Abstract：
This study elucidates the molecular mechanisms by which FABP3 regulates neuronal apoptosis via mitochondrial autophagy in the context of cerebral ischemia-reperfusion (I/R). Employing a transient mouse model of middle cerebral artery occlusion (MCAO) established using the filament method, brain tissue samples were procured from I/R mice. High-throughput transcriptome sequencing on the Illumina CN500 platform was performed to identify differentially expressed mRNAs. Critical genes were selected by intersecting I/R-related genes from the GeneCards database with the differentially expressed mRNAs. The in vivo mechanism was explored by infecting I/R mice with lentivirus. Brain tissue injury, infarct volume ratio in the ischemic penumbra, neurologic deficits, behavioral abilities, neuronal apoptosis, apoptotic factors, inflammatory factors, and lipid peroxidation markers were assessed using H&E staining, TTC staining, Longa scoring, rotation experiments, immunofluorescence staining, and Western blot. For in vitro validation, an OGD/R model was established using primary neuron cells. Cell viability, apoptosis rate, mitochondrial oxidative stress, morphology, autophagosome formation, membrane potential, LC3 protein levels, and colocalization of autophagosomes and mitochondria were evaluated using MTT assay, LDH release assay, flow cytometry, ROS/MDA/GSH-Px measurement, transmission electron microscopy, MitoTracker staining, JC-1 method, Western blot, and immunofluorescence staining. FABP3 was identified as a critical gene in I/R through integrated transcriptome sequencing and bioinformatics analysis. In vivo experiments revealed that FABP3 silencing mitigated brain tissue damage, reduced infarct volume ratio, improved neurologic deficits, restored behavioral abilities, and attenuated neuronal apoptosis, inflammation, and mitochondrial oxidative stress in I/R mice. In vitro experiments demonstrated that FABP3 silencing restored OGD/R cell viability, reduced neuronal apoptosis, and decreased mitochondrial oxidative stress. Moreover, FABP3 induced mitochondrial autophagy through ROS, which was inhibited by the free radical scavenger NAC. Blocking mitochondrial autophagy with sh-ATG5 lentivirus confirmed that FABP3 induces mitochondrial dysfunction and neuronal apoptosis by activating mitochondrial autophagy. In conclusion, FABP3 activates mitochondrial autophagy through ROS, leading to mitochondrial dysfunction and neuronal apoptosis, thereby promoting cerebral ischemia-reperfusion injury.
摘要：
这项研究阐明了FABP3在脑缺血再灌注（I/R）背景下通过线粒体自噬调节神经元凋亡的分子机制。采用细丝法建立的短暂性大脑中动脉闭塞（MCAO）小鼠模型，脑组织样本来自I/R小鼠。在IlluminaCN500平台上进行高通量转录组测序以鉴定差异表达的mRNA。通过将来自GeneCards数据库的I/R相关基因与差异表达的mRNA相交来选择关键基因。通过用慢病毒感染I/R小鼠来探索体内机制。脑组织损伤,缺血半暗带梗死体积比，神经缺陷,行为能力,神经元凋亡，凋亡因子，炎症因子，和脂质过氧化标记物使用H&E染色进行评估，TTC染色,隆加得分,旋转实验,免疫荧光染色，和Westernblot。对于体外验证，使用原代神经元细胞建立OGD/R模型。细胞活力，凋亡率，线粒体氧化应激，形态学,自噬体形成，膜电位，LC3蛋白水平,使用MTT法评估自噬体和线粒体的共定位，LDH释放试验,流式细胞术,ROS/MDA/GSH-Px测量，透射电子显微镜,MitoTracker染色,JC-1方法，蛋白质印迹,和免疫荧光染色。通过整合转录组测序和生物信息学分析，FABP3被鉴定为I/R中的关键基因。体内实验表明，FABP3沉默减轻了脑组织损伤，减少梗死体积比，改善神经功能缺损，恢复行为能力，和减少神经元凋亡，炎症,I/R小鼠的线粒体氧化应激。体外实验表明，FABP3沉默可以恢复OGD/R细胞的活力，减少神经元凋亡，减少线粒体氧化应激。此外，FABP3通过ROS诱导线粒体自噬,被自由基清除剂NAC抑制。sh-ATG5慢病毒阻断线粒体自噬，证实FABP3通过激活线粒体自噬诱导线粒体功能障碍和神经元凋亡。总之,FABP3通过ROS激活线粒体自噬，导致线粒体功能障碍和神经元凋亡，从而促进脑缺血再灌注损伤。
