Effect Of Overexpressed Daxx On Apoptosis Of Raw264.7Cells Induced By Cholesterol: Model Of β-cyclodextrin

Objective: The eukaryotic vector of Daxx was constructed to be stably transfectedinto RAW264.7cells. The aim of the present study was to determine the effect ofDaxx on apoptosis of RAW264.7cells induced by Chol:MβCD.Methods: The eukaryotic vector of Daxx was constructed to be stably transfectedinto RAW264.7cells. We detected the expression of Daxx in cells transfected withreorganized vector using RT-PCR and Western blotting to decide whether theeukaryotic vector is successfully constructed. Then, the recombinant transfectedRAW264.7cells were treated with20mg/L Chol:MβCD for48h. Oil Red O Dye wasused to show the lipid droplets in Chol:MβCD treated cells. Enzyme fluorescentanalysis was performed to determine the content of cellular total cholesterol and freecholesterol. RAW264.7macrophages growth inhibition was measured by MTT assay.The apoptosis effect of RAW264.7murine macrophages induced by Chol:MβCD wasanalyzed by flow cytometric analysis. The expression of ASK1, JNK and Caspase3were assayed by Western blotting.Results: RT-PCR and Western blotting analyses showed that the expression of Daxxwas significantly increased in gene-transfected RAW264.7cells. To explore the effectof Chol:MβCD on cellular growth in RAW264.7cells, the cells were treated with20mg/L Chol:MβCDfor48h. Oil Red O showed that the lipid droplets were more intransfected RAW264.7cells than in control and vector group. Intracellular totalcholesterol (TC) and free cholesterol (FC) significantly increased in transfectedRAW264.7cells. Transfection of Daxx increased obviously the effect of Chol:MβCDon apoptosis in RAW264.7cells and significantly decreased the vitality of RAW264.7cells. WB showed ASK1, JNK and Caspase3protein level significantly elevated whenRAW264.7cells were transfected by Daxx. Conclusion: Daxx mediated the apoptosis of RAW264.7cells induced byChol:MβCD. This may be because Daxx was translocated to the cytoplasm, bind toASK1, and subsequently lead to ASK1oligomerization, then activate JNK andCaspase3pathway.