| Objective: Exercise preconditioning(EP)refers to repeated high-intensity,intermittent aerobic exercise that can lead to myocardial relative or absolute ischemia-hypoxia,inducing endogenous cardioprotection to protect myocardial tissues against subsequent sustained ischemic-hypoxic injury.Autophagy,as one of the mechanisms of endogenous cardioprotection,has attracted great attention from researchers at home and abroad.Autophagy is a biological process in which cells capture aged and damaged proteins or organelles to be degraded or recycled within lysosomes.The formation of autophagosome is the core process of autophagy,and it is inseparable from the regulation of LC3-Ⅱ synthesis-associated proteins,which need to be further studied.In this study,blood indexes,histological indexes and electrocardiogram were used to explore the relationship among them,and to compare the degree of EEP and LEP in alleviating exercise-induced myocardial ischemic-hypoxic injury.Based on EP alleviating myocardial ischemic-hypoxic injury caused by exhaustive exercise and the adaptive response of autophagy to ischemia-hypoxia stress,to explore the possible mechanism of EP of inducing autophagy and promoting autophagosome formation by studying the correlation between Beclin1 and myocardial ischemia-hypoxia and the changes of LC3-Ⅱ synthesis-associated proteins during EP.This study will provide an in-depth theoretical and experimental basis for the study on cardioprotective mechanisms of EP.Methods: A total of 120 healthy male Sprague-Dawley rats were randomly divided into six groups,group C(control),group EEP(early exercise preconditioning),group LEP(late exercise preconditioning),group EE(exhaustive exercise),group EEP+EE(early exercise preconditioning +exhaustive exercise)and group LEP+EE(late exercise preconditioning +exhaustive exercise).The model of EP was established with intermittent exercise of 4 repeated cycle periods,each consisting of a 10-min run at 30m/min and 0% grade,with intermittent rest for 10 min.The model of exercise-induced myocardial injury in rats was established by exercising to exhaustion at 30 m/min.(1)Plasma c Tn I content was detected by chemiluminescence.The changes of myocardial ischemia-hypoxia were observed by HBFP staining and C-2R BG staining.ST-segments and T-waves of ECG were used to reflect myocardial ischemia-hypoxia.The correlation of blood indexes,histological indexes and electrocardiogram was analyzed,and the intensity of EEP and LEP alleviating exercise-induced myocardial ischemic-hypoxic injury was compared.(2)The expression level of Beclin1 was determined by western blotting,and analyzing the correlation between Beclin1 and C-2R BG staining.(3)The expression changes of LC3 and Atg7 in myocardium were observed by laser confocal scanning microscopy with immunofluorescence double-labelling,and the extent of colocalization was analyzed.The protein expression levels of LC3,Atg7,Atg4 B,Atg3 and p62 were determined by immunofluorescence and western blotting,and the gene expression levels of LC3,Atg7,Atg4 B and Atg3 were determined by quantitative real-time polymerase chain reaction.Results:(1)Correlation between blood indexes,histological indexes and electrocardiogram: Both IOD and positive area of C-2R BG staining were positively correlated with c Tn I level in groups EEP,LEP,EE,EEP+EE and LEP+EE(P<0.05).Both IOD and positive area of C-2R BG staining were positively correlated with ST-segment and T-wave in groups EEP,EE,EEP+EE and LEP+EE,respectively(P<0.05).Compared with group EE,the plasma c Tn I level,IOD,positive area and MOD of HBFP staining and C-2R BG staining were significantly decreased in groups EEP+EE and LEP+EE(P< 0.05).Compared with group EE,ST-segments and T-waves in groups EEP+EE and LEP+EE were significantly decreased(P<0.05).(2)Western blotting results showed that compared with group C,the expression level of Beclin1 in groups EEP,EE,EEP+EE and LEP+EE was significantly increased(P<0.05).Both IOD and positive area of C-2R BG staining were positively correlated with Beclin1 in groups EEP,LEP,EE,EEP+EE and LEP+EE(P < 0.05).(3)Immunofluorescence double-labelling results showed that LC3 immunofluorescence images displayed green,Atg7 immunofluorescence images displayed red,and the merged images displayed yellow.Compared with group C,the correlation R of LC3 and Atg7 in group EEP was significantly increased(P<0.05).Compared with group EE,there was no significant difference in the correlation R of LC3 and Atg7 in groups EEP+EE and LEP+EE.Immunofluorescence results showed that Atg4 B immunofluorescence images displayed green,and Atg3 immunofluorescence images displayed red.Compared with group C,Atg4 B and Atg3 fluorescence mean intensity were significantly increased in groups EEP and LEP(P<0.05).Compared with group EE,Atg4 B and Atg3 fluorescence mean intensity in group EEP+EE were significantly increased(P<0.05).Western blotting results showed that compared with group C,the expression levels of LC3-I,LC3-II,LC3-II/I and Atg7 were significantly increased in group EEP(P<0.05),and the expression levels of LC3-I and Atg7 in group EE were significantly increased(P<0.05).The expression levels of LC3-II and LC3-II/I in group EEP+EE were significantly increased compared with group EE(P<0.05).Compared with group C,the expression levels of Atg4 B and Atg3 in groups EEP and LEP were significantly increased(P< 0.05),and the expression levels of Atg4 B in group EE were significantly increased(P<0.05).Compared with group EE,the expression levels of Atg4 B and Atg3 in group EEP+EE were significantly increased(P < 0.05).Compared with group C,the expression level of p62 in group EE was significantly increased(P<0.05).Compared with group EE,the expression level of p62 in groups EEP+EE and LEP+EE was significantly decreased(P<0.05).Quantitative real-time polymerase chain reaction results showed that compared with group C,there was no significant difference in LC3 m RNA level between groups EEP and LEP,while Atg7 m RNA,Atg4 B m RNA and Atg3 m RNA level were significantly increased(P<0.05).Compared with group EE,there was no significant difference in LC3 m RNA level between groups EEP+EE and LEP+EE,while Atg7 m RNA,Atg4 B m RNA and Atg3 m RNA level were significantly increased(P<0.05).Conclusion: Myocardial ischemia-hypoxia can lead to changes in STsegment,T-wave,and morphological structure of cardiomyocyte,and increased plasma c Tn I level in rats.LEP was stronger than EEP in alleviating myocardial ischemic-hypoxic injury induced by exhaustive exercise.LC3-Ⅱsynthesis-associated proteins of autophagosome formation did not play a role,and the reasons for this need to be further explored.EP can induce autophagy through intermittent myocardial ischemia-hypoxia,promote the formation of autophagosomes by increasing the levels of LC3,Atg7,Atg4 B and Atg3,and promote the effective degradation of autophagy substrates by increasing the level of p62,to exert cardioprotection of alleviating the myocardial ischemic-hypoxic injury caused by exhaustive exercise. |
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