The Study Of Ox-LDL On THP-1-derived Macrophages Autophagy And Its Mechanism

Objective: Oxidized low density lipoprotein （ox-LDL） is the production of lowdensity lipoprotein oxidized by reactive oxygen species （ROS） and divalent metal ionssuch as Cu²⁺and Fe²⁺. Macrophage uptake ox-LDL via scavenger receptor and thenconserved to mcarophage-derived foam cells, which mediates the atherosclerotic lesionsformation and progression. Recently, studies showed that macrophage autophagy wasexisted in atherosclerotic lesions.However, the effect of ox-LDL on macrophageautophagy and its regulatory mechanism was not clear.Methods:1、After treatment with Phorbol-12-myristate-13-acetate （PMA） for24h,THP-1macrphages were treated with0,10,20,40or80mg/L Ox-LDL for24h. Reversetranscription polymerase chain reaction （RT-PCR） and Western blot were used to detecteLC3、Beclin1、TET2mRNA and protein expression, respectively. The autofluorescentsubstance monodansylcadaverine （MDC） was used to detect autophagic vacuoles （AVs）.Cell immunofluorescence was used to detecte LC3protein concentration.2、THP-1macrophages were transfected with TET2siRNA or5-A za-2′-deoxycytidinefor24h, and then treated with80mg/L Ox-LDL for24h. RT-PCR and Western blot wereused to detecte LC3、Beclin1、TET2mRNA and protein expression, respectively. Cellimmunofluorescence was used to detecte LC3protein concentration. MDC was used todetect AVs.Results:1、After treated with0,10,20,40or80mg/L Ox-LDL for24h, Beclin1mRNAand protein expression was obviously decreased（P﹤0.05）, LC3mRNA expression hasno obviously changed （Pï¹¥0.05）. However, the protein levels was decreased with aconcentration dependent manner （P﹤0.001）. Cell immunofluorescence results showedthat the concentration of LC3was negative correlation with Ox-LDL concentration. MDC staining showed that Ox-LDL decreased AVs in THP-1macrophage.2、After treated with0,10,20,40or80mg/L Ox-LDL for24h，TET2mRNA and proteinexpression was obviously decreased （P﹤0.05）. When treated with TET2siRNA, bothmRNA and protein levels were further decreased compared with80mg/L （P﹤0.01）.However, both mRNA and protein levels were up-regulated （P﹤0.05） in5-Aza-2′-deoxycytidine group. Consisted with the western blot results, cellimmunofluorescence showed that LC3protein concentration was further decreased inTET2siRNA group. MDC staining showed that THP-1macrophage autophagy wasfurther decreased in TET2siRNA group compared to80mg/L group and5-Aza-2′-deoxycytidine group.Conclusion: Ox-LDL down-regulated the expression of TET2, which contributed to theinhibition of THP-1macrophage autophagy.