四君子汤干预脾气虚骨骼肌线粒体未折叠蛋白反应-自噬-细胞凋亡机制的研究

The effect of Sijunzi Decoction treating limb useless in spleen-qi deficiency is remarkably effective whose mechanism is related to mitochondrial damage repair but the detail is unknown. It is found that mitochondrial DNA is easily mutant under stress. To maintain mitochondrial homeostasis, cell initiates mitochondrial unfolded protein response(UPR), mitochondrial autophagy(mitophagy) and apoptosis to defense stress at molecular, organelle and cellular levels, respectively. All these three mechanism together are referred to “UPR-mitophagy-apoptosis” mechanism. Our previous works showed that mitochondrial of quadriceps femoris with abnormal protein accumulation in spleen-qi deficiency rats, however the expression of corresponding protease was down-regulated; the mitochondrial dynamic disordered and the mitophagy flux reduced; Sijunzi Decoction could increase rat’s grasping force. Therefore, it is proposed that limb useless in spleen-qi deficiency might be caused by the impairment of UPR-mitophagy-apoptosis mechanism in skeletal muscle, which could be repaired by Sijunzi Decoction. To prove this hypothesis, the mitochondria in quadriceps femoris will be focused on in this study, changes of morphology, UPR, mitochondrial dynamics, mitophagy and apoptosis in spleen-qi deficiency and the treating effects of Sijunzi Decoction will be studied by electron microscope and molecular techniques, attempting to reveal the pharmacological mechanism of Sijunzi Decoction.

四君子汤治疗脾气虚四肢不用效果显著，其机理与修线粒体损伤有关，但详细机制不明。线粒体产能是脾主肌肉的重要环节，但线粒体DNA极易诱发突变并产生蛋白毒性应激。对此，细胞可启动线粒体未折叠蛋白反应(UPR)、线粒体自噬和细胞凋亡，从分子、细胞器和细胞水平逐级对抗，维持线粒体稳态，本项目将其合称UPR-自噬-细胞凋亡机制。前期工作发现，脾气虚证大鼠股四头肌线粒体出现异常蛋白积聚，产生蛋白毒性应激，但UPR和自噬均减弱；而四君子汤可提高肌力。推测脾气虚时骨骼肌线粒体稳态维持机制减弱，而四君子汤可能修复该机制。据此提出“四君子汤治疗脾气虚四肢不用与修复骨骼肌线粒体未折叠蛋白反应-自噬-细胞凋亡机制有关”的假说，以股四头肌线粒体为对象，采用电镜和分生技术，从ATF5激活UPR、miR-149调控自噬和细胞凋亡方面观测脾气虚时的变化及四君子汤的治疗作用，揭示四君子汤的药理机制和脾主肌肉的科学内涵。

四君子汤治疗“脾气虚四肢不用”效果显著，其机理与修复线粒体损伤有关，但详细机制不明。线粒体产能是脾主肌肉的重要环节，但线粒体DNA极易诱发突变并产生蛋白毒性应激。对此，细胞可启动线粒体未折叠蛋白反应(UPR)、线粒体自噬和细胞凋亡，从分子、细胞器和细胞水平逐级对抗，维护线粒体稳态，本项目将其合称"UPR-自噬-细胞凋亡"机制。结合前期研究基础，本项目提出“四君子汤治疗脾气虚四肢不用与修复骨骼肌线粒体未折叠蛋白反应-自噬-细胞凋亡机制有关”的科学假说，并采用了相应的实验方案进行验证。. 本项目以四君子汤干预脾气虚证大鼠股四头肌线粒体为研究对象，充分阐明了项目假说。本研究发现，首先，四君子汤治疗“脾气虚四肢不用”线粒体的形态和功能并不依赖于HSP60/70协同机制和LonP/ClpP系统(即UPR)的修复作用；其次，四君子汤维护脾气虚骨骼肌线粒体稳态并未靶向miR-149及SIRT-1/ PGC-1α通路所介导的线粒体生物发生。. 本研究认为，四君子汤治疗“脾气虚四肢不用”线粒体的形态和功能主要与抑制线粒体分裂过程中Drp1-Mff复合体主导的线粒体碎片化进而维护线粒体网络有关，并协同抑制AMPK/ULK1/mTOR通路介导的自噬，从亚细胞水平共同对抗细胞凋亡，通过提升线粒体产能进而提高肌力，治疗“脾气虚四肢不用”。. 不仅如此，本研究还发现，脾气虚骨骼肌线粒体呼吸链功能异常及UPR功能紊乱可能与转录因子CHOP激活不足有关；同时，脾气虚骨骼肌线粒体自噬水平的激活受到PINK1/Parkin通路抑制所介导的线粒体膜电位下降和AMPK/ULK1通路激活的双重调节。因此，本项目也为阐明“脾主肌肉”的科学内涵奠定了实验依据。