自噬参与锰诱导的SH-SY5Y细胞毒性研究

Autophagy was involved in cytotoxicity of Mn(Ⅱ) to SH-SY5Y cells

目的研究不同剂量的锰(Mn)对人神经母细胞瘤细胞SH-SY5Y细胞生长增殖的影响以及诱导自噬的情况。方法体外培养SH-SY5Y细胞,以200～1 000μmol/L MnCl2处理后进行四唑盐比色实验(MTT)观察MnCl2对细胞的抑制作用,MDC染色法检测自噬小泡形成情况,Western blot检测自噬标志蛋白LC3和Bclin-1的表达和活化情况。DCFH-DA染色检测活性氧(ROS)含量。结果 MTT结果显示200～1 000μmol/L MnCl2对细胞的生长增殖有显著的抑制作用。Western blot结果显示MnCl2处理后LC3蛋白表达增加并且活化,Bcelin-1蛋白表达增加,细胞产生自噬小泡。与对照组相比,200、400和800μmol/L MnCl2组ROS含量明显升高。结论一定剂量的MnCl2对SH-SHY5Y细胞的生长增殖有明显的抑制作用,这一作用可能是通过破坏胞内氧化还原平衡导致ROS大量产生继而诱发细胞发生自噬性的死亡。

Objective In this study,we investigated the inhibition of Mn（II） on SH-SY5Y proliferation and whether it causes autophagy.Method Cell viability was tested by MTT assay;autophagic levels were evaluated by MDC staining and west-ern blot of LC-3 and Beclin-1;ROS generation was detected using DCFH-DA.Results 200 ～ 1 000 μmol/L MnCl2 had obvious inhibition on SH-SY5Y cells;MnCl2 induced autophagy which was demonstrated by enhanced fluorescence intensity of monodan-sylcadervarine（MDC）,as well as elevated expression of autophagy-related protein LC3 and Beclin-1.Furthermore,our data in-dicated that cellular ROS levels were increased after treatment of MnCl2.Conclusion MnCl2 has obvious cytotoxicity on SH-SY5Y cells,which may be happened via ROS-triggered autophagic pathway.