Cao Peng, Wang Yi, Zhang Cong, Sullivan Mitchell A, Chen Wen, Jing Xiang, Yu Huifan, Li Fei, Wang Qu, Zhou Zhongshi, Wang Qi, Tian Wen, Qiu Zhenpeng, Luo Lianxiang
Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Hubei Key Laboratory of Wudang Local Chinese Medicine Research, School of Pharmaceutical Sciences, Hubei University of Medicine, Shiyan, Hubei, China; Hubei Province Clinical Research Center for Precision Medicine for Critical Illness, Wuhan, China; Hubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Critical Care Medicine, Sichuan Academy of Medical Science and Sichuan Provincial People's Hospital, Chengdu, China.
J Nutr Biochem. 2023 Oct;120:109414. doi: 10.1016/j.jnutbio.2023.109414. Epub 2023 Jul 7.
The global incidence of nonalcoholic fatty liver disease (NAFLD) has been surging in recent years, however, no drug is currently approved to treat this disease. Quercetin, a natural flavonoid abundant in plants and fruits, has been reported to alleviate NAFLD, however, the exact molecular mechanism remains unclear. This study aims to further elucidate its potential mechanism of action. The beneficial effects and the underlying mechanism of quercetin in alleviating NAFLD were explored both in vitro and in vivo, by employing chemical inhibitors of autophagosomes (3-methyladenine, 3-MA), autolysosomes (chloroquine, CQ), AMPK (Compound C, CC) and SIRT1 (selisistat, EX-527). The levels of intracellular lipids, reactive oxygen species, mitochondria function, autophagy, and mitophagy were assessed by fluorescent labeling and examined using flow cytometry or confocal microscopy. Key protein expressions of autophagy, mitophagy, and inflammation were also determined. In vivo, quercetin was shown to dose-dependently effectively alleviate NAFLD, but intraperitoneal injection of 3-MA could block the beneficial effects of quercetin on body weight, liver weight, serum ALT/AST, hepatic ROS and inflammation. In vitro, quercetin could reduce intracellular lipids (Nile Red staining) and ROS/DHE accumulation, which could be also blocked by 3-MA or CQ. Furthermore, we found that CC could abrogate the protective effects of quercetin on lipid and ROS accumulation in vitro. Also, CC abolished the proautophagic and anti-inflammatory effects of quercetin, as shown by western blot determination and Lyso-Tracker labeling. Importantly, mitophagy, a specific form of mitochondria-targeted autophagy, was enhanced by quercetin, as demonstrated by PINK1/Parkin protein variation and immunofluorescence colocalization of autophagosomes and mitochondria, which could also be blocked by the intervention of CC. This study demonstrates that quercetin prevents NAFLD through AMPK-mediated mitophagy and suggests that promoting mitophagy via an upregulation of AMPK may be a promising therapeutic strategy against NAFLD.
近年来，非酒精性脂肪性肝病（NAFLD）的全球发病率一直在飙升，然而，目前尚无获批用于治疗该疾病的药物。槲皮素是一种在植物和水果中大量存在的天然黄酮类化合物，据报道可缓解NAFLD，但其确切的分子机制仍不清楚。本研究旨在进一步阐明其潜在的作用机制。通过使用自噬体化学抑制剂（3-甲基腺嘌呤，3-MA）、自溶酶体化学抑制剂（氯喹，CQ）、AMPK化学抑制剂（化合物C，CC）和SIRT1化学抑制剂（西利司他，EX-527），在体外和体内探究了槲皮素在缓解NAFLD方面的有益作用及其潜在机制。通过荧光标记评估细胞内脂质、活性氧、线粒体功能、自噬和线粒体自噬水平，并使用流式细胞术或共聚焦显微镜进行检测。还测定了自噬、线粒体自噬和炎症的关键蛋白表达。在体内，槲皮素显示出剂量依赖性地有效缓解NAFLD，但腹腔注射3-MA可阻断槲皮素对体重、肝脏重量、血清ALT/AST、肝脏ROS和炎症的有益作用。在体外，槲皮素可减少细胞内脂质（尼罗红染色）和ROS/DHE积累，3-MA或CQ也可阻断这种积累。此外，我们发现CC可消除槲皮素在体外对脂质和ROS积累的保护作用。同样，如蛋白质印迹测定和溶酶体示踪剂标记所示，CC消除了槲皮素的促自噬和抗炎作用。重要的是，线粒体自噬是一种靶向线粒体的自噬特殊形式，如PINK1/Parkin蛋白变化以及自噬体与线粒体的免疫荧光共定位所示，槲皮素可增强线粒体自噬，CC干预也可阻断这种增强作用。本研究表明，槲皮素通过AMPK介导的线粒体自噬预防NAFLD，并表明通过上调AMPK促进线粒体自噬可能是一种有前景的抗NAFLD治疗策略。
