Effect Of Astragalus Polysaccharide On The Expression Of PCSK9 In THP-1 And HEPG2 Cells

Objective To investigate the effect of Astragalus Polysaccharide(APS) on the expression of Proprotein convertase subtilisin/kexin type 9(PCSK9) and explore the connection with peroxisome proliferator-activated receptor β/δ(PPAR-β/δ).Methods Cultivation of THP-1 and Hep G2 cells in vitro, CCK-8 assay was applied to select the optimal concentration of APS on the THP-1 monocyte-macrophages cells and HepG2 cells. Experiment is divided into two parts, Part one,THP-1 derived macrophages as experiment object,groups: Control group; LDL group; LDL+APS group; LDL+GW0742 group; LDL+GSK0660+APS group. The fluorescent expression quantity of PCSK9 in different groups were detected by immunocytochemistry. The relative mRNA level and protein expression level of PCSK9、LDLR and PPAR-β/δ were examined by fluorescent quantitative real-time PCR(RT-PCR) and Western Blot. Part two,HepG2 cells as experiment object,groups: Control group; APS group; GW0742 group; GSK0660+APS group; The fluorescent expression quantity of pcsk9 and absorbing state of Dil-LDL in each group were dected by confocal laser-scanning microscopy, The relative mRNA and protein expression level of PCSK9、LDLR and PPAR-β/δ were examined by RT-PCR and Western Blot.Results Part one:1)The result of CCK-8 assay was 400ug/ml; 2)Both RT-PCR and Western blot datas validated that the expression of PCSK9 in LDL group was up-regulated VS control group, but down-regulated in LDL+APS and LDL+GW0742 groups, compared with LDL+APS and LDL+GW0742 groups, the LDL+APS+GSK0660 group has higher expression( P<0.05); 3)The protein expression of LDLR was down-regulated in LDL group VS control group, up-regulated in LDL+APS and LDL+GW0742 groups VS LDL group, and the LDL+APS+GSK0660 group reduced expression compared to the former two groups(P<0.05),but the mRNA expression of LDLR in LDL group、LDL+APS group、LDL+GW0742 group 、 LDL+GSK0660+APS group were all lower VS control group, and there,s no significance difference between the four groups. 4)The mRNA and protein expression of PPAR-β/δ was obviously increased in LDL+APS and LDL+GW0742 groups VS control group, and down-regulated in the LDL+APS+GSK0660 group.Part two:1)The result of CCK-8 assay was 600ug/ml; 2) Confocal results suggest that the uptake of Dil-LDL in APS group and GW0742 group were strengthened VS control group, but weakened in GSK0660+APS group. 3)Both RT-PCR and Western blot datas validated that the expression of PCSK9 in APS group and GW0742 group was down-regulated VS control group, but up-regulated in GSK0660+APS VS APS group and GW0742 group. 4) The relative protein expression of LDLR was up-regulated in APS group and GW0742 group VS control group, and down-regulated in GSK0660+APS group VS APS group and GW0742 group.But the mRNA expression has no statistical difference. 5) The expression of PPAR-β/δ was obviously increased in APS and GW0742 groups VS control group at both mRNA and protein levels, and down-regulated in the APS+GSK0660 group.Conclusion Aps could down-regulated the expression of PCSK9 in both THP-1 derived macrophages and HepG2 cells, up-regulated LDLR accordingly,in which PPAR-β/δ may be involved.