盐酸小檗碱对代谢异常引起的线粒体功能紊乱的调节机制研究

Recently, the role of mitochondrial dysfunction in metabolic disorder diseases has gained increasing attention. Epidemiological study of the series of rapid growing metabolic disorders such as type 2 diabetes or insulin resistance all demonstrate significant mitochondrial function down-regulation, indicating that as a critical and basal organelle that is in charge of terminal steps of glucose and lipid metabolism, mitochondrion might be crucial in revealing the mechanism of such metabolic disorders. Hydrochloride (BBR) is a kind of alkaloid compound purified from herbs, which has been used to treat type 2 diabetes for several years with its outstanding features of few side effects. Previous study revealed its effect on ameliorating hyperglycemia, hyperlipidemia and obesity. Our group was the first to verify BBR’s effect on increasing energy expenditure and mitochondrial uncoupling effect in vivo. However, the exact protein targets of BBR and its regulation mechanism in treating metabolic diseases are still elusive. For mechanism study, we find evidence of BBR regulating mitochondrial function through mitochondrial metabolic enzymes or respiratory chain proteins. Epigenetic regulation would be critical to mitochondrial function. As a newly risen field of epigenetic regulation, post-transcriptional modification such as acetylation and succinylation has shed light on the control of target metabolic enzymes according to our recent data. Furthermore, factors which regulate acetylation may also participate in such pathways. SIRT1 (Sirtuin type 1) is a member of the NAD+ dependent histone deacetylase. As our data indicates, it might be an essential target for interpreting effect of BBR by its epigenetic regulation. In this study, SIRT1 would be our research target, and experiments would be conducted to find out the related protein target of BBR in treating metabolic diseases, as well as revealing the regulation of post-transcriptional modification during treatment.

近年来，线粒体功能紊乱在代谢异常疾病发病机制中所起的作用逐渐成为了研究的热点。人群研究显示2型糖尿病或胰岛素抵抗的病人线粒体功能下降。盐酸小檗碱是一种从植物中发现的天然生物碱，本人所在课题组首先在临床上验证其有着降血糖、降血脂、降体重等多种调节代谢作用，副作用少，有良好的药用前景。本课题组已经证实小檗碱在体内有增加能量消耗、提高线粒体解偶联水平的作用，但其对线粒体的具体调节机制和分子靶点仍不明确。本申请人进一步研究了小檗碱在体内调节线粒体功能的机制，发现了其对线粒体关键代谢酶和呼吸链复合物的蛋白有调节作用，并且乙酰化等转录后调控机制也参与其中。本申请人在此研究基础上，计划继续深入研究小檗碱调节线粒体功能的分子机制，寻找其作用的具体靶点蛋白，再以去乙酰化酶SIRT活性为核心，分析乙酰化等转录后调控机制对上述蛋白活性的影响，阐述其在药效中发挥的作用，为代谢性疾病的治疗提供新的分子靶点和思路。

在代谢性疾病研究领域，线粒体功能紊乱在代谢异常疾病发病机制中所起的作用逐渐引起关注。本项目按研究计划执行，以盐酸小檗碱这种天然生物碱为例，以其对线粒体功能的调节机制为研究对象，完成了如下研究工作。1.首先建立了药物干预小鼠模型，观察药物治疗组小鼠的代谢体征，检测其体重、血糖、血脂、饮食量等生化指标并进一步检测胰岛素耐量，通过代谢笼等设备检测小鼠能量消耗，证实了盐酸小檗碱提高能量消耗、线粒体功能。2.收集小鼠肝脏、肌肉、脂肪等多种组织标本，提取蛋白、RNA、DNA等，并利用先进技术提取新鲜线粒体，用Seahorse线粒体功能检测仪检测组织线粒体氧耗量，发现盐酸小檗碱提高多种组织中线粒体的氧耗量，提高肌肉细胞中线粒体的含量和活性；3.进一步检测了盐酸小檗碱对线粒体调控因子的基因水平和蛋白水平的影响。发现了盐酸小檗碱提高多种线粒体合成因子、线粒体功能相关酶、肌纤维相关因子、脂肪酸氧化因子的基因和蛋白水平表达；并且发现其对多种线粒体相关调节因子的调节是剂量依赖性的；4.通过构建PGC-1α的siRNA质粒，敲除PGC-1α基因，发现盐酸小檗碱对于线粒体的调节作用依赖于AMPK-PGC-1α通路，并且增加线粒体最大氧耗量；5.检测盐酸小檗碱对线粒体代谢通路中关键酶转录后修饰调控水平的影响，发现盐酸小檗碱抑制PDHK1及此酶的转录后修饰lipoytion的水平，通过多种机制抑制PDH活性；同时盐酸小檗碱在Sirt1敲除小鼠中提高了去乙酰化酶Sirt1的活性进而激活了FGF21。综上所述，本项目发现了盐酸小檗碱在多种组织内均可以通过调节线粒体合成因子、线粒体功能相关酶来调节线粒体功能，且其调节作用依赖于AMPK- PGC-1α通路，线粒体酶PDH及细胞因子Sirt1的转录后修饰调控也参与其中。.本项目基本按照研究计划进行，证实了盐酸小檗碱对线粒体的调节作用，并深入探讨了相关机制。此研究对代谢性疾病的病理机制探讨及代谢调节药物新靶点的开发具有非常重要的科学价值，在药物研发方面有很好的应用前景。