基于长链非编码RNA uc.222调控KChIP2基因表达探讨电针治疗心肌缺血的机制

Acupuncture in the treatment of myocardial ischemia has distinct advantage and good clinical application, while its mechanism is not clear. With the development of modern science, high-throughput chip screening technology has opened up a new idea for acupuncture research. Our previous studies have shown that LncRNAs play an important role in the process of electroacupuncture against myocardial ischemia. LncRNA uc.222 is one of LncRNAs with highly conserved sequences, which expression is the most obvious. And it is correlated with mRNA expression of KChIP2, which is a cardiac related gene. In this study, the rat model of acute myocardial ischemia administered subcutaneously with isoproterenol will be as observation object, electroacupucture on “Neiguan” point as the intervention measures, changes of ECG, myocardial enzymes and myocardial tissue structure as acupuncture efficacy index, expression changes of LncRNA uc.222 as the starting point, and siRNA will be used to silence uc.222. Using ELISA, Real-time PCR, Western blot, histochemistry, electron microscopy and other technical methods, we’ll analyze the relationship of expression changes among uc.222, KChIP2 and Kv4.2 to investigate the function and mechanism of LncRNA in the treatment of myocardial ischemia by electroacupuncture, and to provide some experimental basis for the clinical application of electroacupuncture.

针刺治疗心肌缺血有整体调节优势，疗效确切，但机制尚不明确。基因组学高通量筛选技术为针刺治疗心肌缺血的机制研究开辟了一条新思路。我们前期研究表明长链非编码RNA（LncRNA）在电针治疗心肌缺血过程中发挥重要作用，其中一条序列高度保守的LncRNA uc.222在电针组中表达差异最明显，并与心肌相关mRNA—KChIP2表达密切相关。本研究用盐酸异丙肾上腺素皮下注射造成急性心肌缺血大鼠模型，电针内关穴为干预措施，以ECG、心肌酶学、心肌组织结构等为针刺疗效指标，以uc.222表达变化为切入点，采用小干扰 RNA（siRNA）沉默uc.222，运用ELISA、Real-time PCR、Western blot、免疫荧光化学、电镜等多种技术，从uc.222、KChIP2、Kv4.2表达变化关系，探讨LncRNA在电针治疗心肌缺血中的功能及作用机制，为电针治疗心肌缺血的临床应用提供部分实验依据。

国内外研究已经证实针刺治疗心肌缺血的有效性，但机制仍未完全阐明，因此在一定程度上限制了针刺治疗心肌缺血的临床应用及推广。前期通过基因组学高通量筛选，课题组发现一条与心肌缺血相关的序列高度保守的LncRNA uc.222。本研究探讨了电针不同部位对心肌缺血的影响，以及LncRNA uc.222在其中的可能机制与相关途径。.本研究通过皮下注射盐酸异丙肾上腺素制作大鼠心肌缺血模型，进而通过电针大鼠心包经内关穴、非经非穴位进行干预治疗，从心电图、血清心肌酶、病理HE染色、电镜微观结构等方面对治疗效果进行评价，分析穴位特异性对治疗效果的影响。结果表明：①心肌缺血后，心电活动QT间期离散率（QTd）、校正QT间期离散率（QTc）延长，ST段偏移幅度增加，心肌酶LDH、CK-MB活性升高，SOD活性降低，心肌细胞肥大、横截面积增加，超微结构呈退行性改变。②电针非经非穴后，心肌缺血上述指标与模型组无差异。③电针内关穴后，心电活动QTd、QTc缩短，ST段偏移幅度减少，心肌酶LDH、CK-MB活性降低，SOD活性升高，肥大的心肌细胞面积减小，超微结构退行性改变减轻。可见循经取穴电针可以改善心肌缺血状况。.通过SiRNA干扰，观察心肌组织LncRNA uc.222、KchIP2、Kv4.2的表达及心肌缺血指标的变化。结果显示：①心肌缺血后，模型组LncRNA uc.222表达升高，KchIP2、Kv4.2表达下调。②电针内关穴后，LncRNA uc.222表达降低，KchIP2、Kv4.2表达升高。③SiRNA干扰后，模型组、电针组LncRNA uc.222表达均降低，KchIP2表达升高，Kv4.2表达下调。④SiRNA干扰后，模型组QTd缩短，心肌酶SOD升高，心肌细胞面积减小；电针组心肌细胞面积减小更为显著，超微结构退行性改变减轻。提示心肌组织LncRNA uc.222、KchIP2的表达参与心肌缺血过程，与电针改善心肌缺血有一定相关性。.本研究为丰富针刺临床疗效提供了很好的实验数据，也为针刺效应机制研究提供了一定的理论依据。