[1]刘 成,赵 娟,贾 茜,等.ACSL4通过AMPK/mTOR通路抑制七氟醚诱导神经元铁死亡机制的实验研究[J].现代检验医学杂志,2024,39(06):67-72.[doi:10.3969/j.issn.1671-7414.2024.06.011]
　LIU Cheng,ZHAO Juan,JIA Qian,et al.Experimental Study on the Mechanism of ACSL4 Inhibition of Sevofluraneinduced Neuronal Iron Death through the AMPK/mTOR Pathway[J].Journal of Modern Laboratory Medicine,2024,39(06):67-72.[doi:10.3969/j.issn.1671-7414.2024.06.011]
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ACSL4通过AMPK/mTOR通路抑制七氟醚诱导神经元铁死亡机制的实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年06期
页码:
67-72
栏目:
论著
出版日期:
2024-11-15

文章信息/Info

Title:
Experimental Study on the Mechanism of ACSL4 Inhibition of Sevofluraneinduced Neuronal Iron Death through the AMPK/mTOR Pathway
文章编号:
1671-7414（2024）06-067-06
作者:
刘 成; 赵 娟; 贾 茜; 谢生春; 罗 彬; 魏官锋
（绵阳市人民医院麻醉科，四川绵阳 621000）
Author(s):
LIU Cheng;  ZHAO Juan;  JIA Qian;  XIE Shengchun;  LUO Bin;  WEI Guanfeng
（Department of Anesthesiology, Mianyang People’s Hospital, Sichuan Mianyang 621000, China）
关键词:
酰基辅酶A 合成酶长链家族成员4; 神经元; 铁死亡; 七氟醚; AMPK/mTOR 信号通路
分类号:
R392-33
DOI:
10.3969/j.issn.1671-7414.2024.06.011
文献标志码:
A
摘要:
目的 探讨酰基辅酶A 合成酶长链家族成员4（acyl-CoA syntbetase long chain family member 4，ACSL4）在七氟醚（sevoflurane，Sev）诱导的神经元细胞损伤中的作用及机制。方法 以人神经母细胞瘤SH-SY5Y 细胞为研究对象，分别设置对照组（二甲基亚砜，10μmol/L）、Sev 组和Sev+ 铁死亡抑制剂Ferrostatin-1（Fer-1，10μmol/L）组，采用CCK-8 法检测细胞活性。体外构建4.1% Sev 暴露的术后认知功能障碍模型，按照转染类别分为Ctrol 组、Sev 组、Sev+si-NC 组、Sev+si-ACSL4 组和Sev+si-ACSL4+compound C 组。采用比色法检测各组细胞中丙二醛（malonaldehyde，MDA）、4- 羟基壬烯醛（4-hydroxynonenal，4-HNE）、谷胱甘肽（glutathione，GSH）和Fe2+ 含量；2’，7’- 二氯荧光素二乙酸盐（DCFH-DA）荧光探针检测活性氧水平；实时荧光定量PCR（qRT-PCR）检测ACSL4，谷胱甘肽过氧化酶4（glutathione peroxidase 4，GPX4）和溶质载体家族7 成员11（solute carrier family 7 member 11，SLC7A11）mRNA 表达；蛋白免疫印迹法（WB）检测ACSL4，GPX4，腺苷酸激活蛋白激酶（adenosine 5’-monophosphate-activated proteinkinase，AMPK）、磷酸化（phosphorylated，p）-AMPK，哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin）mTOR 和p-mTOR 蛋白表达。结果 CCK-8 结果显示，Sev 组细胞活力（0.41±0.11）较对照组（0.98±0.07）明显降低，Sev+Fer-1 组细胞活力（0.83±0.09）较Sev 组显著升高，差异具有统计学意义（t=7.572，5.118，均P<0.01）。Sev 组细胞中Fe2+，MDA，4-HNE，ROS 水平和p-AMPK/AMPK 比率以及ACSL4 的mRNA 和蛋白表达高于Ctrol 组（t=5.900，7.421，4.795，13.517，10.825，9.945，11.334），GSH，p-mTOR/mTOR 比率以及SLC7A11，GPX4 的mRNA 和蛋白表达低于Ctrol 组（t=20.438，3.551，11.460，12.211，6.845，8.287），差异具有统计学意义（均P<0.05）。Sev+si-ACSL4 组Fe2+，MDA，4-HNE，ROS 水平和p-AMPK/AMPK 比率以及ACSL4 的mRNA 和蛋白表达低于Sev+si-NC 组（t=3.818，3.164，3.054，4.465，13.088，7.918，9.737）， 细胞活力、GSH 含量、p-mTOR/mTOR 比率以及SLC7A11，GPX4 的蛋白表达高于Sev+si-NC 组（t=2.912，7.248，7.574，20.092，5.915），差异具有统计学意义（均P<0.05）。Sev+si-ACSL4+compound C 组细胞活力、GSH含量和SLC7A11，GPX4蛋白表达低于Sev+si-ACSL4 组（t=4.435，8.521，4.522，8.767），而Fe2+，MDA，4-HNE 和ROS 水平高于Sev+si-ACSL4 组（t=10.046，4.004，2.957，3.752），差异具有统计学意义（均P<0.05）。结论 抑制ACSL4 表达可通过激活AMPK/mTOR 信号通路减轻Sev 诱导的SHSY5Y细胞铁死亡。
Abstract:
Objective To investigate the role and mechanism of acyl-CoA syntbetase long chain family member 4 (ACSL4) in Sevoflurane (Sev) induced neuronal cell damage. Methods Human neuroblastoma SH-SY5Y cells were used as the research object, and control group (dimethyl sulfoxide, 10 μmol/L), Sev group and Sev+iron death inhibitor Ferrostatin-1 (Fer-1, 10 μmol/ L) group were set up. CCK-8 method was used to detect cell activity in each group.4.1% Sev exposed postoperative cognitive dysfunction model was constructed in vitro and divided into Ctrol group, Sev group, Sev+si-NC group, Sev+si-ACSL4 group, and Sev+si-ACSL4+compound C group according to the transfection category.The contents of Malonaldehyde (MDA), 4-hydroxynonenal (4-HNE), Glutathione (GSH) and Fe2+ in each group were detected by colorimetry. The level of reactive oxygen species was detected using a 2’ , 7’- dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. Real time fluorescence quantitative PCR（qRT-PCR）was used to detect the mRNA expression of ACSL4, glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11).Protein immunoblotting was used to detect the expression of ACSL4, GPX4, adenosine 5’- monophosphate activated protein kinase (AMPK), phosphorylated (p) - AMPK, mammalian target of rapamycin(mTOR) and p-mTOR proteins. Results CCK-8 results showed that the cell viability of Sev group （0.41±0.11）was significantly lower than that of control group (0.98±0.07), and the cell viability of Sev+Fer-1 group（0.83±0.09）was significantly higher than that of Sev group (0.41±0.11), and the differences were statistically significant (t=7.572, 5.118, all P<0.01). The levels of Fe2+, MDA, 4-HNE, ROS and p-AMPK/AMPK ratios, as well as the mRNA and protein expression of ACSL4 in the Sev group cells, were higher than those in the Ctrol group（t=5.900, 7.421, 4.795, 13.517, 10.825, 9.945, 11.334）, the GSH conten, p-mTOR/ mTOR ratio, and mRNA and protein expression of SLC7A11 and GPX4 were lower than those in the Ctrol group (t=20.438, 3.551, 11.460, 12.211, 6.845,8.287), and the differences were statistically significant (all P<0.05)repectively. The levels of Fe2+, MDA, 4-HNE, ROS, and p-AMPK/AMPK ratios, as well as the mRNA and protein expression of ACSL4 in the Sev+si-ACSL4 group, were lower than those in the Sev+si-NC group （t=3.818, 3.164, 3.054, 4.465, 13.088, 7.918, 9.737）, the cell viability, GSH content, p-mTOR/mTOR ratio, and protein expression of SLC7A11 and GPX4 were higher than those in the Sev+si-NC group（t=2.912, 7.248, 7.574, 20.092, 5.915）, and the differences were statistically significant (all P<0.05), respectively. The cell viability, GSH content, SLC7A11, and GPX4 protein expression in the Sev+si-ACSL4+compound C group were lower than those in the Sev+si-ACSL4 group （t=4.435, 8.521, 4.522, 8.767）, while the levels of Fe2+, MDA, 4-HNE, and ROS were higher than those in the Sev+si-ACSL4 group（t=10.046, 4.004, 2.957, 3.752）, and the differences were statistically significant (all P<0.05). Conclusion Inhibiting ACSL4 expression attenuates Sev-induced iron death in SH-SY5Y cells by activating the AMPK/mTOR signaling pathway.

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备注/Memo

备注/Memo:
基金项目：四川省卫生健康委员会科研课题（19SYJS15）。
作者简介：刘成（1986-），男，硕士，主治医师，研究方向：麻醉药学，E-mail:liucheng198611@126.com。
通讯作者：魏官锋(1973-)，男，副主任医师，研究方向：麻醉药学，E-mail：625588509@163.com。
更新日期/Last Update: 2024-11-15