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.
关键词
鱼藤酮/胸主动脉内皮细胞/腺苷酸激活的蛋白激酶-UNC-51样激酶1/自噬
Key words
rotenone/thoracic aorta endothelial cells/AMP-actived proteinkinase-UNC-51 like autophagy activating kinase 1/autophagy
维普
万方数据