The Study Of α-ketoglutarate On High Fat Diet ApoE^-/- Mice Atherosclerotic Lesions And Its Potential Mechanisms

Objective:α–ketoglutarate（α-KG）,an intermediate of the Krebs cycle,has been reported to maintain Embryonic stem cells self-renewal and mediate dietary restriction Caenorhabditis elegans longevity.However,the relationship ofα-KG and atherosclerosis is unclear.Here,the role ofα-KG on high fat diet ApoE^-/-mice atherosclerosis lesions and its potential mechanisms were explored.Methods:After one week of adaptive feeding,thirty male eight-week-old ApoE^-/-mice were randomly divided into two groups:control group（high fat diet）andα-KG treatment group（high fat diet supplement with 2%α-KG）.All mice were free drinking water and sacrificed at 12 week.Automatic biochemical analyzer was used to detect the plasma lipid levels including total cholesterol,triglyceride,high density lipoprotein and low density lipoprotein.SudanⅣstaining was supplied to observed atherosclerotic lesions in whole aorta.The aortic sinus of mice were isolated for frozen sections.Oil red O staining was used to observe the atherosclerotic lesions.Masson staining was used to detect the content of collagen fibers in atherosclerotic lesions.Immunofluorescence was used to investigate the levels of CD68,BECN1,microtubule-associated protein1 light chain 3（LC3）,p62,intercellular cell adhesion molecule-1（ICAM-1）,vascular cell adhesion molecule 1（VCAM-1）,interleukin-1beta（IL-1β）and ten-Eleven-Translocation oncogene family member2（TET2）.Western blot was used to detect the levels of BECN1,(LC3,p62,ICAM-1,VCAM-1,IL-1βand TET2 in cultured human umbilical vein endothelial cells（HUVECs）.The autophagy of HUVECs was detected with transmission electron microscopy and the autophagic flux was observed with dual fluorescence mRFP-GFP-LC3 adenovirus.Results:There was no difference in body weight,total cholesterol,and triglyceride and HDL cholesterol levels among mice in two groups.Oil red O and SudanⅣstaining showed thatα-KG treatment obviously attenuated high fat diet ApoE^-/-mice atherosclerotic lesions.Masson staining showed thatα-KG decreased the content of collagen fibers in atherosclerotic lesions.Inα-KG treatment group,the levels of TET2,autophagy marker Beclin1 and LC3 were obviously increased.Meanwhile,the autophagy dysfunction marker p62,inflammatory factors ICAM-1,VCAM-1 and IL-1β,CD68 positive cells were significantly decreased（p<0.05）.In cultured HUVECs,α-KG increased the levels of TET2,BECLIN 1 and the ratio of LC3II/LC3I with dose-depent manner.Meanwhile,the levels of inflammatory factors ICAM-1,VCAM-1 and IL-1βwere decreased response toα-KG concentration.When pretreatment with TET2 shRNA,the upregulation of autophagy and autophagic flux and the inhibition of inflammatory factors expression induced byα-KG were attenuated.Consisted with these results,TET2overexpression further improvedα-KG-induced endothelial cells autophgy and autophagic flux.The inhibition of inflammatory factors expression was also further strengthened.Conclusion:α-KG improves TET2-mediated endothelial cell autophagy and autophagic flux,which inhibited endothelial cell inflammation factors expression and contritbutes to the prevention of atherosclerosis.