Rotenone Activates the AMPK-ULK1 Signaling Pathway to Induce Autophagy in the Rat Thoracic Aorta Endothelial Cells
Objective To investigate the effect of rotenone on autophagy in rat thoracic aortic endothelial cells(RTAEC)and its preliminary mechanism.Methods Sixty SD rats were selected and randomly divided into a control(Con)group,dimethyl sulfoxide(DMSO)group,and rotenone group[1,2,4 mg·(kg·d)-1],and each group was intervened for 28 d.Body weight,blood pressure,and heart rate were recorded before and after the experiment;HE staining was used to observe the pathological and physiological changes in the thoracic aorta and heart tissue,while transmission electron microscopy was used to observe their ultrastructure and autophagosome formation;Cell experiment grouping:Con group,DMSO group,and rotenone group(20,100,500 nmol·L-1),treated for 24 h respectively.After screening the optimal concentration of rotenone intervention using ROS and ATP levels,500 nmol·L-1 was selected for subsequent intervention.AMP activated protein kinase(AMPK)inhibitor compound C(CC)was intervened in cells 2 h before treatment with rotenone.RT-qPCR and Western blot were used to detect changes in autophagy related factors and adenosine activated protein kinase UNC-51-like protein kinase 1(AMPK-ULK1)pathway in the thoracic aorta.Results Compared with the DMSO group,the rotenone group showed a significant reduce in body mass gain(P all＜0.01);rotenone had no significant effect on blood pressure and heart rate in rats(P all＞0.05);HE results showed that the thoracic aorta endothelial cells in the rotenone group were accompanied by defects,and the heart tissue fibers were arranged disorderly and fractured;Transmission electron microscopy found that rotenone group of thoracic aorta and heart tissue with abnormal structure and the emergence of the autophagosome;With increasing concentrations of RTAEC rotenone intervention,the level of ROS production increased(P＜0.01),while ATP production decreased significantly(P＜0.01).RT-qPCR results showed that compared to the DMSO group,mRNA expression levels of LC3 and P62 were upregulated(P all＜0.01);AMPK and ULK1 signaling pathway-related factors were increased(P all＜0.01);After addition of the AMPK inhibitor CC,Western blot detection of autophagy-related proteins,compared with the 500 nmol·L-1 group,LC3 expression was decreased(P＜0.01),and P62 showed a corresponding upregulation of expression(P＜0.01);AMPK and p-AMPK expression decreased(P all＜0.01),and the expression of ULK1 and p-ULK1,the downstream factors of AMPK significantly reduced(P all＜0.01).Conclusion Rotenone can promote autophagy in RTAEC,and its autophagy mechanism may be mediated through the AMPK-ULK1 signaling pathway.
rotenonethoracic aorta endothelial cellsAMP-actived proteinkinase-UNC-51 like autophagy activating kinase 1autophagy
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