间歇性低氧预适应对力竭运动大鼠心肌自噬蛋白表达及相关通路的影响

Effects of intermittent hypoxia preconditioning on autophagy protein expressions and related pathways in myocardium of exhaustive exercise rats

目的探讨间歇性低氧预适应对力竭运动大鼠心肌自噬相关蛋白表达及单磷酸激活蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/不协调51样自噬激活激酶1(ULK1)信号通路的影响。方法60只SD大鼠随机分为空白组(Blank组)、对照组(Control组)、EE组、EE+1wIH组和EE+3wIH组,EE+1wIH组和EE+3wIH组大鼠在构建EE模型前分别给予1w和3w的IH预处理,除空白组外其余组大鼠进行游泳训练,且EE组、EE+1wIH组和EE+3wIH组构建EE模型;ELISA法检测各组大鼠血浆心肌肌钙蛋白I(cTnI)水平,铬变素2R(C-2R)染色检测大鼠心肌缺血缺氧情况,TUNEL法检测细胞凋亡,电镜下观察心肌组织内自噬小体数量,免疫组化检测抗体微管相关蛋白1轻链3Ⅱ(LC 3Ⅱ)和Beclin 1蛋白表达,Western blot检测蛋白表达水平AMPK-mTORULK1信号通路蛋白的表达。结果在Blank组中,所有心肌细胞的边界清晰,并且呈均匀的绿色染色;在Control组中,心肌细胞边界清晰,仅有少量红染散在组织中;在EE组中,部分心肌细胞的边界不清晰,心肌细胞以红色为主,少数呈绿色,说明大部分的心肌细胞都不同程度地受到缺血和缺氧的影响。EE+1wIH组和EE+3wIH组红染心肌细胞数明显低于EE组。与Blank组相比,EE组血浆cTnI水平、C-2R平均光密度(MOD)、心肌细胞凋亡率、心肌组织自噬数量、Beclin1、LC3 II、p-AMPK、ULK1蛋白明显升高,p-mTOR蛋白明显降低(P<0.05),与EE组相比,EE+1wIH组和EE+3wIH组上述指标均有明显改善(P<0.05)。结论EE可导致大鼠心肌自噬水平明显升高,而IH可能通过调控AMPK-mTOR-ULK1信号通路改善EE诱导的大鼠心肌细胞的自噬水平。

AIM To investigate the effects of intermittent hypoxia(IH)preconditioning on the expressions of autophagy-related proteins and the monophospho-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/uncoordinated 51 autophagy-activated kinase 1(ULK1)signaling pathways in cardiomyocytes of exhaustive exercise(EE)rats.METHODS Sixty SD rats were randomly divided into blank group,control group,EE group,EE+1wIH group and EE+3wIH group.EE+1wIH group and EE+3wIH group were pretreated with 1w and 3w IH before constructing EE model,respectively.All groups,except blank group,received swimming training,and EE models were constructed in EE group,EE+1wIH group and EE+3wIH group.The levels of cardiac troponin I(cTnI)in plasma were detected by ELISA,the sensitivity and ischemia and hypoxia of rats were detected by C-2R staining,apoptosis was detected by TUNEL method and the number of autophagosomes in myocardial tissue was observed by electron microscopy.Immunohistochemistry was used to detect the expressions of antibody microtubule-associated protein 1 light chain 3 II(LC3 II)and Beclin 1 protein,and Western blot was used to detect the expression of AMPK-mTOR-ULK1 signaling pathway protein.RESULTS In the blank group,the boundaries of all myocardium cells were clear and uniformly green.In the control group,myocardial cells had clear boundaries and only a small amount of red staining was scattered in the tissue.In the EE group,the boundary of some cardiomyocytes was not clear,and the majority of cardiomyocytes were red,while a few were green,indicating that most of the cardiomyocytes were affected by ischemia and hypoxia to varying degrees.The number of red stained cardiomyocytes in EE+1wIH and EE+3wIH groups was significantly lower than that in EE group.Compared with those in blank group,plasma cTnI level,C-2R average optical density(MOD),myocardial cell apoptosis rate,autophagy number of myocardial tissue,Beclin1,LC3 II,p-AMPK and ULK1 proteins were significantly increased in EE group,while p-mTOR protein was significantly decreased,with statistically significant differences(P<0.05).The above indicators were significantly improved in EE+1wIH group and EE+3wIH group and the difference was statistically significant(P<0.05).CONCLUSION EE can significantly increase the level of cardiac autophagy in rats and IH may improve the level of cardiac autophagy induced by EE by regulating AMPK-mTOR-ULK1 signaling pathway.