【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。更多还原

【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.更多还原