丹皮酚对MPP^+诱导的SH-SY5Y细胞线粒体自噬和死亡的保护作用

Protective effects of paeonolon MPP^+-induced mitophagy and death in SH-SY5Y cells

目的研究线粒体自噬在MPP^+损伤SH-SY5Y细胞中的发生及丹皮酚的干预作用。方法以1 mmol·L^(-1) MPP^+诱导SH-SY5Y细胞损伤建立帕金森病模型。采用MTT和LDH法分别检测不同浓度的丹皮酚对MPP^+诱导的SH-SY5Y细胞存活率和损伤度的影响;MDC染色荧光显微镜下检测自噬空泡聚集;流式细胞术定量分析细胞内酸性自噬泡的形成;透射电镜观察线粒体自噬现象;激光共聚焦检测溶酶体和线粒体共定位;Western blot检测线粒体自噬相关蛋白parkin、LC3和Beclin-1的表达变化。结果在1～100μmol·L^(-1)浓度范围内,预先加入丹皮酚能拮抗MPP^+诱导的SH-SY5Y细胞损伤,并呈一定的量效关系;SH-SY5Y细胞经MPP^+处理24 h,观察到自噬现象,出现自噬空泡增多,自噬水平增加,并存在较多的线粒体与溶酶体共定位的现象,LC3-Ⅱ和Beclin-1蛋白表达增加,parkin表达降低。预先加入丹皮酚后能逆转这些现象。结论丹皮酚抑制MPP^+诱导的SH-SY5Y细胞线粒体自噬的发生,阻止神经细胞的死亡。

Aim To study the occurrence of mitophagy in MPP^＋-induced SH-SY5Y cells and the involvement of paeonol. Methods Being pretreated with different concentrations of paeonol, SH-SY5Y cells were damaged by 1 mmol·L^-1 MPP^＋. Cell viability and cell toxicity were evaluated by MTT and LDH assay. The autophagic vacuoles were observed by fluorescence microscopy and measured by flow cytometry with monodansylcadaverine staining. Mitophagy was observed with transmission electron microscopy. Co-localization of mitochondria and lysosome were detected with a confocal laser-scanning microscopy. The expression of mitophagy associated-proteins were determined by Western blotting. Results After incubation with MPP^＋ for 24 h, the autophagic vacuoles in SH-SY5Y cells remarkably increased and autophagic activity significantly increased. MPP^＋ incubation transferred LC3 from LC3-Ⅰ to LC3-Ⅱ, increased Beclin 1, but decreased parkin expression. Critically, the co-localization of mitochondria and lysosome increased in SH-SY5Y cells after MPP^＋ incubation. However, pretreatment with 10 μmol·L^-1 paeonol effectively reversed these phenomena. Conclusions Paeonol can inhibit MPP^＋-induced mitophagy and cell death in SH-SY5Y cells, suggesting that paeonol may have potential target for neuroprotection in PD.