丹酚酸D对MPP+损伤SH-SY5Y细胞线粒体功能和生物合成的影响

摘要

目的 研究丹酚酸 D ( salvianolic acid D, SalD)对MPP+损伤SH-SY5Y细胞线粒体功能和生物合成的影响及机制.方法 采用 MPP+损伤 SH-SY5Y细胞模型,MTT法检测MPP+对细胞的毒性作用,MTT和LDH法考察SalD对SH-SY5Y细胞活力的影响,AO/EB法检测细胞凋亡.应用DCFH-DA和MitoSOX分别检测细胞 ROS及线粒体超氧化物水平,通过测定细胞内ATP水平以及线粒体膜电位变化,考察线粒体功能. qPCR检测MPP+损伤细胞后,PGC-1α及其下游调控基因NRF1和TFAM的mRNA水平,Western blot及免疫荧光测定细胞中PGC-1α、NRF1 和TFAM蛋白含量.结果 MPP+可损伤SH-SY5Y细胞,导致细胞存活率明显降低为51.34% . 0.1、1、5 μmol·L-1SalD 和 5 mmol·L-1 NAC能够减轻MPP+诱导的SH-SY5Y细胞损伤及LDH释放,其细胞存活率分别增加到67.98% 、71.79% 、76.91% 、77.55% .并且,SalD能减少MPP+诱导的细胞内ROS及线粒体超氧化物的升高,降低线粒体膜电位,改善线粒体功能.同时,SalD能够明显抑制MPP+损伤导致的PGC-1α、NRF1、TFAM mRNA转录及表达降低.结论 SalD能够抑制MPP+诱导的SH-SY5Y细胞损伤,保护线粒体功能和线粒体生物合成.%Aim To investigate the effects of salvianol-ic acid D ( SalD) on mitochondrial function and bio- synthesis in SH-SY5Y cells after MPP+injury and the possible mechanisms. Methods The cell model was established by MPP+injury in SH-SY5Y cells. The cytotoxicity of MPP+was detected by MTT assay. The effects of SalD on viability of SH-SY5Y cells were ex-amined by MTT and LDH assay. The apoptosis of SH-SY5Y cells was detected by AO/EB assay. The levels of ROS and mitochondrial superoxide were determined using DCFH-DA and MitoSOX probes, respectively. Mitochondrial function was examined by measuring ATP level and mitochondrial membrane potential. The levels of PGC-1α and its downstream regulatory genes NRF1 and TFAM mRNA were detected by qPCR. The protein levels of PGC-1α, NRF1 and TFAM in cells were detected by Western blot and immunofluorescence assays. Results MPP+injury resulted in a significant reduction of cell viability to 51.34%. 0.1, 1, 5 μmol ·L-1SalD and 5 mmol·L-1NAC could reduce MPP+-induced SH-SY5Y cell injury and LDH release. The cell viability increased to 67.98% , 71.79% , 76.91% and 77.55% , respectively. Moreover, SalD could reduce the increase of intracellular ROS and mi-tochondrial superoxide induced by MPP+, decrease mitochondrial membrane potential and improve mito-chondrial function. SalD also significantly increased both the transcription and expression levels of PGC-1α, NRF1 and TFAM. Conclusion SalD could in-hibit MPP+-induced SH-SY5Y cell injury and improve mitochondrial function and mitochondrial biosynthesis.