基于LncRNA-MALAT1/miR-124/BDNF轴探讨疏肝方调控肝郁气滞证大鼠行为的表观遗传学机制

Stagnation of liver qi syndrome is a common syndrome in traditional Chinese medicine, which shares a close relationship with social-living-environmental factors. The miRNA and LncRNA related epigenetic researches are focus on the interaction of the phenotype and the environment, which is more suitable for the exploration of this syndrome’s objective mechanism. Previous study found that in the stagnation of liver qi syndrome rats, the level of hippocampus BDNF protein and mRNA are decreased. The Shugan Decoction can adjust the abnormal and improve the rats’ behavior performance. BDNF is regarded as a core behavior regulatory proteins in stagnation of liver qi syndrome. Through literature search and bioinformatics methods, the miRNA-124 and LncRNA MALAT1 have being found to be a significant epigenetic regulative factor on BDNF, they can form a regulation axis with the miRNA-124 as the core node, BDNF as the ultimate target. This research uses the qRT-PCR and Western Blot test method, miRNA-124 over-expression, inhibition technology, observes the Shugan Decoction’s effects on the rat behavior, test the MALAT1/miR-124/BDNF level’s change. The purpose of this research is to study stagnation of liver qi syndrome rats’ epigenetic features, explore the epigenetic regulation mechanism and available epigenetic targets of Shugan Decoction’s effects on stagnation of liver qi syndrome rats, which can enrich the epigenetic scientific connotation of traditional Chinese medicine.

肝郁气滞证是中医常见证型，与社会环境因素关系密切，miRNA及LncRNA等表观遗传学研究强调表型与环境交互影响，能更好探寻该证的客观实质。前期研究发现肝郁气滞证大鼠海马BDNF蛋白及mRNA水平下降，疏肝方能上调该蛋白及mRNA的水平以改善模型大鼠行为学表现。BDNF是公认肝郁气滞证抑郁样行为核心调控蛋白，文献及生物信息学研究发现miRNA-124及LncRNA-MALAT1为BDNF重要表观遗传调控因子，三者能组成以miRNA-124为核心节点、BDNF为最终靶点的调控轴。本研究采用qRT-PCR及Western-Blot等方法，运用miRNA-124过表达、抑制技术，观察疏肝方对肝郁气滞证大鼠行为影响，测定MALAT1/miR-124/BDNF轴的改变，研究肝郁气滞证大鼠表观遗传特征，并揭示疏肝方改善该证大鼠行为的表观遗传学机制及有效靶点，进一步丰富中医表观遗传科学内涵。

肝郁气滞证是中医常见证型，与社会环境因素关系密切，前期研究发现肝郁气滞证大鼠海马BDNF蛋白及mRNA水平下降，疏肝方能上调该蛋白及mRNA的水平以改善模型大鼠行为学表现。本研究采用慢性不可预知温和应激法制备肝郁气滞证大鼠模型，采用侧脑室置管注射miRNA-124 agomir及miRNA-124 antagomir技术制备miR-124过表达及低表达的肝郁气滞证大鼠模型；观察疏肝方对肝郁气滞证大鼠行为学及体重的影响，采用Western-blot及qRT-PCR方法测定海马BDNF蛋白表达、海马LncRNA-MALAT1、miR-124、BDNF mRNA表达。结果发现肝郁气滞证大鼠海马miRNA-124 mRNA表达降低，LncRNA-MALAT1表达明显上升，BDNF mRNA及蛋白水平明显下降，其与抑郁样行为有关。采用agomir技术进行miRNA-124过表达处理肝郁气滞证大鼠其海马miRNA-124显著上升，LncRNA-MALTA1水平下调，BDNF蛋白水平上升，以糖水偏好试验为主的大鼠抑郁样行为明显改善。高剂量组大鼠海马抑郁样症状明显改善，其与miRNA-124表达上升，LncRNA-MALAT1下调，BDNF mRNA及蛋白表达水平明显升高有关。以上结果初步说明肝郁气滞证大鼠模型抑郁样行为与海马miR-124低表达导致LncRNA-MALAT1表达水平上调、BDNF表达水平下降有关，过表达miRNA-124能通过上调MALAT1表达及BDNF蛋白水平来改善抑郁样行为；疏肝方通过上调miR-124表达水平来下调MALAT1及上调BDNF mRNA及蛋白表达水平，从而达到改善肝郁气滞证大鼠抑郁行为的作用。本研究探索肝郁气滞证的表观遗传学特征，解释疏肝方改善肝郁气滞证大鼠行为学表现分子机制，为该方临床应用及推广提供科学依据。