[1]宋志远,武一平,杨华,等.褪黑素通过下调SLC7A11表达来诱导胶质瘤细胞SH-SYSY铁死亡的机制研究[J].卒中与神经疾病杂志,2022,29(05):416-421.[doi:10.3969/j.issn.1007-0478.2022.05.004]
　Song Zhiyuan,Wu Yiping,Yang Hua,et al.Study on the mechanism of melatonin inducing iron death of SH-SYSY glioma cells by down-regulating the expression of SLC7A11[J].Stroke and Nervous Diseases,2022,29(05):416-421.[doi:10.3969/j.issn.1007-0478.2022.05.004]
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褪黑素通过下调SLC7A11表达来诱导胶质瘤细胞SH-SYSY铁死亡的机制研究()
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《卒中与神经疾病》杂志[ISSN:1007-0478/CN:42-1402/R]

卷:
第29卷
期数:
2022年05期
页码:
416-421
栏目:
论著
出版日期:
2022-10-10

文章信息/Info

Title:
Study on the mechanism of melatonin inducing iron death of SH-SYSY glioma cells by down-regulating the expression of SLC7A11
文章编号:
1007-0478(2022)05-0416-06
作者:
宋志远; 武一平; 杨华; 孙艳艳; 曲大成; 任洪波
056001 河北省邯郸市中心医院神经外三科［宋志远 武一平 杨华 曲大成 任洪波（通信作者）］，心内二科（孙艳艳）
Author(s):
Song Zhiyuan;  Wu Yiping;  Yang Hua;  et al.
Department of Neurosurgery, Handan Central Hospital, Handan 056001
关键词:
褪黑素; 溶质载体家族7成员11; 胶质瘤; 铁死亡
Keywords:
Melatonin; Solute carrier family 7 member 11; Glioma; Iron death
分类号:
R739.41
DOI:
10.3969/j.issn.1007-0478.2022.05.004
文献标志码:
A
摘要:
目的 探讨褪黑素（Melatonin，MT）通过下调溶质载体家族7成员11（Solute carrier family 7member 11，SLC7A11）表达对胶质瘤细胞（Glioma cell，SH-SYSY）铁死亡的影响。 方法 使用不同水平的MT处理人胶质瘤SH-SY5Y细胞，采用细胞计数试剂盒-8（Cell counting kit-8，CCK-8）法检测细胞活性，进而选择合适的MT水平；实验分组为二甲基亚砜（Dimethyl sulfoxide，DMSO）组（加入常规培养基）、MT组（加入1 mmol/L的MT）、凋亡抑制剂氟甲基酮（Z-VAD-FMK）+MT组（加入1 mmol/L的MT和20 μmol/L的凋亡抑制剂Z-VAD-FMK）、坏死性凋亡抑制剂Necrosulfonamide（Nec-1）+MT组（加入1 mmol/L的MT和1 μmol/L的坏死性凋亡抑制剂Nec-1）、自噬抑制剂Chloroquine +MT组（加入1 mmol/L的MT和5 mmol/L的自噬抑制剂Chloroquine）、特异性铁死亡抑制剂Deferoxamine（DFO）对照组（加入20 μmol/L的特异性铁死亡抑制剂DFO）及DFO+MT组（加入1 mmol/L的MT和20 μmol/L DFO）；CCK-8法检测细胞活性；碘化丙啶（Propidium iodide，PI）染色法评价细胞死亡情况；Phen Green SK荧光指示剂法测定细胞Fe2+水平；活性氧（Reactive oxygen，ROS）检测试剂盒测定细胞总ROS水平；C11-BODIPY染色法检测细胞脂质活性氧（Lipid reactive oxygen species，lipid ROS）水平；总谷胱甘肽（Glutathione，GSH）检测试剂盒测定细胞GSH水平；Western blot法检测细胞铁蛋白重链（Ferritin heavy polypeptide，FTH）、铁蛋白轻链（Ferritin light chain，FTL）、膜铁转运蛋白（Ferroportin，FPN）、转铁蛋白受体1（Transferrin receptor 1，TFR1）和SLC7A11蛋白表达水平；实时定量反转录聚合酶链反应（Real-time quantitative reverse transcription polymerase chain reaction，qRT-PCR）检测细胞SLC7A11 mRNA表达水平。 结果 随着MT水平的增高，SH-SY5Y细胞活性逐渐降低（P<0.05），由于1 mmol/L处理的SH-SY5Y细胞活性最低，因此选择1 mmol/L MT诱导细胞。与DMSO组比较，DFO+MT组SH-SY5Y细胞活性显著升高（P<0.05），而Z-VAD-FMK+MT组、Nec-1+MT组和Chloroquine+MT组SH-SY5Y细胞活性无明显差异（P>0.05）。此外，MT能显著提高SH-SY5Y细胞中Fe2+水平、ROS和lipid ROS水平以及TFR1蛋白表达水平，降低GSH水平和FPN1，FTH，FTL蛋白及SLC7A11基因表达水平，促进细胞铁死亡（P<0.05），而DFO则能逆转MT对上述指标水平的影响（P<0.05），但DFO本身对上述指标水平无明显影响（P>0.05）。 结论 MT可能通过下调SLC7A11表达来提高细胞内Fe2+水平，进而诱导胶质瘤细胞铁死亡。
Abstract:
Objective To investigate the effect of melatonin (MT) on the iron death of SH-SYSY glioma cells by down-regulating the expression of solute carrier family 7 member 11 (SLC7A11). Methods Different concentrations of MT were used to treat human glioma SH-SY5Y cells, and the CCK-8 method was used to detect cell viability to select the appropriate MT concentration. The experimental groups were: DMSO group (added conventional medium), MT group (added 1 mmol/L MT), Z-VAD-FMK+MT group (added 1 mmol/L MT and 20 μmol/L apoptosis inhibitor Z-VAD-FMK), Nec-1+MT group (added 1 mmol/L MT and 1 μmol/L necroptosis inhibitor Nec-1), chloroquine+MT group (added 1 mmol/L MT and 5 mmol/L autophagy inhibitor chloroquine), DFO control group (added 20 μmol/L specific iron death inhibitor DFO), and DFO+MT group (added 1 mmol/L MT and 20 μmol/L DFO). CCK-8 method was used to detect cell viability; propidium iodide (PI) staining method was used to evaluate cell death; Phen Green SK fluorescent indicator method was used to measure the level of cell Fe2+; reactive oxygen species (ROS) detection kit was used to measure the total level of ROS; C11-BODIPY staining method was used to detect the level of lipid ROS (lipid ROS); the total glutathione (GSH) detection kit was used to measure the level of cellular GSH; Western blot method was used to detect the expression levels of cell ferritin heavy polypeptide (FTH), ferritin light chain (FTL), ferroportin (FPN), transferrin receptor 1 (TFR1) and SLC7A11 proteins; qRT-PCR was used to detect the expression level of SLC7A11 mRNA in the cells. Results With the increase of MT concentration, the activity of SH-SY5Y cells gradually decreased (P<0.05), since the activity of SH-SY5Y cells treated with 1 mmol/L was the lowest, 1 mmol/L MT was selected to induce cells. Compared with DMSO group, SH-SY5Y cell activity in DFO+MT group was significantly increased (P<0.05), while SH-SY5Y cell activity was different in Z-VAD-FMK+MT group, Nec-1+MT group and chloroquine+MT group was not statistically significantly different (P>0.05). In addition, MT could significantly increase Fe2+ level, ROS and lipid ROS levels, and TFR1 protein expression in SH-SY5Y cells, reduce GSH level and FPN1, FTH, FTL proteins and SLC7A11 gene expression, and promote cell iron death (P<0.05), while DFO can reverse the effects of MT on the above indicators (P<0.05), but DFO itself had no significant effect on the above indicators (P>0.05). Conclusion MT may reduce the expression of SLC7A11 and increase the intracellular Fe2+ level, thereby inducing iron death of glioma cells.

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备注/Memo

备注/Memo:
基金项目：邯郸市科学技术资助项目（编号为1723208069-1）
更新日期/Last Update: 2022-10-10