Correlation Between Daxx Mediated Apoptosis Of RAW264.7 Cells And Cellular Lipid Accumulation Induced By Ox-LDL

OBJECT: To explore correlation between Daxx mediated apoptosis of RAW264.7 macrophages and cellular lipid accumulation induced by oxidized low density lipoprotein (ox-LDL).METHODS: RAW264.7 macrophages growth inhibition was measured by MTT assay. The apoptosis effect of RAW264.7 murine macrophages induced by ox-LDL was analyzed by flow cytometric analysis and DNA agarose electrophoresis. After expression of Daxx was silenced by special siRNA, the macrophages apoptosis was observed by AO/EB fluorescence staining. Oil Red O Dyeing experiment was used to show the cellular lipid droplets in lipid-loaded cells. High performance liquid chromatography (HPLC) analysis was performed to determine the content of cellular cholesterol. Real time RT-PCR was used to detect the mRNA expressions of Daxx and SCAP. Western-Blot analysis was used to detect caveolin-1 protein expression.RESULTS: To explore the effect of ox-LDL on cellular growth in RAW264.7 cells, the cells were treated with ox-LDL at different concentration for 48h. The results markedly showed that ox-LDL inhibited RAW264.7 growth in a dose-dependent manner. When 0 rag/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 75mg/L ox-LDL were applied to the cells, the growth rates were 100±7.28%, 103.2±16.35%, 76.27±16.54%, 56.49±11.94% and 40.65±8.76% respectively, with IC50 value 50.6mg/L. Flow cytometry analysis showed that when RAW264.7 cells were exposed to different concentration of ox-LDL (0 mg/L, 12.5 mg/L, 25 mg/L,50 mg/L, 75 mg/L) for 48h, the apoptosis indexes were 2.3%, 4.9%, 8.6%, 14.7%, 33.1% respectively. Furthermore, after treated with 50mg/L ox-LDL for different time, the apoptotic rate of RAW264.7 cells increased in a time-dependent manner. The apoptotic "Ladder" bands were observed dearly after macrophages were exposure to 50mg/L ox-LDL for 48h. Real time RT-PCR revealed that ox-LDL increased expression of Daxx mRNA in a dose-dependent and time-dependent manner with a peak at 50mg/L ox-LDL. Daxx silenced by special siRNA resulted in decrease of apoptosis rat in RAW264.7 cells. After 48h exposure to 50mg/L ox-LDL, under the fluorescent microscope partial cells Showed typical apoptotic morphological changes such as nacarat fluorescent body shrinking and fragment of the nucleus. At the same time, the cellular cholesterol content also increased obviously, and many lipid droplets accumulated in cells. Transfection of Daxx siRNA attenuated the effect of ox-LDL on apoptosis and decreased lipid droplets in RAW264.7 cells. The apoptotic rate decreased obviously, and typical morphological change of apoptosis also descended markedly. The cellular cholesterol content and lipid droplets renewed. WB showed caveolin-1 protein level increased in a time-dependent manner, but decreased after transfection of Daxx siRNA. Real time RT-PCR revealed that ox-LDL inhibited the expression of SCAP mRNA and the SCAP mRNA expression rised after RNA interfering.CONCLUSION:1. Daxx mediates apoptosis of RAW264.7 macrophages induced by ox-LDL.2. Daxx exerts its pro-apoptotic effects by interfering in cholesterol uptake,which is associated with down-regulation of SCAP and up-regulation of caveolin-1.