The Research On The Mechanism Of Squalene Based On SCAP-SREBP Sterol Regulation Pathway

Objective:The aim of this study was to investigate the mechanism of squalene on up-regulating the expression of LDLR by activating the SCAP/SREBP pathway to promote the cell uptake and metabolism of cholesterol.Methods:We have established a traditional Chinese medicine monomer library,then members of the library were docked with SSD named sterol binding site from the pathway of SCAP/SREBP,cholesterol as a reference,using SYBYL-X 2.0 computer simulation analysis software,and we can find the traditional Chinese medicine monomer who have a good binding ability on SSD comparing with cholesterol.Using lentiviral transfection method and picking cells under fluorescent microscope method we establish a stable CFPSCAP/YFPInsig2/HepG2 cell line.Viability of HepG2 cell treated by squalene was measured by MTT assay to select the reasonable medication scope.Applying flow cytometer(FCM)and fluorescence microscope to detect the effect on activity of LDLR in HepG2 cells treated by different concentration of squalene.To explore squalene whether can develop the influence of the expression of LDLR in HepG2 cells though activating the pathway of SCAP/SREBP by using FRET technology and CFPSCAP/YPInsig2/HepG2 reporter gene system.Results:We have filtered squalene which is a herbal monomer molecules from traditional Chinese medicine monomer library by using SYBYL-X 2.0 molecular docking software.We have established a stable CFPSCAP/YFPInsig2/HepG2 cell line.MTT experiments show that squalene have little cytotoxicity and 0-400μmol/L is a reasonable medication scope of squalene for HepG2 cells.We validate that squalene has raised the expression activity of LDLR in HepG2 cells using flow cytometer(FCM)and fluorescence microscope.FRET experiment preliminary confirmed from cells and protein levels that squalene can up-regulate the expression of LDLR in HepG2 cells though activating the pathway of SCAP/SREBP.Conclusion:Squalene can up-regulate the expression of LDLR in HepG2 cells by activating the pathway of SCAP/SREBP.