摘要
目的探讨鱼藤酮对大鼠胸主动脉内皮细胞(RTAEC)自噬的影响及其初步机制。方法选用60只雄性SD大鼠,随机分为对照(Con)组、二甲基亚砜(DMSO)组和鱼藤酮组[1、2、4 mg·(kg·d)^(-1)],各组分别干预28 d。实验前后分别记录大鼠体质量、血压和心率;HE染色观察胸主动脉和心脏组织病理生理改变,透射电镜观察其超微结构及自噬小体发生。细胞实验分组:Con组、DMSO组和鱼藤酮组(20、100和500 nmol·L^(-1)),分别处理24 h,用活性氧(ROS)和三磷酸腺苷(ATP)水平筛选鱼藤酮干预最佳浓度后,选取500 nmol·L^(-1)进行后续干预。腺苷酸激活的蛋白激酶(AMPK)抑制剂化合物C(CC)在鱼藤酮处理前2 h干预细胞;RT-qPCR和Western blot检测胸主动脉自噬相关因子及腺苷酸激活的蛋白激酶-UNC-51样激酶1(AMPK-ULK1)通路的改变。结果与DMSO组相比,鱼藤酮组体质量增长量减少(P均<0.01);鱼藤酮对大鼠的血压和心率没有影响(P均>0.05);HE结果显示,鱼藤酮组胸主动脉内皮细胞伴有缺损,且心脏组织纤维排列紊乱、断裂;透射电镜结果发现,鱼藤酮组胸主动脉和心脏组织伴有结构异常及自噬小体出现;随着RTAEC鱼藤酮干预浓度增大,ROS产生水平增加(P<0.01),而ATP的生成减少(P<0.01);RT-qPCR结果显示,与DMSO组相比,LC3和P62的mRNA表达水平均上调(P均<0.01);信号通路相关因子AMPK和ULK1的mRNA表达水平均增加(P均<0.01);加入AMPK抑制剂CC,Western blot检测自噬相关蛋白,与500 nmol·L^(-1)组相比,LC3表达降低(P<0.01),P62表达上调(P<0.01);AMPK和p-AMPK表达减少(P均<0.01),AMPK的下游因子ULK1及p-ULK1表达均减少(P均<0.01)。结论鱼藤酮可促进RTAEC发生自噬,而其自噬机制可能是通过AMPK-ULK1信号通路介导。
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.
作者
常笑语
薛姝婧
曹红亭
吴阳
朱玲勤
李光华
CHANG Xiaoyu;XUE Shujing;CAO Hongting;WU Yang;ZHU Lingqin;LI Guanghua(School of Public Health,Ningxia Medical University,Yinchuan 750004,China;Department of Physiology and Neurobiology,School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;Department of Ultrasound,Ningxia Women and Children's Hospital,Peking University First Hospital,Yinchuan 750001,China)
出处
《宁夏医科大学学报》
2024年第9期865-872,共8页
Journal of Ningxia Medical University
基金
宁夏自然科学基金项目(2024A1978)。
