Mitophagy-mitochondrial Iron Overload Induces Warburg Effect To Promote The Proliferation Of Human Aortic Vascular Smooth Muscle Cells Induced By Apelin-13

Objective: One of the main features of atherosclerosis is the abnormal proliferation of vascular smooth muscle.Studies have found that Apelin/APJ system can regulate the occurrence and development of cardiovascular diseases such as atherosclerosis and hypertension.Previous studies in our laboratory have found that both mitophagy and Warburg effect can mediate Apelin-13 to promote HA-VSMCs proliferation.The purpose of this study is to explore whether mitophagy causes mitochondrial iron overload and regulates Warburg effect,which mediates Apelin-13 to promote HA-VSMCs proliferation,and reveals a novel molecular mechanism of mitophagy-mitochondrial iron overload-Warburg effect mediated by Apelin-13/APJ system to promote HA-VSMCs proliferation.Methods:1.Western Blot: Detected the expression of PINK1,Parkin,PKM2,LDHA,MCT1,MCT4 in HA-VSMCs;2.RNA interference: The chains were designed to interfere with the expression of PINK1,Parkin in human aortic vascular smooth muscle cells(HA-VSMCs)respectively;3.Plasmid overexpression: Constructed overexpression of plasmid to increase the expression of PINK1 and Parkin in human aortic vascular smooth muscle cells(HA-VSMCs);4.CCK8 Kit: CCK8 Kit detected the proliferation of HA-VSMCs;5.Lactic acid kit: Lactic acid kit was used to detect the content of lactic acid in HA-VSMCs;6.Glucose kit: Glucose content in HA-VSMCs was detected by glucose kit;7.BCA protein quantitation kit: Protein content of HA-VSMCs was measured by BCA cell quantitative kit;8.Treatment with mitochondrial iron chelator(DXZ)and mitochondrial targeted antioxidants(Mito-TEMPO)inhibited the expression of PKM2,LDHA,MCT1,MCT4 in HA-VSMCs promoted by Apelin-13;9.RPA and Mito-SOX red fluorescent probes were used to observe the contents of chelated iron and ROS in mitochondria of living cells.Results: 1.SiRNA interfering PINK1 inhibited the expression of PINK1,Parkin,PKM2,LDHA,MCT1 and MCT4 in HA-VSMCs induced by apelin-13;2.SiRNA interfering Parkin inhibited the expression of Parkin,PKM2,LDHA,MCT1 and MCT4 in HA-VSMCs promoted by apelin-13;3.Plasmid overexpression of PINK1 induced Apelin-13 to promote the expression of PKM2,LDHA,MCT1 and MCT4 in HA-VSMCs;4.Plasmid overexpression of Parkin induced Apelin-13 to promote the expression of Parkin,PKM2,LDHA,MCT1 and MCT4 in HA-VSMCs;5.SiRNA interfering with PINK1,Parkin inhibited the proliferation of HA-VSMCs promoted by Apelin-13,decreased the content of extracellular lactic acid and increased the amount of extracellular glucose;6.Plasmid overexpressing PINK1 and Parkin induced Apelin-13 to promote the proliferation of HA-VSMCs,increased the content of extracellular lactic acid and inhibited the increase of extracellular glucose;7.Apelin-13 caused mitochondrial iron overload and promoted mitochondrial ROS production observed by Delta Vision living cell imaging microscope;8.DXZ and Mito-TEMPO treatment inhibited the expression of PKM2,LDHA,MCT1,MCT4 in HA-VSMCs promoted by Apelin-13.Conclusion: Mitophagy-mitochondrial iron overload induces Warburg effect to promote Proliferation of Human Aortic Vascular smooth muscle cells induced by Apelin-13.