expression of Becline-1,LC3-1,LC3-2,Dynein,SQSTM,ATG3,ATG5,ATG9,ATG12 in mRNA level.Meanwhile,significantly increased the expression of Becline-1 in protein level,and significantly decreased the expression of LC3-1,LC3-2,mTOR in protein level.It indicates that Trx knockdown significant autophagy in chicken cardiomyocytes.Trx knockdown significantly decreased the expression of Bax,Caspase-3,Fas in mRNA level,and significantly increased the expression of Caspase-6,Caspase-7,Caspase-8,Caspase-9 in mRNA level.Meanwhile,significantly decreased the expression of Bax,Caspase-3 in protein level,and significantly increased the expression of Bcl-2 in protein level.It indicates that Trx knockdown activated apoptosis pathway.However,due to the inhibition of caspase-3 gene,we believe that this apoptosis response is not the main reason of cardiomyocyte injury,we regard it as an early protective mechanism.?4?Trx knock down can significantly decreased the expression of HSP40 in mRNA level,and significantly increased the expression of HSP27,HSP60,HSP70,HSP90 in mRNA level.It indicates that Trx knockout can induce the reaction of heat shock to resist cell death under adverse conditions.?5?Trx knock down can significantly increase the expression of XBP1,GRP78,GRP94,IRE?ATF4,ATF,EIF2?in mRNA level.Meanwhile,significantly increased the expression of GRP78,XBP1,ATF4,ATF6 in protein level.It indicates that Trx knockout can induce ER instress in cardiomyocytes through ATF6 pathway.?6?Trx knock down can significantly decrease the expression of TNT,TNC in mRNA and protein levels.and significantly increase the expression of MYL2,MYL3 in mRNA and protein levels.It indicates that Trx knockout can influence the contraction of cardiomyocytes.?7?Trx knock down can significantly decreased the expression of IL-7,IL-17 in mRNA level,and significantly increased the expression of iNOS,NF-?B,COX-2,TNF-?,PTGEs,HO-1,IL-1?,IL-6,IL-8,IL-10 in mRNA level.Meanwhile,significantly increased the expression of COX-2,HO-1,iNOS,NF-?B in protein level.It indicates that Trx knockout induces a significant inflammatory reaction in cardiomyocytes,and some corresponding cytokines change,thus regulating other metabolic responses of the body.?8?Trx knock down can significantly decreased the expression of Dio1,Dio2,Gpx1,Gpx2,Gpx3,Gpx4,TrxR1,TrxR2,TrxR3,Selt,Selw,Selk,Sepx1 in mRNA level,and significantly increased the expression of Dio3,Selm,Selp15,Selh,Selu,Seli,Seln,Sepp1,Selo,Sels,Sels2,Selp in mRNA level.It indicates that Trx knock down can induce the change of selenoproteins expression.To sum up,Trx suppression can cause dysfunction of insulin-modulated cardiac energy metabolism and increase insulin resistance through PI3K-AKT pathway inhibition.The disturbance of energy metabolism can induce the overload of Ca²⁺in the cytoplasm of cardiomyocytes and induce endoplasmic reticulum stress.In addition,cardiomyocytes also had strong autophagy and inflammation,and heat shock proteins played an important protective role in the whole process of injury.