CyPA Enhances The Ox-LDL-induced Activation And Apoptosis In RAW264.7 Cells Via Regulating Autophagy

Objective To explore whether Cy PA enhance the ox-LDL-induced activation and apoptosis in Raw264.7 Cells via regulating autophagy.Methods RAW264.7 cells were cultured in vitro. The levels of Cy PA、β-actin、LC3、beclin-1、bcl-2、ABCA1 and CD36 were determined by Western blot. The level of Cy PA in supernatant was measured by ELISA. Small interfering RNA(si RNA) was used to silence the expression of Cy PA and Western blot was used to examine efficiency of gene silence. Autophagosome were observed by electron microscope. Autophagy flux was observed by GFP-RFP-LC3 adenovirus. Lipid deposition was identified by oil red o staining. The positive rates of CD80, CD86 and MHCII were determined by Flow Cytometry(FCM).Apoptosis was examined by Tunel.Results(1) The level of Cy PA was significantly increased at 24 h after stimulated by ox-LDL(100mg/L) in RAW264.7 cells with the peak at 6h(p<0.05).The level of Cy PA in supernatant was increased in time dependent way(p<0.05).(2) RAW264.7 cells were incubated with ox-LDL for 0, 6, 12 and 24 h after gene silencing of Cy PA, the ratio of LC3II/I was decreased in Cy PA silent cells compared with the control cells(p<0.05), but there is no difference of the level of beclin-1. Typical autophagic vacuoles were observed in the cytoplasm by electron micropy, Cy PA silencing caused a marked decrease of autophagosome compared with the control group(p<0.05).(3) RAW264.7 cells were pretreated with GFP-RFP-LC3 adenovirus and then incubated with ox-LDL, Cy PA silent cells presented more GFP—RFP+ punct(autophagolysosome) than the control cells(p<0.05).(4) Cy PA silencing markedly decreased the lipid accumulation in RAW264.7 cells. We further found ATP-binding cassette transporter A1(ABCA1) was significantly increased while the scavenger receptor CD36 was decreased in Cy PA silent cells compared to the control cells(p<0.05)(5) Gene silencing of Cy PA significantly decreased the level of CD80, CD86 and MHC class 2 in RAW264.7 cells when incubating with ox-LDL for 24h(p<0.05). Further, when pretreated with 1m M 3-MA for 1h, the difference between two groups was abolished with the cell marks increased.(6) TUNEL assay were determined in RAW264.7 cells under ox-LDL stimulation. The results demonstrated that gene silencing of Cy PA caused a marked decraease of apoptotic RAW264.7 cells compared with the control group. Further, when pretreated with 3-MA, the apoptotic cells decreased but there was no significant difference between Cy PA silent cells and the control cells.Conclusions(1) Cy PA was up-regulated and released by ox-LDL stimulating in RAW264.7cells.(2) Cy PA enhances the expression markers of autophagy but blocks autophagy flux in RAW264.7 cells.(3) Cy PA regulates lipid accumulation in macrophage foam cells.(4) Cy PA enhances ox-LDL-induced activation and apoptosis via regulating autophagy in RAW264.7 cell.