NCLX介导的线粒体钙超载在CRF骨骼肌萎缩发生中的作用及健脾益肾方干预研究

Muscle atrophy is a common complication of chronic renal failure (CRF), but the underlying mechanism has not been fully elucidated. Mitochondrial stress and apoptosis induced by the imbalance of mitochondrial calcium homeostasis is considered as one of the important mechanisms of mitochondrial injury. Our previous study found that CRF muscle atrophy was closely associated with mitochondrial damage. Recently studies demonstrated that mitochondrial calcium overload resulted from down-regulation of NCLX expression. Our pretest study found that the expression of NCLX was decreased in skeletal muscle of CRF rats. We hypothesized that decreased NCLX induced mitochondrial calcium overload and subsequently caused mitochondrial stress and apoptosis might be a new mechanism for muscle atrophy in CRF. This research aims to observe the dynamic changes in mitochondrial calcium overload, mitochondrial stress and apoptosis and to analysis effect of mitochondrial calcium overload induced NCLX on muscle atrophy in CRF model of rats, supplemented with disproof treatment by Jian-Pi-Yi-Shen decoction. The research also aims to focus on molecular events related to muscle mass loss in the down-stream of NCLX pathway, using overexpression and siRNA of NCLX technology together with medicated serum.Then the research plans to reveal the therapeutic mechanism of Jian-Pi-Yi-Shen decoction for muscle atrophy in CRF from a modern medical standpoint of mitochondrial calcium ion transport，and provide a support in theory for the treatment of CRF with Jian-Pi-Yi-Shen decoction.

骨骼肌萎缩是慢性肾衰竭（CRF）最常见的并发症之一，发生机制尚未阐明。线粒体钙稳态失衡引起线粒体应激与凋亡，被认为是线粒体损伤的重要机制之一。本课题组前期研究发现，CRF骨骼肌萎缩与线粒体损伤密切相关。新近报道，NCLX下调引起线粒体钙超载，导致线粒体应激与凋亡。我们发现，CRF大鼠骨骼肌组织NCLX表达减少。我们推测：NCLX下调介导的线粒体钙超载，通过线粒体应激和细胞凋亡造成线粒体损伤，这可能是CRF骨骼肌萎缩发生的新机制。本项目拟复制CRF大鼠模型，以健脾益肾方干预，动态观察线粒体钙超载与线粒体应激和凋亡的变化规律，探索NCLX介导的线粒体钙超载导致线粒体应激与凋亡对CRF骨骼肌萎缩的影响；体外用NCLX过表达和RNA干扰技术，结合含药血清的验证，重点阐明NCLX下游导致骨骼肌质量减少的分子事件，从线粒体钙离子转运的角度揭示健脾益肾方的作用靶点和环节，为其防治CRF提供新的理论依据。

骨骼肌萎缩是慢性肾脏病(Chronic kidney disease,CKD)最常见的并发症之一，可影响患者生存质量，增加死亡率及其他合并症危险，是患者预后不良和高病死率的独立危险因素之一。近些年来的研究表明，线粒体钙稳态失衡导致的线粒体应激与凋亡被认为是线粒体损伤的重要机制。最新研究发现，NCLX下调引起线粒体钙超载，造成线粒体氧化应激和细胞凋亡，导致骨骼肌萎缩。健脾益肾方是传统中医治疗CRF的经验方，广泛应用于临床，临床改善肾功能和营养不良的效果显著。本研究分别以5/6肾切除小鼠和肿瘤坏死因子α（TNF-α）诱导C2C12肌管萎缩作为CKD骨骼肌萎缩研究的体内及体外模型。首先，我们从动物水平上证实健脾益肾方可以有效改善CKD小鼠的肌肉萎缩情况，抑制线粒体氧化应激和细胞凋亡，保护CKD小鼠骨骼肌免受线粒体钙超载损害，并激活了NCLX相关通路。其次，体外实验研究中，我们从细胞水平上证明健脾益肾方改善TNF-α诱导的肌管萎缩，减少线粒体氧化应激、细胞凋亡和线粒体钙超载，同时激活NCLX信号通路。最后，从分子层面对NCLX进行敲低，能证实NCLX通路在健脾益肾方调控线粒体氧化应激和细胞凋亡改善骨骼肌萎缩发挥的作用。以上研究表明，健脾益肾方通过调控NCLX介导的线粒体钙超载抑制线粒体氧化应激和凋亡，从而延缓CKD骨骼肌萎缩。