藏红花素调控lncRNA TTTY15/let-7c-5p 通路保护帕金森病细胞损伤模型机制研究
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R285.5;R744.8

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Study on Mechanism of Crocin Protecting Cell Injury Model of Parkinson Disease by Regulating lncRNA TTTY15/let-7c-5p Pathway
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    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、100 μmol/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。

    Abstract:

    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- 7c- 5p were detected by real time fluorescence quantitative PCR(RT-qPCR). The target relationship between TTTY15 and let-7c-5p was identified by dual luciferase reporter assay and RT- qPCR. TTTY15 small interfering RNA(si- TTTY15) and let- 7c- 5p mimics were transfected into human neuroblastoma cell(SK-N-SH) respectively;the effect of down-regulated TTTY15 or over-expression of let-7c-5p 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-7c-5p 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-7c-5p 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-7c-5p 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-7c-5p 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- 7c- 5p of SK- N- SH cell was increased(P<0.05). Compared with those in the MPP ++ si- NC group, in the MPP ++ si- TTTY15 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-7c-5p group,the expression level of let-7c-5p 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-7c-5p pathway.

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黄宁宁,喻跃国,王晓蓉.藏红花素调控lncRNA TTTY15/let-7c-5p 通路保护帕金森病细胞损伤模型机制研究[J].新中医,2020,52(19):14-20

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  • 在线发布日期: 2020-10-12
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