亚健康疲劳骨骼肌基因表达的miR-324调控机制及维康颗粒的干预效应

(FSH) has become a serious public health problem. However, the pathogenesis is still less known. In our previous studies, we interfered the FSH mice by the traditional Chinese medicine Weikang granule which is effective in the treatment of FSH, and with the combination of miRNA microarray and iTRAQ proteomics technologies, we made the association analysis between miRNA and FSH by bioinformatics methods. Finally, miR-324 was shown to be closely related to the differently expressed genes in FSH. Therefore, we hypothesized that miR-324 may play a significant role in the development of FSH and it would be an important way to prevent and treat it by Weikang granules. The plans of this project are as following. Firstly, we aim to cause the miR-324 overexpression or inhibition by the use of specificity agonist and inhibitor respectively in the FSH mice to make sure the action of miR-324 in the development of FSH. Secondly, we establish the cell model in vitro to explore the critical target gene of miR-324 by the techniques of gene transfection and dual luciferase reporter gene assay. Finally, we use the method of serum pharmacology to clarify the effects of Weikang granules in the regulation of miR-324 and its target genes.This could not only advance the the prevention and treatment of FSH, but also deepen the understanding of the biological functions of miRNA and open up new thread and way in the TCM-WM research meanwhile.

亚健康疲劳(FSH)已成为严重的公共卫生问题，然而目前对其知之甚少。我们前期以防治FSH有确切疗效的维康颗粒为干预手段，利用miRNA表达谱芯片结合iTRAQ定量蛋白组学技术，通过生物信息学方法对miRNA与FSH进行关联分析，结果发现miR-324与FSH多个基因表达异常密切相关，因此推测miR-324可能在FSH发生中起到重要作用，而调节miR-324亦可能是维康颗粒防治FSH的重要途径。本项目拟首先利用FSH小鼠模型，给予特异性激动剂或抑制剂，分别地过表达或抑制miR-324，以明确miR-324在FSH发生中的作用；其次采用体外细胞模型，应用基因转染、双荧光素酶报告基因检测方法，确定miR-324的关键靶基因；最后应用血清药理学方法，阐明维康颗粒对miR-324及其靶基因的干预效应。这不仅将深化理解miRNA的生物学功能，还会促进亚健康的防治和中医"治未病"的发展。

本项目利用亚健康疲劳小鼠模型和亚健康疲劳骨骼肌细胞体外模型，采用负重力竭游泳时间测定小鼠疲劳程度，酶标仪测定小鼠血浆中氧化应激损伤指标，透射电镜观察组织细胞超微结构，CCK-8法检测细胞活力，HPLC检测骨骼肌细胞线粒体能量负荷值，流式细胞仪检测细胞凋亡情况，荧光定量RT-PCR检测miR-324表达变化，双荧光素酶报告基因方法验证miR-324靶基因，Western blot检测miR-324靶基因蛋白表达情况。结果表明：miR-324激动剂能够改善亚健康疲劳状态，减轻氧化应激损伤，提高亚健康疲劳骨骼肌细胞活力和能量负荷值，而降低细胞凋亡水平，其靶基因细胞色素C（CYCS）明显下调；维康颗粒能显著减轻模型小鼠亚健康疲劳状态，提高亚健康疲劳骨骼肌细胞活力和保护线粒体，细胞凋亡明显减少，同时上调miR-324而下调CYCS表达水平。结论：miR-324/CYCS参与了亚健康疲劳的发生发展过程，而维康颗粒可通过调节miR-324以发挥防治亚健康疲劳的作用，为其临床应用提供了实验依据和进一步研发奠定了基础。