基于细胞周期蛋白CDK1介导的线粒体稳态研究GC方防治非酒精性脂肪肝效应的机理

Our previouse work has shown that the compounds Geniposide and Chologenic acid (GC) in Chinese herbal are the key compound that can be used to prevent and treat non-alcoholic fatty liver disease (NAFLD). Via collarabration with Dr. Jian jian Liat University of California Davis Medical Center, USA, we found that GC may reduce saturated fatty acid-induced liver cell injury by enhancing mitochondrial homeostasis. Recent findings from his lab revealed that the cell cycle regulator CDK1 can relocate into the inner membrane and matrix of the mitochondria and improve the cell energy biosynthesis and antioxidant ability, thus improve the mitochondrial bioenergetics and homeostasis, which may play a key role in GC-mediated protection against NAFLD. We hypothesized that “GC could improve mitochondria energy biosynthesis and hemostasis, thus significanly reduce free fatty acid induced hepatocyte damage by inducing mitochondrial CDK1 activation”.In the proposed study, we will further elucidate the molecular mechanism underlgying GC-mediated CDK1 activation and mitochondrial homeostasis in NAFLD. We will use a specific mitochondrial gene transfection model with mitochondria-trageted CDK1 or its mutant form (MTS-CDK1-mut) in mouse liver cells. This model will be tested with saturated fatty acid induced hepatic mitochondria damage and high fat-enriched diet NAFLD mouse model. These results will help to elucidate the molecularmechnisms, therapeutic targets and compatibility of GC, which will provide a critical platform for the further development of new effective combination of herval compounds to treat NAFLD.

本课题组已发现并证实中药成分复方（京尼平苷合绿原酸，GC方）对非酒精性脂肪肝（NAFLD）有很好的防治效应。近与美国加州大学合作研究发现GC方对饱和性脂肪酸诱导的小鼠肝细胞线粒体损伤具有良好的保护作用。近发现，细胞周期蛋白CDK能够移位至线粒体内膜和基质从而促进线粒体能量代谢和氧自由基清除能力, 发挥保护肝细胞线粒体稳态的生物学效应，在NAFLD发生发展中具重要意义。鉴此，提出“GC方可能通过影响肝细胞线粒体CDK1的表达促进线粒体能量代谢和稳态平衡，进而改善游离性脂肪酸介导的肝细胞损伤”的假说。拟通过建立线粒体导向野生型和失活突变型CDK1肝细胞模型, 结合运用饱和性脂肪酸诱导线粒体损伤的细胞模型和高脂饮食诱导的NAFLD动物模型，针对GC方促进线粒体能量代谢降低肝细胞氧化应激损伤等关键环节，探讨GC方影响CDK1及其线粒体稳态，抗线粒体损伤等防治NAFLD的机制、靶点以及配伍特

背景目的：肝脏细胞线粒体功能障碍是非酒精性脂肪性肝炎(NASH)的关键发病机制。基于前期有关中药成分复方GC方（京尼平苷和绿原酸组合）有效防治非酒精性脂肪病（NAFLD）病理进程的工作基础，本课题着重研究该方促进线粒体能量代谢和降低肝细胞氧化应激损伤的关键药理环节和分子学机制，为进一步研发治疗NAFLD显效、机制明确的新型中药制剂提供科学坚实的理论依据。.内容方法：机制研究方面：运用CRISPR/Cas9基因编辑技术构建线粒体导向野生型和失活突变型CDK1、SIRT3和CPT2小鼠肝细胞株，通过免疫共沉淀蛋白印迹、C14同位素标记、脂肪酸氧化测定系统分析CDK1/SIRT3调控线粒体脂肪酸代谢、能量合成和氧化应激损伤的作用。药理效应研究方面：通过体外棕榈酸(Palm)诱导的脂毒性细胞模型和体内高脂饮食(HFD)介导的大鼠脂肪肝模型以及四氯化碳（CCL4）介导的C57BL/6小鼠急性肝损伤模型，系统评价GC对肝脏脂肪酸氧化、肝细胞功能和形态学变化的影响。利用UHPLC/MS生物信息学分析GC潜在的线粒体稳态调控机制。细胞抗氧化剂维生素E作为体内研究的阳性对照。.研究结果：本课题研究发现：①细胞线粒体导向CDK1蛋白可以介导SIRT3蛋白Ser17位点的磷酸化，从而使线粒体内膜脂肪酸转运调控蛋白CPT2 内链K453和K457发生去乙酰化并形成空间二聚体，从而使CPT2活性和线粒体脂肪酸氧化能力增强；②GC可以有效改善高剂量脂肪酸和CCL4介导的肝细胞线粒体能量代谢障碍和氧化应激损伤。线粒体蛋白质组学研究发现GC方干预后可以使参与线粒体氧化还原调节和脂肪酸降解代谢的线粒体蛋白簇增强。同时，GC方可显著增加CDK1-T161和SIRT3磷酸化形式的表达以及CDK1/SIRT3蛋白的结合，同时，GC方促进SIRT3下游线粒体脂肪酸转运蛋白CPT2、呼吸链亚基NDUFA9和氧自由基清除酶MnSOD去乙酰化水平综合调控。相比之下，CDK1突变细胞系Palm损伤实验中GC方的保护作用较小或丧失。.结论意义：GC方可有效改善肝细胞线粒体能量代谢障碍和氧化应激损伤。线粒体CDK1-SIRT3通路是GC方有效调节线粒体稳态治疗NAFLD的重要途径。为进一步研发治疗NAFLD显效、机制明确的新型中药研发提供科学依据。

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