β分泌酶抑制剂对冈田酸诱导PC12细胞淀粉样蛋白前体蛋白代谢的影响

目的 探索β分泌酶抑制剂对冈田酸(OA)诱导的PC12细胞淀粉样蛋白前体蛋白(APP)代谢的影响. 方法 10 nmol/LOA作用4 h、8 h、16 h、24h及48 h诱导PC12细胞tau磷酸化,加β分泌酶抑制剂干预,MTT法测定细胞抑制率,免疫细胞化学法及Western blot检测全长APP和β-C末端片段(βCTF). 结果 10 nmol/L OA对PC12细胞的抑制作用呈时间依赖性,β分泌酶抑制剂可明显减轻该作用.OA诱导PC12细胞内全长APP和13CTF含量增加,B分泌酶抑制剂进一步增加细胞内全长APP含量,并减少βCTF含量. 结论 OA诱导PC12细胞中APP主要经β分泌酶途径代谢,生成具有神经毒性的βCTF.β分泌酶抑制剂通过维持细胞存活和减少13CTF从而减轻OA诱导的神经毒性,但使细胞内APP进一步增多.

Effect of β-secretase inhibitor on amyloid protein precursor metabolism in okadaic acid-induced PC12 cells

Objective To observe the effect of β-secretase inhibitor on amyloid protein precursor (APP) metabolism in okadaic acid (OA)-induced PC12 cells. Methods PC12 cells were pretreated with β-secretase inhibitor followed 30 min later by treatment with 10 nmol/L OA for 4, 8, 16, 24 or 48 h to induce tau phosphorylation. The viability of the PC12 cells was measured by MTT assay, and APP metabolism in the cells was examined by immunocytochemistry and Western blotting with specific antibodies against the APP-N-terminus (NT) and APP-C-terminus (CT). Results OA at the concentration of 10 nmol/L time-dependently inhibited PC12 cell viability, and this effect was obviously attenuated by pretreatment of the cells with β-secretase inhibitor. After OA treatment, both APP and β-C-terminal fragment (βCTF) were significantly increased in PC12 cells, and pretreatment with β-secretase inhibitor further increased the intracellular APP level and reduced the level of βCTF. Conclusion OA treatment causes a shift in APP metabolism to the β-secretase pathway that produces neurotoxic βCTF in PC12 cells. Inhibition of β-secretase may alleviate OA-induced cytotoxicity by decreasing the level of βCTF, but the application of β-secretase inhibitor may further increase APP level in the cells.