The Role And Mechanism Of Macrophage-specific Pdha1 Knockout In Atherosclerosis

Background Atherosclerosis is a chronic cardiovascular disease that harms human health and is one of the most common causes of death in the elderly.There are many mechanisms of atherosclerosis,such as the theories of lipid infiltration,smooth muscle cell cloning,endothelial injury response,insulin resistance and so on.Modified lipoproteins,mainly oxidize low-density lipoproteins（Ox LDL）,were previously regarded as the major contributors to the pathogenesis,development,and immune response during atherosclerosis.While behind these mechanisms,there is a common mechanism — “disturbance of cellular energy metabolism”.Cell energy metabolism disorder refers to the generation and utilization of energy supply substances by cells,which leads to a series of pathophysiological changes in the body.During atherosclerosis,changes in cell metabolism and respiration lead to excessive production of reactive oxygen species（ROS）,leading to oxidative stress response.While,as the core part of the innate immune system,macrophages are closely related to the pathogenesis of obesity and atherosclerosis.Macrophages are one of the key cell types that maintain homeostasis,regulate inflammatory and regenerative processes.Macrophages can exhibit different phenotypes and functions depending on changes in their microenvironment.Pyruvate dehydrogenase（PDH）catalyzes the oxidative decarboxylate of pyruvate to form acetyl-Co A,CO2 and NADH,and occupies a key position in glucose oxidation,which connects glycolysis and tricarboxylic acid cycles and plays a key role in supporting mitochondrial energy production and cell survival.It is the key link of anabolism and catabolism,such as oxidative metabolism,gluconeogenesis,lipid synthesis,cholesterol synthesis and maintenance of carbon flux in tricarboxylic acid cycle,and important in the occurrence and development of cardiovascular diseases.PDH activity is reversibly regulated by PDHE1α subunit（PDHA1）phosphorylation.PDHA1 is the main regulatory site of PDH.It is a key factor affecting the metabolic flow of tricarboxylic acid cycle and glycolysis in mitochondria.Our previous study found that the expression level and activity of macrophage pyruvate dehydrogenase（PDHA1）increased significantly in patients with coronary heart disease.In order to clarify the relationship of macrophage pyruvate dehydrogenase in atherosclerosis,in this study,we further explored the role and mechanism of macrophage-specific Pdha1 konckout on atherosclerosis.Part I Construction of ApoE^-/-Pdha1fl/flLyz2-Cre mice and Pdha1fl/flLyz2-Cre micePurpose: In this study,we intend to construct macrophage specific knockout Pdha1 mice,and atherosclerosis with macrophage-specific knockout of Pdha1(ApoE^-/-Pdha1fl/flLyz2-Cre)model mice.Methods: Cas9 nickase,Pdha1-L1 and Pdha1-R1 were translated into RNA in vitro,and injected into the fertilized eggs of mice with Donor DNA to obtain gene knockout mice efficiently.Then Pdha1-loxp mice were mated with Lyz2-Cre mice to achieve the objective of Pdha1 gene conditional knockout.The above macrophage Pdha1-specific knockout mice were mated with ApoE^-/-mice for further breeding,and finally ApoE^-/-Pdha1fl/flLyz2-Cre mice were obtained.Results: The results showed that Pdha1fl/flLyz2-Cre mice carried lyz-Cre gene and Pdha1-loxp gene successfully.Genotypic identification of ApoE^-/-Pdha1fl/flLyz2-Cre model mice showed that Apo E gene were knockout,Lyz2-Cre gene and Pdha1-loxp gene were carried,simultaneously.Conclusion: 1.Pdha1fl/flLyz2-Cre mouse model was constructed.Pdha1 gene was specifically knocked out in mouse macrophage.2.ApoE^-/-Pdha1fl/flLyz2-Cre homozygous mice were bred,laying foundation for further explore the role of macrophage-specific Pdha1 konckout on atherosclerosis process.Part II Role of Macrophage-specific knockout of Pdha1 in atherosclerosisPurpose: This study aims to further clarify the effect of macrophage-specific knockout of Pdha1 on the progression of atherosclerosis.Methods: 8 ApoE^-/-Pdha1fl/flLyz2-Cre mice and 8 ApoE^-/-mice（8-12 weeks,male）were selected.After high fat diet for 3 months,mice were harvested for use.Results: The blood glucose of ApoE^-/-Pdha1fl/flLyz2-Cre group was significantly increased（P<0.01）.The levels of TC（P<0.05）,TG（P<0.01）and LDL-C（P<0.001）in serum of ApoE^-/-Pdha1fl/flLyz2-Cre were significantly increased,but there was no significant difference in HDL-C level.Some steatosis hepatocytes were found in liver sections of ApoE^-/-group.Obvious steatosis was found in the liver of ApoE^-/-Pdha1fl/flLyz2-Cre group.Oil red O staining was performed on aorta.The total aorta area of atherosclerotic lesions in ApoE^-/-Pdha1fl/flLyz2-Cre group was significantly increased（P<0.01）and the aortic sinus plaque area was significantly increased（P<0.05）.The expression of NLRP3,Caspase-1 and IL-1β in plaque of macrophage-specific knockout of Pdha1 mice was increased.Conclusion: 1.Macrophage-specific knockout of Pdha1 promotes atherosclerosis in mice and may be associated with activation of inflammation。 2.Macrophage-specific knockout of Pdha1 can affect the metabolism of the mice.Part III Molecular mechanism of Macrophage-specific knockout of Pdha1 in atherosclerosisPurpose: In this study,si RNA technology was used to silence Pdha1 in THP-1 mononuclear macrophages,and the effect of foamification of macrophage by Ox LDL was observed.The effect of Pdha1 knockout on transcription levels of other genes was observed,especially genes related to atherosclerosis.Methods: In this study,the expression of PDHA1 and the formation of foam cells were detected after siPdha1 transfection of THP-1 monocytes/macrophages in vitro.ApoE^-/-Pdha1fl/flLyz2-Cre and ApoE^-/-were selected and fed high fat diet for 3 months.Abdominal macrophages were extracted and transcriptomic sequencing was performed.The results of transcriptome were verified by q PCR.Results: After siPdha1 transfection,PDHA1 protein expression was decreased.The cellular foam was obvious.The results of transcriptome sequencing analysis showed that 2054 genes were differentially expressed in the two groups.Genecards database was applied to search atherosclerosis related genes with “atherosclerosis” as the keyword,a total of 4343 genes were found.Then,intersection was taken with the differential genes in this study,548 genes were obtained for further enrichment analysis.The significantly differential expression genes（Pdha1,Timp1,Tlr4,Mmp9,Vcam1,Hsp90aa1,Slc2a1,Kras）in ApoE^-/-Pdha1fl/flLyz2-Cre and ApoE^-/-Cre macrophages were selected.Differential expression genes were detected by q PCR,and the results of q PCR were consistent with those of transcriptome detection.Conclusions: The transcription levels of several genes（cell adhesion,chemotaxis,extracellular matrix,etc.）associated with atherosclerosis have changed in macrophages of ApoE^-/-Pdha1fl/flLyz2-Cre mice.It is possible to regulate HSP90 mediated TLR4 receptor activation to induce downstream responses.