辛伐他汀对转β分泌酶HEK293细胞RhoA/ROCK途径及Aβ_(42)生成的影响

Effects of simvastatin on RhoA / ROCK pathway and secretion of Aβ_(42) in β-secretase transferred HEK293 cells

目的观察辛伐他汀(Sim)通过RhoA/ROCK途径对转β分泌酶(BACE1)-HEK293细胞分泌β-淀粉样蛋白(Aβ)的影响。方法体外培养BACE1-HEK293细胞,在保证培养环境胆固醇充足的条件下分为对照组、1μmol/L Sim组、1μmol/L Sim+250μmol/L甲羟戊酸(Mev)组、5μmol/L Sim组、5μmol/L Sim+250μmol/L Mev组对细胞进行处理。MTT检测细胞存活率;ELISA检测Aβ42分泌量;Western blotting检测细胞膜RhoA及细胞浆磷酸化肌球蛋白磷酸酶肌球蛋白结合亚基(p-MYPT1)表达量。结果 MTT显示各组细胞存活率无差异(P>0.05);1μmol/L Sim组和5μmol/L Sim组细胞分泌Aβ42较对照组减少(P<0.05);两组细胞膜上RhoA及细胞浆p-MYPT1含量较对照组显著降低(P<0.01),分别加入250μmol/L Mev后能对抗Sim的作用(P<0.01)。结论 Sim可以通过降低胆固醇以外的途径减少RhoA蛋白的细胞膜定位和下游激酶ROCK的活化,抑制细胞Aβ42的分泌。

Objective To observe the effect of simvastatin （Sim） on the production of beta-amyloid peptide （Aβ） via RhoA/ROCK pathway. Methods HEK293 cells transferred with β-secretase （BACE1） were cultured in vitro with cholesterol sufficient culture medium and divided into five groups： control group, 1 μmol/L Sim group, 1 μmol/L Sim ＋250 μmol/L mevalonic acid（Mev） group, 5 μmol/L Sim group, 5 μmol/L Sim ＋ 250μmol/L Mev group. MTT was employed to identify the vitality of the cells ; ELISA was used for detecting extracellular Aβ42 ; Western blotting was employed to detect the expression of RhoA in cell membrane and phosphorylated myosin-binding subunit of myosin phosphatase （p-MYPT1） in cytoplasm. Results All treatment groups had no effects on the vitality of BACE1- HEK293 cells（ P 〉 0.05 ）. Compared with control group, the secreted Aβ42 was decreased in 1 μmol/L and 5 μmol/L Sim groups （ P 〈 0.05 ）. The expression of RhoA in cell membrane and p-MYPT1 in cytoplasm were both decreased in 1 μmol/L and 5 p, mol/L Sire groups （ P 〈 0.01 ）, and 250 μmol/L Mev could reversed the effect of Sim （ P 〈 0.01 ）. Conclusion Simvastatin decreases not only the RhoA in cell membrane together with p-MYPT1 in cytoplasm, but also the secretion of Aβ42 via cholesterol-independent mechanism.