Effects Of NIX-mediated Mitophagy On Ox-LDL-induced Macrophage Pyroptosis

Background & Objective: Pyroptosis is a novel form of cell death which is uniquely dependent on caspase-1.The pyroptosis involved in ox-LDL induced human macrophages death through promoting caspase-1 activation,which was important for formation of unstable plaques in atherosclerosis progress.Emerging evidence has demonstrated that autophagy occurs in atherosclerotic plaques and macrophage autophagy exerts a protective effect in advanced atherosclerosis.The absence of autophagy leads to the activation of NLRP3 inflammatory bodies.The production of reactive oxygen species ROS is closely related to cell death,which can activate caspase-1 and induce cell death.Several studies have reported that the BNIP3 gene family might play important roles in mediating mitophagy and mitochondrial quality control.The BNIP3 gene family is a unique subfamily of the BH3-only members of the Bcl-2 family,which includes BNIP3?NIX(BNIP3-like protein X,also called BNIP3L).NIX is able to homodimerize and localize to mitochondria.To date,multiple lines of evidence supports the essential role of NIX in developmental removal of mitochondria during erythrocyte maturation.NIX directly interacts with the Atg8 family of homologous proteins(LC3 and GABARAP),an indicator protein localized on isolation membrane,and mediates the subsequent binding and sequestration of mitochondria into autophagosomes.Our study showed that NIX-mediated mitochondrial autophagy is involved in the clearance of damaged mitochondria.However,how the effect of NIX on ox-LDL induced the macrophage pyroptosis is still unclear.Methods:1.By different concentrations of ox-LDL to select the appropriate concentration and time.The expression of NIX m RNA level was observed by PCR on mouse macrophage J774 A.1,and the protein level of NIX by Western-blot.2.Detection of mitochondrial autophagy.The detection of mitochondrial autophagy was observed by confocal laser scanning of LC3 ? NIX and colocalization of mitochondria.The expression of LC3-II / I and NIX by Western-blot after ox-LDL.The effect of silencing NIX expression on mitochondrial autophagy induced by ox-LDL was detected;3.The detection of cell death was performed.LDH and AO/ EB was used to detect;The detected the expression of TOMM20,caspase-1p10 and IL-1? by Western-blot;The effect of silencing NIX expression on macrophages pyroptosis induced by ox-LDL was detected;4.Detect the level of ROS in the cells before and after the action of ox-LDL by DCFH-DA staining,The expression of NLRP3 and NIX were detected by Western-blot.Detected above experimental results expression on the level of ROS and NLRP3 by the silencing NIX;Results:1.As the increase of concentration and time after ox-LDL,the expression level of NIX m RNA and protein increased with the increase the maximum at 100 ?g / ml and the top of 6 hours.2.Western-blot showed that the expression of LC3-? /? was increased after ox-LDL treatment in J774 A.1.Confocal microscopy results showed that there were co-localization relationship between NIX?LC3 and mitochondria.The expression of autophagy LC3 protein in autophagy pathway was lower after NIX silenced.3.LDH release assay results indicate that LDH release was increased in using si RNA-mediated silencing NIX than normal cell.AO/EB staining show that using si RNA-mediated silencing NIX of cell was higher normal cell.Western-blot results suggest that caspase-1 p10 and IL-1? protein increased.4.Ox-LDL-induced macrophages,the results found that inhibition of NIX.The expression of intracellular ROS levels and NLRP3 levels was increased.Conclusions: These results demonstrate that NIX could inhibit the ox-LDL-induced macrophages pyroptosis via autophagy.