The OxLDL/?2GPI/anti-?2GPI Complex Inhibits Autophagy In Macrophages And The Investigation On Its Mechanism

Objective: Autophagy is a highly conserved lysosomal-dependent intracellular material degradation process that is closely related to diseases such as cancer,autoimmune diseases and cardiovascular diseases.Atherosclerosis(AS)is the leading cause of death from cardiovascular disease.It has been reported that patients with systemic autoimmune diseases such as antiphospholipid syndrome(APS)and systemic lupus erythematosus(SLE)have a tendency to accelerate the development of atherosclerosis.Our previous studies have proved that the oxidized low density lipoprotein(oxLDL)/?2 glycoprotein I(?2GPI)/anti-?2GPI complex is one of the main causes of the disease.The complex can lead to a series of atherosclerosis-related factors events,such as the inflammation,secretion of adhesion factors and apoptosis in endothelial cells,the proliferation,migration,apoptosis and lipid accumulation in smooth muscle cells,and the forming of macrophages,etc.In addition,given that macrophage autophagy plays an important role in the instability of atherosclerotic vulnerable plaques,whether the oxLDL/?2GPI/anti-?2GPI complex influences the autophagy in AS is still not clear currently.Therefore,this study aims to explore the effect of oxLDL/?2GPI/anti-?2GPI complex on macrophage autophagy in AS,and the role of the major inflammation-related pathway TLR4/NF-?B and the autophagy classic pathway PI3K/AKT/ mTOR in them.Methods:(1)After the stimulation with media,oxLDL,oxLDL/anti-?2GPI complex,oxLDL/?2GPI complex,?2GPI/anti-?2GPI complex,oxLDL/?2GPI/anti-?2GPI complex in mouse macrophagic cell line RAW264.7 and human monocytic cell line THP-1 derived macrophages,Western blot was used to detect the expression of autophagy-associated proteins including LC3,P62 and Beclin1 in the cells;The tandem mRFP-GFP-LC3 adernovirus was used to detect the patency of autophagosomes and autophagy flow in macrophages;(2)After treatment with chloroquine(50 ?M),Western blot was used to detect the expression of autophagy-associated proteins including LC3 and P62 in different groups;The tandem mRFP-GFP-LC3 adernovirus was used to detect the patency of autophagosomes and autophagy flow in macrophages;(3)After treatment with TLR4 inhibitor(TAK242,5 ?M)or NF-?B inhibitor(PDTC,20 ?M),Western blot was used to detect the expression of autophagy-associated proteins including LC3 and P62 in different groups;The tandem mRFP-GFP-LC3 adernovirus was used to detect the patency of autophagosomes and autophagy flow in macrophages;(4)Western blot was used to detect the phosphorylation of PI3 K,AKT and mTOR proteins in macrophages stimulated by media,oxLDL,oxLDL/anti-?2GPI complex,oxLDL/?2GPI complex,?2GPI/anti-?2GPI complex,oxLDL/?2GPI/anti-?2GPI complex;(5)After treatment with PI3 K pathway inhibitor(LY294002,50 ?M),Western blot was used to detect the expression of autophagy-associated proteins including LC3,P62,Beclin1 and the expression level of phosphorylated PI3 K,AKT,mTOR proteins in different groups;The tandem mRFP-GFP-LC3 adernovirus was used to detect the patency of autophagosomes and autophagy flow in macrophages.Results:(1)After stimulation with oxLDL/?2GPI/anti-?2GPI complex,LC3-?,Beclin1 proteins and autophagosomes were decreased in the cells,but the P62 protein was increased and the autophagy flow was blocked,which was significantly different from the media group(p<0.05).(2)After treatment with chloroquine,the expression level of LC3-? protein in macrophages and the number of autophagosomes were increased in the oxLDL/?2GPI/anti-?2GPI complex group,but the change caused by oxLDL/?2GPI/anti-?2GPI complex group was not as large as that by chloroquine alone(p<0.05).However,there was no significant change in the expression level of P62 protein compared to the oxLDL/?2GPI/ anti-?2GPI complex group alone(p>0.05).(3)After pretreatment with TLR4 inhibitor or NF-?B inhibitor,the autophagy-associated protein changes caused by the oxLDL/?2GPI/anti-?2GPI complex could be partially restored,and the autophagy flow which was blocked had also been alleviated(p<0.05).(4)After stimulation with oxLDL/?2GPI/anti-?2GPI complex,the phosphorylation levels of PI3 K,AKT and mTOR proteins in macrophages were up-regulated compared to the media group(p<0.05).(5)After pretreatment with PI3 K pathway inhibitor LY294002,the macrophage autophagy inhibited by oxLDL/?2GPI/anti-?2GPI complex could be partially restored,and the autophagy flow which was blocked could be alleviated(p<0.05).Conclusions:(1)The oxLDL/?2GPI/anti-?2GPI complex can inhibit the autophagy and block the autophagy flow in macrophages,and TLR4 or NF-?B inhibitors can alleviate its inhibitory effects,suggesting that TLR4/NF-?B is involved in the process of macrophage autophagy regulated by oxLDL/?2GPI/anti-?2GPI complex.(2)The oxLDL/?2GPI/anti-?2GPI complex can up-regulate the phosphorylation level of PI3 K,AKT and mTOR in macrophages,and the pre-treatment of PI3 K pathway inhibitor can reduce its phosphorylation level and partially restore the macrophage autophagy,suggesting that the autophagy classic pathway PI3K/AKT/mTOR is involved in the process of macrophage autophagy regulated by oxLDL/?2GPI/anti-?2GPI complex.