目的 观察雌马酚(equol, Eq)干预对棕榈酸（palmitic acid,PA）诱导的L6细胞线粒体稳态紊乱的影响,并探讨其相关机制。方法 体外培养L6细胞,将细胞分为对照组、模型（PA）组、PA+Eq组、PA+Eq+SR18292、PA+ Eq+Phttp组、PA+Eq+RM02812组。检测作用不同时间点各组细胞活性氧（reactive oxygen species,ROS）水平、细胞活力、葡萄糖摄取能力;以及干预24 h各组线粒体膜电位、自噬强度变化;荧光探针标记线粒体,观察各组线粒体形态变化;透射电镜观察各组线粒体形态结构和自噬小体;检测各组同源性磷酸酶张力蛋白诱导激酶1（PTEN induced putative kinase 1,PINK1）、帕金森蛋白（Parkin）、SQSTM1蛋白（sequestosome l,P62)、微管相关蛋白1Ⅰ轻链3 ( microtubule-associated protein 1Ⅰlight chain 3, LC3Ⅰ)、微管相关蛋白1Ⅱ轻链3（ microtubule-associated protein 1Ⅱlight chain 3, LC3Ⅱ)、线粒体融合蛋白2（mitochondrial fusion protein 2,MFN2）、动力相关蛋白1（dynaminrelatedprotein 1,DRP1）和过氧化物酶体增殖物激活受体γ共激活因子-1α（peroxisome proliferator-activated receptor γ co-activator-1α,PGC-1α）表达。结果 0.25 mmol/L棕榈酸处理细胞6 h后活性氧水平显著增高（P<0.05）,细胞活力下降（P<0.05）,葡萄糖摄取量降低 (P<0.05);与模型组相比,雌马酚能明显降低活性氧水平（P<0.05）,提高细胞活力和葡萄糖消耗;棕榈酸干预24 h后,线粒体膜电位明显下降（P<0.05）,自噬水平降低（P<0.05）;与模型组相比,雌马酚能改善线粒体膜电位下降,促进线粒体融合,减少裂变（P<0.05）; PINK1、Parkin、P62、LC3Ⅱ、MFN2和PGC-1α的表达明显增高（P<0.05）,DRP1表达降低（P<0.05）结论 雌马酚能够改善棕榈酸诱导的L6细胞线粒体稳态紊乱,减轻线粒体裂变,其部分机制可能与雌马酚促进线粒体自噬有关。
Abstract
Objective To investigate the effects of equol (Eq) on palmitic acid (PA) -induced mitochondrial homeostasis in L6 cells, and to explore the related mechanisms. Methods L6 cells were cultured in DMEM (containing 10% FBS) and divided into control group, model group, PA+Eq group, PA+Eq+SR18292 group, PA+Eq+Phttp group and PA+Eq+RM02812 group. The levels of reactive oxygen species, cell viability, and glucose uptake ability at different times were measured. Additionally, changes of mitochondrial membrane potential and autophagy intensity after 24 hours of intervention were examined. We used fluorescence to label the mitochondria and observed the morphological changes of mitochondria. Transmission electron microscopy was used to observe mitochondrial morphology and autophagosome. The proteins of PINK1, PARKIN, P62, LC3Ⅰ, LC3Ⅱ, MFN2, DRP1, and PGC-1ɑ were detected by Westen blotting. Results After treatment with 0.25 mmol/L PA for 6 h, ROS level was significantly increased (P<0.05), cell viability was decreased (P<0.05), and glucose intake was decreased (P<0.05). Compared with the model group, Eq could significantly reduce ROS level, increase cell viability and glucose consumption (P<0.05). After 24 hours of PA exposure, mitochondrial membrane potential and autophagy level were significantly decreased (P<0.05). Compared with the model group, Eq could improve the decrease of mitochondrial membrane potential, promote mitochondrial fusion, and reduce mitochondrial fission (P<0.05). The protein expression of Parkin, PINK1, P62, LC3Ⅱ, MFN2 and PGC-1ɑ was significantly increased (P<0.05), and the expression of DRP1 protein was significantly decreased (P<0.05). Conclusion Eq can improve mitochondrial homeostasis disorder induced by palmitic acid in L6 cells. Eq reduces mitochondrial fission, which may be partially related to the promotion of mitophagy.
关键词
雌马酚 /
L6细胞 /
活性氧 /
线粒体稳态 /
线粒体自噬
Key words
equol /
L6 cells /
ROS /
mitochondrial homeostasis /
mitophagy
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基金
国家自然基金面上项目（No.81973040）
