【摘要】 目的:探讨藏红花素对帕金森病细胞损伤模型的影响和可能机制。方法:不同浓度(10、50、100μmol/L)的藏红花素作用于1-甲基-4-苯基-吡啶离子(MPP+)诱导SK-N-SH细胞,试剂盒检测细胞中丙二醛(MDA)和还原性谷胱甘肽(GSH)的含量,流式细胞术检测细胞凋亡,实时荧光定量PCR (RT-qPCR)检测长链非编码RNA (lncRNA)睾丸特异性转录Y-连锁15 (TTTY15)和微小RNA (miRNA) let-7c-5p的表达水平。双荧光素酶报告实验和RT-qPCR确定TTTY15和let-7c-5p之间的靶向关系。分别转染TTTY15小干扰RNA (si-TTTY15)、let-7c-5p模拟物至人神经母细胞瘤细胞(SK-N-SH),采用上述方法检测敲低TTTY15或过表达let-7c-5p对MPP+诱导的SK-N-SH细胞损伤的影响。结果:与Con组比较,MPP+组SK-N-SH细胞MDA含量升高,GSH含量显著降低(P<0.05);与MPP+组比较,MPP++crocin-L组、MPP++crocin-M组、MPP++crocin-H组SK-N-SH细胞MDA含量降低,GSH含量升高(P<0.05)。MPP++crocin-L组、MPP++crocin-M组、MPP++crocin-H组3组MDA、GSH变化与藏红花素剂量相关,MDA随剂量增加而降低,GSH随剂量增加而升高(P<0.05)。与Con组比较,MPP+组SK-N-SH细胞凋亡率、Bax蛋白表达升高,Bcl-2蛋白表达降低(P<0.05);与MPP+组比较,MPP++crocin-L组、MPP++crocin-M组、MPP++crocin-H组SK-N-SH细胞凋亡率、Bax蛋白表达降低,Bcl-2蛋白表达升高(P<0.05)。MPP++crocin-L组、MPP++crocin-M组、MPP++crocin-H组之间各检测指标比较,差异有统计学意义(P<0.05)。与Con组比较,MPP+组SK-N-SH细胞TTTY15表达升高,let-7c-5p表达降低(P<0.05);与MPP+组比较,MPP++crocin-L组、MPP++crocin-M组、MPP++crocin-H组SK-N-SH细胞TTTY15表达降低,let-7c-5p表达升高(P<0.05)。MPP++crocin-L组、MPP++crocin-M组、MPP++crocin-H组各指标组间比较,差异有统计学意义(P<0.05)。与miR-NC组比较,let-7c-5p组WT-TTTY15的SK-N-SH细胞荧光素酶活性降低(P<0.05);miR-NC组与let-7c-5p组MUT-TTTY15的SK-N-SH细胞荧光素酶活性比较,差异无统计学意义(P>0.05)。与pcDNA组比较,pcDNA-TTTY15组SK-N-SH细胞let-7c-5p表达降低(P<0.05);与si-NC组比较,si-TTTY15组SK-N-SH细胞let-7c-5p表达升高(P<0.05)。与MPP++si-NC组比较,MPP++si-TTTY15组SK-N-SH细胞TTTY15的表达水平降低,凋亡率、Bax蛋白表达、MDA含量降低,Bcl-2蛋白表达、GSH含量升高(P<0.05)。与MPP++NC组比较,MPP++let-7c-5p组SK-N-SH细胞let-7c-5p的表达水平升高,凋亡率、Bax蛋白表达、MDA含量降低,Bcl-2蛋白表达、GSH含量升高(P<0.05)。结论:藏红花素对帕金森病细胞损伤模型具有保护作用,其机制可能与下调lncRNA TTTY15/let-7c-5p通路有关。更多还原

【Abstract】 Objective: To discuss the effect of crocin on cell injury model of Parkinson disease and its possible mechanism. Methods:1-methyl-4-phenyl pyridinium cation(MPP+) was acted on by different concentrations of crocin(10,50 and 100μmol/L) to induce SK-N-SH cell. The contents of malondialdehyde(MDA) and reduced glutathione(GSH) in cells were detected by kits. Cell apoptosis was detected by flow cytometry. The expression levels of long non-coding RNA(lncRNA)testis-specific transcript Y-linked 15(TTTY15) and micro RNA(miRNA) let-7 c-5 p were detected by real time fluorescence quantitative PCR(RT-qPCR). The target relationship between TTTY15 and let-7 c-5 p was identified by dual luciferase reporter assay and RT-qPCR. TTTY15 small interfering RNA(si-TTTY15) and let-7 c-5 p mimics were transfected into human neuroblastoma cell(SK-N-SH) respectively;the effect of down-regulated TTTY15 or over-expression of let-7 c-5 p on SKN-SH cell injury induced by MPP+was tested by the above methods. Results:Compared with those in the Con group,in the MPP+group,the MDA content in SK-N-SH cell was increased,and GSH content was significantly decreased(P<0.05).Compared with those in the MPP+group,in the MPP++crocin-L,MPP++crocin-M and MPP++crocin-H groups,the MDA contents in SK-N-SH cell were decreased,and GSH contents were increased(P<0.05). In the MPP++crocin-L,MPP++crocin-M and MPP++crocin-H groups,the changes in MDA and GSH were associated with the dose of crocin;MDA was decreased as the dose was increased,and GSH was increased as the dose was increased(P<0.05). Compared with those in the Con group,in the MPP+group,the apoptosis rate of SK-N-SH cell and the expression of Bax protein were increased,and the expression of Bcl-2 protein was decreased(P<0.05). Compared with those in the MPP+group,in the MPP++crocin-L,MPP++crocin-M and MPP++crocin-H groups,the apoptosis rate of SK-N-SH cell and the expression of Bax protein were decreased,and the expression of Bcl-2 protein was increased(P<0.05). When compared each detection index between the MPP++crocin-L,MPP++crocin-M and MPP++crocin-H groups,there was significance in the difference(P<0.05). Compared with those in the Con group, in the MPP+group, the expression of TTTY15 of SK-N-SH cell was increased, and the expression of let-7 c-5 p was decreased(P<0.05). Compared with those in the MPP+group,in the MPP++crocin-L,MPP++crocin-M and MPP++crocin-H groups,the expression of TTTY15 of SK-N-SH cell was decreased,and the expression of let-7 c-5 p was increased(P<0.05). When compared each index between the MPP++crocin-L,MPP++crocin-M and MPP++crocin-H groups, there was significance in the difference among the three groups(P<0.05). Compared with that in the miR-NC group,in the let-7 c-5 p group,luciferase activity of SK-N-SH cell of WT-TTTY15 was decreased(P<0.05);when compared luciferase activity of SK-N-SH cell of MUT-TTTY15 between the two groups,there was no significance in the difference(P>0.05). Compared with that in the pcDNA group,in the pcDNA-TTTY15 group,the expression of let-7 c-5 p of SK-N-SH cell was decreased(P<0.05). Compared with that in the si-NC group,in the si-TTTY15 group,the expression of let-7 c-5 p of SK-N-SH cell was increased(P<0.05). Compared with those in the MPP++si-NC group,in the MPP++siTTTY15 group,the expression level of TTTY15 of SK-N-SH cell was decreased;the apoptosis rate,the expression of Bax protein and MDA content were decreased,and the expression of Bcl-2 protein and GSH content were increased(P<0.05).Compared with those in the MPP++NC group,in the MPP++let-7 c-5 p group,the expression level of let-7 c-5 p of SK-NSH cell was increased; the apoptosis rate, the expression of Bax protein and MDA content were decreased, and the expression of Bcl-2 protein and GSH content were increased(P<0.05). Conclusion:Crocin has protective effect on cell injury model of Parkinson disease,whose mechanism may be related to down-regulation of lncRNA TTTY15/let-7 c-5 p pathway.更多还原