Zeng Youjie, Guo Ren, Chen Songhua, Lin Yuxin, Cao Si, Wang Xia, Zhang Siyi, Xu Huilin, Qing Wenxiang, Yang Heng, Ouyang Wen
Department of Anesthesiology, Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Yuelu District, Changsha, 410013, Hunan, China.
Department of Pharmacy, Third Xiangya Hospital, Central South University, Changsha, 410013, Hunan, China.
Mol Med. 2025 May 15;31(1):191. doi: 10.1186/s10020-025-01255-w.
Diacylglycerol O-acyltransferase 1 (DGAT1) is crucial for triglyceride synthesis, yet its role in ischemic stroke remains unclear. This study investigated DGAT1 in ischemic stroke using middle cerebral artery occlusion (MCAO) rat models and highly differentiated PC12 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R).
The therapeutic effects of DGAT1 inhibition in MCAO rats were assessed using the Zea-Longa score and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. The effects on highly differentiated PC12 cells subjected to OGD/R were evaluated using the Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays. Ferroptosis-related mitochondrial damage was evaluated using transmission electron microscope. Additionally, the mechanisms by which DGAT1 inhibition regulates ferroptosis were further explored via immunohistochemistry, immunofluorescence, Western blotting, qPCR, JC-1 assay, and reactive oxygen species (ROS) detection.
DGAT1 expression was elevated in both MCAO and OGD/R models. The DGAT1 inhibitor A 922500 improved neurological deficits, reduced infarct volume, and minimized neuronal loss in MCAO rats, while also enhancing cell viability and reducing LDH levels in OGD/R-treated PC12 cells. DGAT1 inhibition significantly alleviated ferroptosis in MCAO rats, as indicated by (i) reduced mitochondrial shortening and cristae disruption, (ii) decreased 4-HNE levels, (iii) reduced MDA and increased SOD, and (iv) lowered levels of inflammatory factors (IL-6, MCP-1, and TNF-α). Moreover, both in vivo and in vitro experiments showed that DGAT1 inhibition significantly increased Gpx4 levels, whereas lentiviral delivery of Gpx4 shRNA markedly reversed its beneficial effects. In MCAO rats, Gpx4 shRNA significantly elevated 4-HNE levels and exacerbated ferroptosis-related mitochondrial damage. In vitro, DGAT1 inhibition increased mitochondrial membrane potential and reduced ROS, whereas rotenone, a mitochondrial function inhibitor, decreased Gpx4 and impaired cell viability. Furthermore, DGAT1 inhibition significantly upregulated the key β-oxidation gene Cpt1a, whereas etomoxir, a β-oxidation inhibitor, reduced cell viability and mitochondrial membrane potential, increased ROS, and downregulated Gpx4.
Our study suggests that DGAT1 inhibition may enhance β-oxidation and mitochondrial function, thereby increasing Gpx4 levels, suppressing ferroptosis, and ultimately exerting neuroprotective effects in ischemic stroke.
二酰甘油O-酰基转移酶1（DGAT1）对甘油三酯合成至关重要，但其在缺血性卒中中的作用仍不清楚。本研究使用大脑中动脉闭塞（MCAO）大鼠模型和经氧葡萄糖剥夺/复氧（OGD/R）处理的高分化PC12细胞，对缺血性卒中中的DGAT1进行了研究。
使用Zea-Longa评分和2,3,5-三苯基氯化四氮唑（TTC）染色评估DGAT1抑制对MCAO大鼠的治疗效果。使用细胞计数试剂盒-8（CCK-8）和乳酸脱氢酶（LDH）测定评估对经OGD/R处理的高分化PC12细胞的影响。使用透射电子显微镜评估铁死亡相关的线粒体损伤。此外，通过免疫组织化学、免疫荧光、蛋白质免疫印迹、定量聚合酶链反应、JC-1测定和活性氧（ROS）检测，进一步探索DGAT1抑制调节铁死亡的机制。
DGAT1表达在MCAO和OGD/R模型中均升高。DGAT1抑制剂A 922500改善了MCAO大鼠的神经功能缺损，减少了梗死体积，并使神经元损失最小化，同时还提高了经OGD/R处理的PC12细胞的活力并降低了LDH水平。DGAT1抑制显著减轻了MCAO大鼠的铁死亡，表现为：（i）线粒体缩短和嵴破坏减少；（ii）4-羟基壬烯醛（4-HNE）水平降低；（iii）丙二醛（MDA）减少，超氧化物歧化酶（SOD）增加；（iv）炎症因子（白细胞介素-6、单核细胞趋化蛋白-1和肿瘤坏死因子-α）水平降低。此外，体内和体外实验均表明，DGAT1抑制显著增加了谷胱甘肽过氧化物酶4（Gpx4）水平，而慢病毒递送Gpx4短发夹RNA（shRNA）显著逆转了其有益作用。在MCAO大鼠中，Gpx4 shRNA显著升高了4-HNE水平并加剧了铁死亡相关的线粒体损伤。在体外，DGAT1抑制增加了线粒体膜电位并降低了ROS，而线粒体功能抑制剂鱼藤酮降低了Gpx4并损害了细胞活力。此外，DGAT1抑制显著上调了关键的β-氧化基因肉碱棕榈酰转移酶1A（Cpt1a），而β-氧化抑制剂依托莫昔降低了细胞活力和线粒体膜电位，增加了ROS，并下调了Gpx4。
我们的研究表明，DGAT1抑制可能增强β-氧化和线粒体功能，从而增加Gpx4水平，抑制铁死亡，并最终在缺血性卒中中发挥神经保护作用。
