Exercise Preconditioning Plays A Protective Role In Exhaustive Exercise Rats By Activating The PI3K-Akt Signaling Pathway

Objective:Exhaustive exercise can lead to severe myocardial ischemia and hypoxia,resulting in exercise-induced myocardial injury.However,exercise preconditioning can enhance myocardial tolerance to ischemia and hypoxia,reduce myocardial injury,and improve cardiac function.The protective effect on the heart is clear,and the mechanism is related to the reduction of apoptosis.Activation of the PI3K-Akt signaling pathway plays an important role in inhibiting apoptosis of cardiomyocyte in the mechanism of exercise-induced cardiac protection.This study through the establishment of exercise in rats model,to have a one-time exhaustive exercise for traumatic stimulus,and use the PI3 K inhibitor LY294002 as a comparison.This concern from myocardial structure,serum myocardial enzymes and electrocardiogram,cardiac function and myocardial apoptosis index,mitochondrial permeability transition pore opening level and cardiomyocyte apoptosis related proteins,to explore exercise preconditioning in activated PI3K-Akt signaling pathway to the influence and mechanism of exhaustive exercise rats heart.Methods:1 Groups and models of experimental animals84 healthy male SD rats were randomly divided into 6 groups(n=14): the control group(Con group),the exhaustive exercise group(EE group),the exercise preconditioning group(EP group),the exercise preconditioning + exhaustive exercise group(EP+EE group),the LY294002(PI3K inhibitor)+ exercise preconditioning + exhaustive exercise group(LY+EP+EE),and the LY294002 group(LY group).Theanimal model was established by reference to Domenech and Lennon's exercise training methods.Con group did not run on the treadmill.EE group ran on the rats electric treadmill in the slope of 0 °,at a speed of 25 to 30 m/min to exhaustion.EP group underwent intermittent movement in the slope of 0 °,at the speed of 30 m/min,15 min running,5 min rest,and repeat 3 times,exercise 6 days/week.It lasts for 3 weeks.EP+EE group exercised in the same way as EP group.After the end of the next day in exercise training,for the same way as EE group to exhaustion.LY+EP+EE group rats were intraperitoneal injection of LY294002(10mg/kg)before exercise,and the training method was the same as EP+EE group.LY294002 was intraperitoneally injected into LY group.The injection method was the same as before,and LY group was not involved in training.2 The microstructure and ultrastructure of rat myocardium were observed by light microscopy and transmission electron microscopy.3 Serum levels of creatine kinase isoenzyme(CK-MB)and cardiac troponin I(cTn-I)were determined by enzyme-linked immunosorbent assay(Elisa)to evaluate myocardial injury.4 Electrocardiogram(ECG)was recorded by physiological recorder,and the ECG parameters of each group were recorded: heart rate,QRS duration,and ST segment,and the changes of the ECG was observed.5 Hemodynamic parameters of rats were recorded with Millar pressure-volume catheter: stroke work(SW),cardiac output(CO),stroke volume(SV),heart rate(HR),end-systolic volume(Ves),end-diastolic volume(Ved),end-systolic pressure(Pes),end-diastolic pressure(Ped),ejection fraction(EF),peak rate of presure rise(dp/dt max),peak rate of presure decline(dp/dt min),potential energy(PE),cardiac efficiency(CE),end-systolic pressure volume relationship(ESPVR),end-diastolic pressure volume relationship(EDPVR),relaxation time constant(Tau),to observe the changes of cardiac function.6 TUNEL assay was used to detect the apoptosis index of ratcardiomyocyte and evaluate the degree of apoptosis.7 The open level of mitochondrial permeability transition pore(mPTP)was determined by double-antibody sandwich method,and mitochondrial damage was evaluated.8 Western blot was used to detect the levels of PI3 K,p-PI3 K,Akt,p-Akt,Bad,p-Bad,Bcl-2,Bax and caspase-3 proteins in the left ventricular myocardium of rats,and to evaluate the changes of the above proteins.Results:1 Observation of myocardial histopathology in ratsCompared with Con group,the damage of myocardial microstructure in EE group,EP+EE group and LY+EP+EE group was obvious.Compared with EE group,the myocardial injury was reduced in EP+EE group.The myocardial injury degree of LY+EP+EE group was higher than that of EP+EE group.There was no significant injury change in LY group and EP group comparied to Con group.2 Comparison of CK-MB and cTn-I contents in rat serumCompared with Con group,contents of CK-MB and cTn-I in EE group,EP+EE group and LY+EP+EE group were significantly increased,with statistically significant differences(P<0.05).Compared with EE group,CK-MB and cTn-I in EP+EE group decreased,and the difference was statistically significant(P<0.05).The contents of CK-MB and cTn-I in LY+EP+EE group were significantly higher than those in EP+EE group(P<0.05).CK-MB and cTn-I levels of LY group and EP group were not statistically different from those of Con group(P>0.05).3 Comparison of ECG parameters of rats in each groupCompared with the Con group,the increase in HR,extension of QRS duration and elevation of ST segment in EE group showed statistically significant differences(P<0.05).The ST segment elevation of LY+EP+EE group and EP+EE group was significantly higher than that of Con group(P<0.05).Compared with EE group,EP+EE group showed astatistically significant difference in QRS duration shortening and ST segment elevation(P<0.05),but no statistical difference in HR(P>0.05).Compared with EP+EE group,LY+EP+EE group showed a significant difference in ST segment elevation(P<0.05).There was no significant difference between the electrocardiogram of LY group,EP group and Con group(P>0.05).4 Comparison of cardiac function indexes in rats4.1 Comparison of cardiac systolic function indexes in ratsCompared with Con group,CO,SV,Pes,EF,dp/dt max and ESPVR in EE group decreased,while HR and Ves increased,the difference was statistically significant(P<0.05).In comparison with Con group,LY+EP+EE group,CO,SV,Pes,EF,dp/dt max and ESPVR all decreased,while Ves increased,with statistically significant difference(P<0.05).Compared with EE group,EP+EE group showed increased CO,SV,Pes,EF,dp/dt max and ESPVR,and decreased Ves,with statistically significant differences(P<0.05),decreased HR,had no statistically significant differences(P>0.05).Compared with EP+EE group,LY+EP+EE group showed decreased CO,SV,EF,dp/dt max and ESPVR,and increased Ves,with statistically significant difference(P<0.05).Compared with Con group,EP group increased CO,SV,EF,dp/dt max and ESPVR,but decreased Ves,the difference was statistically significant(P<0.05).There was no difference between LY group and Con group(P>0.05).4.2 Comparison of cardiac diastolic function indexes in ratsCompared with Con group,Ved,Ped,EDPVR and Tau in EE group and LY+EP+EE group all increased,while-dp/dt min decreased,and the difference was statistically significant(P<0.05).Compared with EE group,EP+EE group showed a decrease in Ved,EDPVR and Tau,and an increase in-dp/dt min,with statistically significant differences(P<0.05),Ped decreased,and no statistically significant differences(P>0.05).Compared with EP+EE group,Tau of LY+EP+EE group was increased,while-dp/dt min was decreased,and the difference was statistically significant(P<0.05).Compared with Con group,Ved and EDPVR in EP group all decreased,while-dp/dt min increased,and the difference was statistically significant(P<0.05).There was no difference between LY group and Con group(P>0.05).4.3 Comparison of mechanoenergetics indexes in ratsCompared with Con group,PE increased in EE group and LY+EP+EE group,while SW and CE decreased,and the difference was statistically significant(P<0.05).Compared with EE group,PE decreased in EP+EE group,while SW and CE increased,and the difference was statistically significant(P<0.05).Compared with EP+EE group,LY+EP+EE group showed decreased SW and CE,with statistically significant difference(P<0.05).Compared with Con group,PE decreased in EP group,while SW and CE increased,and the difference was statistically significant(P<0.05).There was no significant difference between LY group and Con group(P>0.05).4.4 Comparison of P-V loop in ratsCompared with Con group,P-V loop of EE group,EP+EE group and LY+EP+EE group shifted to right.Compared with Con group,the P-V loop of EP group shifted to left.Compared with EE group,the P-V loop of EP+EE group shifted to left.The P-V loop of LY+EP+EE group shifted slightly to right compared with EP+EE group.5 Comparison of apoptosis index of rat cardiomyocyteCompared with Con group,the apoptosis index of cardiomyocyte in EE group,EP+EE group and LY+EP+EE group was significantly increased(P<0.05).Compared with EE group,the apoptosis index of EP+EE group decreased,and the difference was statistically significant(P<0.05).Cardiomyocyte apoptosis index of LY+EP+EE group was significantly higher than that of EP+EE group(P<0.05).Cardiomyocyte apoptosis indexes of LY group and EP group were not statistically different from those of Con group(P>0.05).6 Comparison of open levels of mitochondrial permeability transition pore(mPTP)in rat myocardiumCompared with Con group,the open level of mPTP was significantly increased in EE group,EP+EE group and LY+EP+EE group,and the difference was statistically significant(P<0.05).Compared with EE group,the open level of mPTP in EP+EE group decreased,and the difference was statistically significant(P<0.05).The open level of mPTP in LY+EP+EE group was significantly higher than that in EP+EE group(P<0.05).The open level of mPTP in LY group and EP group was not statistically different from that in Con group(P>0.05).7 Western blot analysis of PI3 K,p-PI3 K,Akt,p-Akt,Bad,p-Bad,Bcl-2,Bax and caspase-3 in rat left ventricular myocardium7.1 The expression levels of PI3 K,p-PI3 K,Akt and p-Akt were compared in each group7.1.1 Comparison of expression levels of p-PI3 K and p-Akt in rat myocardiumCompared with Con group,p-PI3 K and p-Akt protein contents in myocardium of EE group and LY+EP+EE group were significantly decreased,and the difference was statistically significant(P<0.05).Compared with EE group,the levels of p-PI3 K and p-Akt proteins in the myocardium of EP+EE group were increased,and the differences were statistically significant(P<0.05).The contents of p-PI3 K and p-Akt in LY+EP+EE group were significantly lower than those in EP+EE group(P<0.05).Compared with Con group,p-PI3 K and p-Akt protein contents were significantly increased in EP group(P<0.05).There was no significant difference in p-PI3 K and p-Akt protein content between LY group and Con group(P>0.05).7.1.2 Comparison of PI3 K and Akt protein expression in rat myocardiumCompared with Con group,the contents of PI3 K and Akt in myocardium of EE group and LY group showed no statistical significance(P>0.05),while the contents of PI3 K and Akt in myocardium of EP group,EP+EE group and LY+EP+EE group were significantly increased(P<0.05).Compared with EP+EE group,LY+EP+EE group showed no significant difference in the levels of PI3 K and Akt in rat myocardium(P>0.05).7.2 Comparison of the contents of Bad,Bcl-2,p-Bad,Bax and caspase-3 in cardiomyocyte apoptosis-related protein in rats in each group7.2.1 Comparison of the contents of Bad,Bax and caspase-3proteins that promote cardiomyocyte apoptosis.Compared with Con group,the contents of Bad,Bax and caspase-3proteins in the myocardium of EE group,EP+EE group and LY+EP+EE group were significantly increased(P<0.05).Compared with EE group,the content of Bad,Bax and caspase-3 protein in EP+EE group decreased,and the difference was statistically significant(P<0.05).The content of Bad,Bax and caspase-3 protein in LY+EP+EE group was significantly higher than that in EP+EE group(P<0.05).There were no significant differences in Bad,Bax and caspase-3 protein levels between LY group and EP group compared with Con group(P>0.05).7.2.2 Comparison of the contents of p-Bad and Bcl-2 proteins in myocardium.Compared with Con group,p-Bad and Bcl-2 protein contents in myocardium of EE group and LY+EP+EE group were significantly decreased(P<0.05),the content of Bcl-2 of EP+EE group was significantly reduced(P<0.05).Compared with EE group,p-Bad and Bcl-2 protein content increased in EP+EE group,and the difference was statistically significant(P<0.05).Compared with EP+EE group,p-Bad and Bcl-2 protein content in LY+EP+EE group decreased,and the difference was statistically significant(P<0.05).p-Bad and Bcl-2 protein levels in EP group were higher than those in Con group,and the difference was statistically significant(P<0.05).There was no significantdifference in p-Bad and Bcl-2 protein levels between LY group and Con group(P>0.05).Conclusion:1 Exhaustive exercise can destroy the myocardial structure of rats,impair cardiac systolic and diastolic function,reduce cardiac efficiency,inhibit the PI3K-Akt signaling pathway to play an anti-apoptosis role,and increase the apoptosis of cardiomyocyte.2 Exercise preconditioning makes the myocardial structure undergo adaptive changes,reduces the degree of myocardial injury and ECG activities obstacles,and improve myocardial systolic and diastolic function.The mechanism is activated PI3K-Akt signaling pathway inhibited the mitochondrial pathway,resist cardiomyocyte apoptosis,and thus play a protective role in exhaustive exercise rats heart.3 Exercise preconditioning can up-regulate the expression of PI3-Akt signaling pathway,and it has an anti-apoptotic effect on myocardium.