葛根素对MPP^+诱导的SH-SY5Y细胞凋亡过程中p53 mRNA及相关基因表达的影响

Role of p53 in Neuroprotective Effects of Puerarin against MPP^+-induced Human Neuroblastoma SH-SY5Y Cell Death

目的:探讨1-甲基-4-苯基-吡啶离子(MPP+)诱导人神经母细胞瘤(SH-SY5Y)细胞凋亡过程中p53及其上下游相关基因的变化情况,并探讨葛根素干预的保护作用。方法:体外培养SH-SY5Y细胞株,待细胞密度达到70%融合后,用终浓度分别为50,100,150μmol·L-1的葛根素预先作用3 h后加入1 mmol·L-1的MPP+继续培养,第48 h采用噻唑蓝(MTT)法测定细胞存活率,第24 h采用Annexin-V/PI流式细胞分析术检测细胞凋亡率,实时荧光定量PCR(RT-PCR)技术分析p53及其相关基因mRNA表达量变化。结果:MTT结果显示葛根素预处理的细胞比单纯MPP+处理的细胞存活率有显著性增加(P<0.05),说明葛根素对MPP+诱导的SH-SY5Y细胞具有保护作用且呈一定的剂量依赖性;Annexin-V/PI流式细胞分析术检测结果表明葛根素可以降低MPP+诱导的SH-SY5Y细胞的凋亡率;RT-PCR结果显示MPP+诱导可以增加SH-SY5Y细胞的p53mRNA表达;葛根素预处理下调了p53及其相关基因p21,Bax,Caspase-3的mRNA的表达,上调了p53负调节因子MDM2和凋亡抑制基因Bcl-2的mRNA的表达。结论:MPP+诱导可以导致SH-SY5Y细胞的p53 mRNA增加;葛根素可以通过调节p53及其相关基因的表达对MPP+诱导的SH-SY5Y细胞发挥保护作用。

Objective： To investigate the expression changes of p53 mRNA and its related genes in 1-methyl-4-phenylpyridinium iodide （MPP＋） -induced SH-SY5Y cells and observe the protective effects of puerarin against the cell death. Method： SH-SY5Y cells were incubated to 70% confluence, the medium containing 50, 100, 150 μmol .L-1 puerarin was added to SH-SY5Y cells, at 3 h after drug application 1 mmol -L-i MPP＋ was added to culture. The viability of MPP＋ -induced SH-SY5Y cells under different doses of puerarin was assayed by 3- （4, 5-dimethyhhiazol-2-yl） -2, 5-diphenyhetrazolium bromide （MTT） at 48 h. The apoptosis rates were detected by flow cytometry with Annexin-V/PI fluorescence staining. The expression of p53 mRNA and its related genes was assayed by real-time quantitative reverse transcription-PCR （RT-PCR） at 24 h. Result：The puerarinpretreatment could apparently increase the viability and inhibit the early apoptosis of the MPP＋- induced SH-SY5Y cells in the experiment （P 〈 0.05 ）. Annexin-V/PI fluorescence staining results showed that puerarin could reduce the apoptosis rate of MPP＋-induced SI-I-SYSY cells. Real-time PCR results showed that the MPP＋ could increase the expression of p53 mRNA, but the expression of p53, p21, Bax, Caspase-3 mRNA was down-regulated in puerarin pretreatment groups compared with the MPP ＋ group, and the expression of MDM2 and Bcl-2 mRNA was up-regulated simultaneously. Conclusion： The expression of p53 mRNA in SH-SY5Y cells can increase by MPP＋ and puerarin can regulate the p53 and its related genes and play a protective role against the death of MPP＋-induced SH-SYSY cells.