三七总皂苷调控巨噬细胞sortilin表达及胆固醇代谢的研究

Effects of Panax notoginseng saponins on the expression of sortilin and cholesterol metabolism in macrophages

目的探讨三七总皂苷(PNS)对巨噬细胞分拣蛋白(sortilin)、腺苷三磷酸结合盒转运子A1(ABCA1)表达的影响和抑制泡沫细胞形成的作用及机制。方法将巨噬细胞分为A组(常规培养)、B组(转染空载慢病毒)、C组(转染过表达sortilin慢病毒)、D组(转染过表达sortilin慢病毒+60μg·mL^(-1)PNS)和E组(转染过表达sortilin慢病毒+10μmol·L^(-1)PD98059+60μg·mL^(-1)PNS)。用蛋白质印迹法检测各组巨噬细胞中sortilin、ABCA1、细胞外调节蛋白激酶(ERK)和磷酸化ERK(p-ERK)蛋白的表达水平;用50μg·mL^(-1)氧化低密度脂蛋白刺激巨噬细胞形成泡沫细胞,用油红O染色评估各组巨噬细胞的脂质含量。结果A、B、C、D、E组的sortilin蛋白相对表达水平分别为1.00±0.08、0.91±0.15、2.28±0.13、1.62±0.09和2.01±0.08,ABCA1蛋白相对表达水平分别为1.00±0.01、0.92±0.07、0.29±0.04、0.66±0.09和0.44±0.07,p-ERK/ERK比值分别为1.00±0.09、0.92±0.05、1.03±0.12、2.00±0.12和1.64±0.14,脂质含量分别为(4.82±2.19)%、(6.70±0.88)%、(44.56±4.15)%、(27.66±3.25)%和(41.67±5.45)%。除p-ERK/ERK外,C组的其他指标与A组相比,在统计学上差异均有统计学意义(均P<0.01);D组的上述指标与C组、E组比较,在统计学上差异均有统计学意义(P<0.01,P<0.05)。结论PNS通过抑制sortilin表达和上调ABCA1表达,从而减少泡沫细胞的形成;ERK是参与PNS调节sortilin和ABCA1表达的主要信号通路之一。

Objective To explore the role of Panax notoginseng saponins(PNS)in regulating the expression of sortilin and ATPbinding cassette transporter A1(ABCA1)in macrophages,and the effect of PNS on inhibiting formation of foam cells and the potential mechanism of PNS adjusting sortilin expression and cholesterol metabolism.Methods The macrophages were divided into five groups as follows:group A(only added with cell culture),group B(transfected with negative control lentivirus),group C(transfected with lentivirus-mediated sortilin overexpression),group D(transfected with lentivirus-mediated sortilin overexpression+60μg·mL^(-1)PNS),group E(transfected with lentivirus-mediated sortilin over expression+10μmol·L^(-1)mitogenactivated protein kinase kinase(MEK)inhibitor PD98059+60μg·mL^(-1)PNS).The protein contents of sortilin,ABCA1,extracellular signal-regulated kinase(ERK)and phosphorylated-ERK(p-ERK)were evaluated with Western blot.All the cells in five groups were cultured with 50μg·mL^(-1)ox-LDL to form foam cells.The lipid in macrophages was investigated with red O assay.Results The relative expression levels of sortilin protein were1.00±0.08,0.91±0.15,2.28±0.13,1.62±0.09 and 2.01±0.08;the relative expression levels of ABCA1 protein were 1.00±0.01,0.92±0.07,0.29±0.04,0.66±0.09 and 0.44±0.07;the ratios of p-ERK/ERK protein were1.00±0.09,0.92±0.05,1.03±0.12,2.00±0.12 and 1.64±0.14;the contents of lipid in macrophages were(4.82±2.19)%,(6.70±0.88)%,(44.56±4.15)%,(27.66±3.25)%and(41.67±5.45)%.Except the ratios of p-ERK/ERK,the other parameters between group C and group A were statistically significant difference(all P<0.01).Meanwhile,there were also statistically significant difference between group D and group C as well as group D and group E(P<0.05,P<0.01).Conclusion PNS inhibits the lipid accumulation in macrophages through upregulating ABCA1 and downregulating sortilin,and ERK signaling pathway may be as one of important mechanisms influencing the expression of sortilin and ABCA1 mediated by PNS.