The Role And Mechanism Of Insulin-like Growth Factor 1 (IGF1) In Chicken Myocardial Injury

Insulin-like growth factor 1?IGF1?,a multifunctional cytokine,regulates cell growth and development by mediating growth hormone stimulation.In the previous study,we found that selenium deficiency induces damage in chicken cardiomyocytes and inhibits the expression of IGF1in the myocardium.Therefore,we speculated that there was some relationship between IGF1 and chicken myocardial injury,but the mechanism is still unclear.We used RNA interference technology to establish the model of IGF1 knockdown chicken cardiomyocytes and chicken embryos.Subsequently,we used western blot,real-time quantitative PCR,electron microscope and fluorescence dyeing to detect the expressions of energy metabolism,endoplasmic reticulum stress,inflammation,calcium levels,calcium ion channels,heat shock proteins?HSPs?,myocardial contraction,autophagy and apoptosis.Our purpose is to explore the role and mechanism of IGF1 in chicken myocardial injury and provide important evidence for a better and more comprehensive understanding of IGF1 function.The results showed that:?1?The pathological observation showed that the growth density of chicken cardiomyocytes significantly decreased,the volume of cardiomyocytes and intercellular connections significantly decreased,and intercellular networks were not interwoven in the IGF1 knockdown group.H.E.staining showed that there were many elongated cardiomyocytes in the IGF1 knockdown group,which indicating dysplasia of cardiomyocytes.Similarly,in chicken embryos,H.E.staining showed that the blood vessels in IGF1 knockdown group were ruptured and the myocardial tissue space increased.Furthermore,ROS,H₂O₂,MDA,T-AOC levels and GSH,GSH-Px,SOD,CAT,i NOS activities in cardiomyocytes were detected in vivo and in vitro.The results showed that the antioxidant capacity of IGF1 knockdown group was significantly reduced compared with the control group.The m RNA and protein expressions of PI3K/Akt pathway and insulin-related genes were further detected.The results showed that IGF1 knockdown significantly increased the expressions of AMPK,JNK and FOXO,while the expressions of PI3K,Akt and 14-3-3 were significantly decreased,and the expressions of insulin-related genes IGF1R,GLUT1,GLUT3,GLUT8,IGFBP1,IGFBP2,IGFBP3,IGFBP4,IGFBP5,IGFBP7,IR and IRS1 were significantly decreased.At the same time,the oxygen consumption and ATP content of cardiomyocytes in IGF1 knockdown group were significantly decreased.From these results,we concluded that IGF1 knockdown activated FOXO expression by inhibiting the PI3K/Akt pathway.Activated FOXO further leads to insulin dysfunction and energy metabolism disorders to aggravate myocardial damage in chickens.?2?We detected the intracellular Ca²⁺and Ca²⁺-ATPase activity,meanwhile,the m RNA and protein expressions of calcium-channel related genes,endoplasmic reticulum stress,inflammation,heat shock proteins and myocardial contraction were detected in vivo and in vitro.The results showed that IGF1 knockdown significantly increased the expressions of calcium channel-related genes ORAI1,TRPC1,TRPC3,CACNA1S,STIM1,PMCA1,NCX,CALM,TRP1,TRP2,TRP3,Ry R1,Ry R2,Ry R3 and CASR,while significantly decreased the expressions of SERCA,and significantly increased the expressions of ER stress-related genes XBP1,LAMP,GRP78,GRP94,CHOP,ATF4and ATF6.In addition,the expressions of inflammatory factors PTGEs,COX-2,i NOS,NF-?B and TNF-?were significantly increased,while the expression of HO-1 was significantly decreased,the expressions of heat shock proteins HSP27,HSP40,HSP60,HSP70 and HSP90 were significantly increased,and the expressions of MYL2,MYL3,TNNT2,TNNC1,MYH11 and MYH15 were significantly increased.From the above results,we concluded that IGF1 knockdown induces Ca²⁺concentration overload in the myocardial cytoplasm,further triggering endoplasmic reticulum stress,myocardial systolic disorder and inflammation,and HSPs could play a potential protective role in the entire process.?3?By means of transmission electron microscopy,AO/EB staining,Hoechst/PI staining and MDC staining,the results showed that the apoptosis rate and autophagy rate of cardiomyocytes in IGF1 knockdown group were higher than that of the control group.To further verify the above results,we used q RT-PCR and western blot analysis to detect the expressions of apoptosis and autophagy related genes in vivo and in vitro respectively.q RT-PCR results showed that IGF1 knockdown significantly increased the m RNA expressions of Bid,Fas,Bak,cytc,Caspase-2,Caspase-3,Caspase-6,Caspase-9,Caspase-10,Becline1,LC3-1,LC3-2,ATG-4,ATG-5,ATG-7,ATG-10,ATG-12 and ATG-13 in vivo and in vitro.The expressions of Bax,Bcl2,Caspase-1,Caspase-7 and m TOR were significantly decreased.Western blot analysis showed that IGF1 knockdown significantly increased the protein expression s of Bax,Bcl2,Caspase-3,Caspase-8,Caspase-9,Caspase-12,Fas,ATG-7 and Becline1 in vivo and in vitro,while the expressions of Bak,LC3 and m TOR were significantly decreased.From the above results,we concluded that IGF1 knockdown induces myocardial injury in chickens through apoptosis and autophagy pathway.In summary,IGF1 knockdown leads to the dysfunction of myocardial antioxidant function and induces a large amount of ROS production in cardiomyocytes,thereby activating FOXO expression by inhibiting the PI3K/Akt pathway.Activated FOXO further causes insulin dysfunction,energy metabolism and myocardial damage.IGF1 knockdown causes Ca²⁺concentration overload in the myocardial cytoplasm,further triggering endoplasmic reticulum stress,myocardial systolic disorder and inflammation,while HSPs play a potential protective role in the whole process.IGF1knockdown induces excessive oxygen free radicals in cardiomyocytes,which induces apoptosis of cardiomyocytes and myocardial injury.Meanwhile,autophagy pathway is also activated,which are thought to be a protective mechanism in the body.The aims are exploring the mechanism of IGF1knockdown in myocardial injury,and providing new ideas for studying the selenium-deficient myocardial injury.