目的 基于长链非编码RNA（long non coding RNA，LncRNA）心肌梗死相关转录本（myocardial infarction associated transcript，MIAT）/miR-384-5p探讨九龙藤黄酮（Bauhinia championii flavones，BCF）抑制自噬抗心肌缺氧损伤的作用及机制。方法 培养H9c2心肌细胞及敲低LncRNA MIAT稳转株，建立心肌细胞氧糖剥夺（oxygen-glucose deprivation，OGD）模型模拟心肌缺血缺氧，给予BCF预处理；选取SD雄性大鼠，沉默miR-384-5p基因，建立急性心肌梗死（acute myocardial infarction，AMI）模型，给予BCF预处理。以qRT-PCR法检测LncRNA MIAT、miR-384-5p及自噬相关基因表达；采用双荧光素酶报告基因实验及RNA反义纯化（RNA antisense purification，RAP）实验验证LncRNA MIAT与miR-384-5p的靶向关系；采用ELISA法检测细胞上清液及大鼠血清中肌钙蛋白-I（cardiac troponin-I，cTn-Ⅰ）水平；采用CCK-8法检测细胞活力；采用透射电镜观察自噬小体数量；以自噬双标腺病毒Ad-mRFP-GFP-LC3感染细胞检测自噬流；采用TTC染色法检测大鼠心肌梗死面积；采用Western blotting法检测自噬相关蛋白表达。结果 与OGD组比较，BCF能够下调LncRNA MIAT并上调miR-384-5p基因表达（P＜0.05、0.01），提高心肌细胞活力（P＜0.01），降低cTn-I水平（P＜0.001），下调Beclin1、Cathepsin D、LC3基因及蛋白表达（P＜0.05、0.01），减少自噬小体数量（P＜0.01），减轻自噬流（P＜0.01）。双荧光素酶报告基因及RAP实验结果显示，LncRNA MIAT靶向负调控miR-384-5p表达。敲低LncRNA MIAT可抑制自噬，减轻心肌缺氧损伤；与单纯BCF预处理相比，敲低LncRNA MIAT可显著增强BCF抑制自噬和保护心肌的作用（P＜0.05、0.01、0.001）。与AMI＋BCF组比较，沉默miR-384-5p后给予BCF处理，大鼠心肌梗死面积显著增加（P＜0.01），血清中cTn-I水平显著升高（P＜0.01），心脏组织中自噬相关基因及蛋白表达均显著上调（P＜0.01），心肌缺血损伤加重。结论 BCF通过LncRNA MIAT/miR-384-5p抑制自噬，从而减轻心肌缺氧损伤。

Objective To investigate the effect of Bauhinia championii flavones (BCF) on myocardial hypoxia injury by inhibiting autophagy based on long non-coding RNA (LncRNA) myocardial infarction associated transcript (MIAT)/miR-384-5p. Methods H9c2 cardiomyocytes and stable LncRNA MIAT knockdown strain were cultured, and cardiomyocyte oxygen-glucose deprivation (OGD) model was established to simulate myocardial ischemia and hypoxia, cells were pretreated with BCF. Male SD rats were selected to establish acute myocardial infarction (AMI) model by silencing miR-384-5p gene expression, and were pretreated with BCF. qRT-PCR was used to detect the expressions of LncRNA MIAT, miR-384-5p and autophagy-related genes; Dual luciferase reporter gene experiment and RNA antisense purification (RAP) experiment were used to verify that LncRNA MIAT and miR-384-5p targeting relationship; ELISA method was used to detect cardiac troponin-I (cTn-I) levels in supernatant of cells and serum of rats; CCK-8 method was used to detect cell viability; Transmission electron microscopy (TEM) was used to observe the number of phagosomes; Autophagy flux was detected by infecting cells with autophagy double-labeled adenovirus Ad-mRFP-GFP-LC3; Myocardial infarction size was detected by TTC staining; Expressions of autophagy-related proteins was detected by Western blotting. Results Compared with OGD group, BCF down-regulated LncRNA MIAT and up-regulated miR-384-5p gene expressions (P < 0.05, 0.01), increased myocardial cells viability (P < 0.01), decreased cTn-I level (P < 0.001), down-regulated Beclin1, Cathepsin D, LC3 gene and protein expressions (P < 0.05, 0.01), decreased the number of autophagosomes (P < 0.01), and reduced autophagic flux (P < 0.01). The results of dual-luciferase reporter gene and RAP assay showed that LncRNA MIAT targeted and negatively regulated the expression of miR-384-5p. Knockdown of LncRNA MIAT could inhibit autophagy and alleviate myocardial hypoxia injury; Compared with BCF pretreatment, knockdown of LncRNA MIAT significantly enhanced the effect of BCF on autophagy inhibition and myocardial protection (P < 0.05, 0.01, 0.001). Compared with AMI + BCF group, after silencing miR-384-5p and then giving BCF treatment, myocardial infarction area of rats was significantly increased (P < 0.01), cTn-I level in serum was significantly increased (P < 0.01), autophagy-related gene and protein expressions in cardiac tissue were significantly up-regulated (P < 0.01), and myocardial ischemia injury was aggravated. Conclusion BCF alleviates myocardial hypoxic injury by inhibits autophagy through LncRNA MIAT/miR-384-5p.

R285.5

国家自然科学基金资助项目（81760726）；国家自然科学基金资助项目（82060659）