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20μmol/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1μmol/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20μmol/L的特异性铁死亡抑制剂DFO)及DFO+MT组(加入1 mmol/L的MT和20μmol/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Fe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Fe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Fe水平,进而诱导胶质瘤细胞铁死亡。
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(SLC7 A11). Methods Different concentrations of MT were used to treat human glioma SH-SY5 Y 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 Fe;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 SLC7 A11 proteins;qRT-PCR was used to detect the expression level of SLC7 A11 mRNA in the cells. Results With the increase of MT concentration, the activity of SH-SY5 Y cells gradually decreased(P<0.05), since the activity of SH-SY5 Y cells treated with 1 mmol/L was the lowest, 1 mmol/L MT was selected to induce cells. Compared with DMSO group, SH-SY5 Y cell activity in DFO+MT group was significantly increased(P<0.05), while SH-SY5 Y 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 Felevel, ROS and lipid ROS levels, and TFR1 protein expression in SH-SY5 Y cells, reduce GSH level and FPN1, FTH, FTL proteins and SLC7 A11 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 SLC7 A11 and increase the intracellular Felevel, thereby inducing iron death of glioma cells.
作者
宋志远
武一平
杨华
孙艳艳
曲大成
任洪波
Song Zhiyuan;Wu Yiping;Yang Hua(Department of Neurosurgery,Handan Central Hospital,Handan 056001)
出处
《卒中与神经疾病》
2022年第5期416-421,共6页
Stroke and Nervous Diseases
基金
邯郸市科学技术资助项目(编号为1723208069-1)。
