Lysosome is the principal organelle for the ultimate degradation of cellular macromolecules, which are delivered through endocytosis, phagocytosis, and autophagy. The lysosomal functions have been found to be impaired by fatty foods and aging, and more importantly, the lysosomal dysfunction in macrophages has been reported as a risk of atherosclerosis development. In this study, we searched for dietary polyphenols which possess the activity for enhancing the lysosomal degradation in J774.1, a murine macrophage-like cell line. Screening test utilizing DQ-BSA digestion identified isorhamnetin (3'-O-methylquercetin) as an active compound. Interestingly, structural comparison to inactive flavonols revealed that the chemical structure of the B-ring moiety in isorhamnetin is the primary determinant of its lysosome-enhancing activity. Unexpectedly isorhamnetin failed to inhibit mTORC1-TFEB signaling, a master regulator of lysosomal biogenesis and function. Our data suggested that the other molecular mechanism might be critical for the regulation of lysosomes in macrophages.Abbreviations: ANOVA: analysis of variance; ApoE: apolipoprotein E; ATP6V0D2: ATPase H+ transporting V0 subunit d2; BAF: bafilomycin A1; BODIPY: boron dipyrromethene; BSA: bovine serum albumin; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified eagle medium; DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LC-MS/MS: liquid chromatography tandem mass spectrometry; MITF: microphthalmia-associated transcription factor; MRM: multiple reaction monitoring; mTORC1: mechanistic target of rapamycin complex 1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor γ; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SDS: sodium dodecyl sulfate; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TBS: Tris-buffered saline; TFA: trifluoroacetic acid; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcriptional factor EB; TFEC: transcription factor EC; V-ATPase: vacuolar-type proton ATPase.

中文翻译：

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Isorhamnetin 是一种 3'-甲氧基化黄酮醇，以不依赖 TFEB 的方式增强 J774.1 小鼠巨噬细胞中的溶酶体蛋白水解作用。


溶酶体是细胞大分子最终降解的主要细胞器，通过内吞作用、吞噬作用和自噬传递。人们发现，脂肪食物和衰老会损害溶酶体功能，更重要的是，巨噬细胞中的溶酶体功能障碍已被报道为动脉粥样硬化发展的风险。在这项研究中，我们寻找具有增强小鼠巨噬细胞样细胞系 J774.1 溶酶体降解活性的膳食多酚。利用 DQ-BSA 消化进行的筛选测试将异鼠李素（3'-O-甲基槲皮素）鉴定为活性化合物。有趣的是，与无活性黄酮醇的结构比较表明，异鼠李素中 B 环部分的化学结构是其溶酶体增强活性的主要决定因素。出乎意料的是，异鼠李素未能抑制 mTORC1-TFEB 信号传导（溶酶体生物合成和功能的主要调节因子）。我们的数据表明，其他分子机制可能对于巨噬细胞中溶酶体的调节至关重要。缩写：ANOVA：方差分析； ApoE：载脂蛋白E； ATP6V0D2：ATPase H+ 转运 V0 亚基 d2； BAF：巴弗洛霉素A1； BODIPY：二吡咯亚甲基硼； BSA：牛血清白蛋白； CTSD：组织蛋白酶D； CTSF：组织蛋白酶F； DMEM：Dulbecco改良的Eagle培养基； DMSO：二甲亚砜； EGCG：表没食子儿茶素-3-没食子酸酯； FBS：胎牛血清； GAPDH：3-磷酸甘油醛脱氢酶； HPLC：高效液相色谱法； LAMP1：溶酶体相关膜蛋白1； LAMP2A：溶酶体相关膜蛋白2A； LC-MS/MS：液相色谱串联质谱法； MITF：小眼症相关转录因子； MRM：多反应监测； mTORC1：雷帕霉素复合物 1 的机制靶点； PBS：磷酸盐缓冲盐水； PPARγ：过氧化物酶体增殖物激活受体γ； RT-qPCR：逆转录定量聚合酶链式反应； SDS：十二烷基硫酸钠； SNARE：可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体； TBS：Tris 缓冲盐水； TFA：三氟乙酸； TFE3：与 IGHM 增强子 3 结合的转录因子； TFEB：转录因子EB； TFEC：转录因子EC； V-ATP酶：液泡型质子ATP酶。