[1]曹明月,王 巍,周美宁.LPIN1/PPARA 通过抑制SLC47A1 介导的神经元铁死亡缓解帕金森病模型大鼠病情进展的机制研究[J].现代检验医学杂志,2024,39(04):63-71.[doi:10.3969/j.issn.1671-7414.2024.04.012]
　CAO Mingyue,WANG Wei,ZHOU Meining.Mechanism Study on LPIN1/PPARA Alleviating the Progression of Parkinson’s Disease in Rats by Inhibiting SLC47A1-Mediated Ferroptosis of Neurons[J].Journal of Modern Laboratory Medicine,2024,39(04):63-71.[doi:10.3969/j.issn.1671-7414.2024.04.012]
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LPIN1/PPARA 通过抑制SLC47A1 介导的神经元铁死亡缓解帕金森病模型大鼠病情进展的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年04期
页码:
63-71
栏目:
论著
出版日期:
2024-07-15

文章信息/Info

Title:
Mechanism Study on LPIN1/PPARA Alleviating the Progression of Parkinson’s Disease in Rats by Inhibiting SLC47A1-Mediated Ferroptosis of Neurons
文章编号:
1671-7414（2024）04-063-09
作者:
曹明月1; 王 巍1; 周美宁2
（1. 大庆龙南医院/ 齐齐哈尔医学院第五附属医院检验科，黑龙江大庆 163453；2. 西安医学院第三附属医院神经内科，西安 710068）
Author(s):
CAO Mingyue1; WANG Wei1; ZHOU Meining2
（1.Department of Clinical Laboratory，Daqing Longnan Hospital/ the Fifth Affiliated Hospital of Qiqihar Medical College，Heilongjiang Daqing 163453, China；2.Department of Neurology, the Third Affiliated Hospital of Xi’an Medical University, Xi’an 710068, China)
关键词:
帕金森病; 脂蛋白1; 过氧化物酶增殖物激活受体A; 溶质载体蛋白家族47 成员A1; 铁死亡
分类号:
R-332
DOI:
10.3969/j.issn.1671-7414.2024.04.012
文献标志码:
A
摘要:
目的 探究脂蛋白1（lipin1，LPIN1）对帕金森病（Parkinson’s disease，PD）模型大鼠病情进展的影响及其调控的可能分子机制。方法 采用6- 羟基多巴胺（6-hydroxydopamine hydrobromide，6-OHDA）注射大鼠单侧的内侧前脑束构建PD 大鼠模型，并稳定转染LPIN1 过表达腺病毒，评估大鼠行为学变化；检测大鼠脑组织中Fe2+ 和还原型谷胱甘肽（glutathione，GSH）含量及酪氨酸羟化酶（tyrosine hydroxylase，TH）蛋白水平；HE 染色观察大鼠脑组织病理变化。构建体外PD 细胞模型，检测细胞中TH，α- 突触核蛋白(α-synuclein，α-syn)，LPIN1 蛋白水平及细胞活力；转染LPIN1 小干扰siRNA 序列和过表达载体及过氧化物酶增殖物激活受体A（peroxisome proliferator-activated receptor A，PPARA）小干扰RNA（small interfering RNA，siRNA）和过表达载体，或用铁死亡诱导剂Erastin 处理细胞24 h，后用6-OHDA 处理细胞48 h。检测各组细胞中Fe2+ 含量、活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、GSH 和炎症因子水平以评估铁死亡；CCK-8 检测细胞增殖活力，Western blot 法检测铁死亡相关蛋白表达。通过STRING 数据库预测LPIN1 的互作蛋白PPARA，利用Co-IP 分析进行验证；JASPAR 生物信息学网站预测PPARA与溶质载体蛋白家族47 成员A1（solute carrier family 47 member 1，SLC47A1）启动子的结合位点，利用Ch-IP 分析进行验证。结果 模型组大鼠皮毛悚立，表现出身体持续震颤、动作迟缓、活动能力减弱等PD 症状；LPIN1 组大鼠运动行为及PD 症状较模型组改善/ 减轻。与假手术组相比，模型组大鼠运动总路程缩短、平均速度降低、步长减小、总静止时间延长、步宽增宽、步态变异率增大，差异具有统计学意义（t=6.816，7.026，26.556，7.454，8.503，7.971，均P<0.05）；模型组大鼠脑组织中Fe2+ 含量升高，GSH 含量及TH 蛋白表达降低，差异具有统计学意义（t=8.305，13.305，7.709，均P<0.05）。LPIN1 组大鼠行为学评估、各指标水平及脑组织病理改变较模型组明显改善/ 减轻。6-OHDA呈剂量依赖性降低PC-12 细胞活力及TH，LPIN1 蛋白水平，升高α-syn 蛋白水平，差异具有统计学意义（F=31.023，7.350，9.124，15.841，均P<0.05）。沉默LPIN1 加剧了6-OHDA 对PC-12 细胞活力的抑制作用（t=2.209，P<0.05），过表达LPIN1 则可抵消6-OHDA 的作用（t=4.989，P<0.05）。过表达LPIN1 降低IL-1β，IL-6 分泌，升高SLC47A1 和GPX4蛋白水平，降低Fe2+，MDA 含量和ROS 水平，升高GSH 含量（t=3.013 ～ 11.639，均P<0.05）；Erastin 可逆转LPIN1过表达对铁死亡的抑制作用，降低PC-12 细胞活力（t=3.087 ～ 7.581，均P<0.05）。LPIN1 与PPARA 蛋白互作并促进PPARA 表达，PPARA 与SLC47A1 启动子结合并促进SLC47A1 转录激活。过表达PPARA 可抵消LPIN1 沉默对PC-12细胞的影响。结论 过表达LPIN1 可能通过抑制PPARA/SLC47A1 轴介导的铁死亡，减少神经元细胞凋亡，进而缓解PD 模型大鼠的病情进展。
Abstract:
Objective To investigate the effect of lipin1 (LPIN1) on the progression of Parkinson’s disease (PD) in rats and the possible molecular mechanism of its regulation. Methods The PD rat model was established by injection of 6-Hydroxydopamine hydrobromide (6-OHDA) into the medial forebrain tract of rats, and the LPIN1-overexpressing adenovirus was stably transfected to evaluate the behavioral changes of rats. The content of Fe2+ and Glutathione (GSH) and the protein level of tyrosine hydroxylase (TH) in rat brain were detected, and HE staining was used to observe the pathological changes in rat brains. The PD cell model was constructed in vitro, and TH, α-synuclein (α-syn), LPIN1 protein levels and cell viability were detected. LPIN1 small interfering siRNA sequence and overexpression vector and peroxisome proliferator-activated receptor (PPARA) small interfering (siRNA) and overexpression vector were transfected, or ferroptosis inducer erastin was used to treat cell for 24 h, then cells were treated with 6-OHDA for 48h. The levels of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA), GSH and inflammatory factors in cells were detected to evaluate ferroptosis. Cell viability was detected with CCK-8, and the expressions of ferroptosis related proteins were detected with Western blot. The interacting protein PPARA of LPIN1 was predicted by STRING database and verified by Co-IP analysis. The binding site of PPARA to the promoter of solute carrier family 47 member 1(SLC47A1) was predicted by JASPAR bioinformatics and verified by Ch-IP analysis. Results The fur of the rats in the model group was frightened, and PD symptoms such as continuous tremor, slow movement and weakened activity were shown. The motor behavior and PD symptoms of LPIN1 group were improved/alleviated compared with the model group. Compared with the sham operation group, the total distance of the model group was shortened, the average speed was reduced, and the step length was reduced, while the total resting time was prolonged, the step width was widened, and the gait variation rate was increased, and the differences were significant (t=4.470 ～ 26.556, all P<0.05). Compared with sham operation group, Fe2+ content in brain tissue of model group was increased, while GSH content and TH protein expression were decreased, with significance differences (t=8.305, 13.305, 7.709, all P<0.05). Compared with the model group, the behavioral evaluation, the level of indexes and the pathological changes of brain tissue in LPIN1 group were improved/alleviated. In addition, 6-OHDA decreased PC-12 cell viability, reduced the levels of TH and LPIN1 protein, and increased the level of α-syn protein in a dosedependent manner, and the differences were significant (F=31.023, 7.350, 9.124, 15.841, all P<0.05). Silencing LPIN1 intensified the inhibitory effect of 6-OHDA on the viability of PC-12 cells (t=2.209, P<0.05), and overexpression of LPIN1 could counteract the effect of 6-OHDA. Overexpression of Lpin1 decreased the secretion of IL-1β and IL-6, increased the protein levels of SLC47A1 and GPX4, decreased the levels of Fe2+, MDA and ROS, and increased GSH content (t=3.013 ～ 11.639, all P<0.05). Erastin reversed the inhibitory effect of Lpin1 overexpression on ferroptosis, and reduced the viability of PC-12 cells (t=3.087 ～ 7.581, all P<0.05). LPIN1 interacted with PPARA protein and promoted PPARA expression, while PPARA bound to SLC47A1 promoter and promoted SLC47A1 transcriptional activation. Overexpression of PPARA counteracted the effect of Lpin1 silencing on PC-12 cells. Conclusion Overexpression of LPIN1 may reduce neuronal cell apoptosis by inhibiting ferroptosis mediated by PPARA/SLC47A1 axis, thus alleviating the progression of PD model rats.

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备注/Memo

备注/Memo:
作者简介：曹明月（1981-），女，学士，主管技师，研究方向：免疫临床基础检验技术，E-mail：8099783@qq.com。
通讯作者：周美宁（1977-），女，学士，副主任医师，研究方向：脑血管疾病，帕金森疾病，E-mail：349053938@qq.com。
更新日期/Last Update: 2024-07-15