Emodin protects H9c2 cells from hypoxia-induced injury by up-regulating miR-138 expression

Myocardial infarction (MI) is a common presentation for ischemic heart disease, which is a leading cause of death. Emodin is a Chinese herbal anthraquinone used in several diseases. However, the effect of emodin in hypoxia-induced injury in cardiomyocytes has not been clearly elucidated. Our study aimed to clarify the functions of emodin in hypoxia-induced injury in rat cardiomyocytes H9c2 and explore the underlying mechanism. The effects of emodin on cell viability and apoptosis were analyzed by the Cell counting kit-8 assay and flow cytometry assay, respectively. The cell proliferation- and cell apoptosis-related proteins were detected by western blot. qRT-PCR was used to determine the relative expression of miR-138. Cell transfection was performed to alter miR-138 and MLK3 expression. miR-138 target was performed by dual luciferase activity assay. Sirt1/AKT and Wnt/β-catenin pathways-related factors phosphorylation were analyzed by western blot. Emodin inhibited hypoxia-induced injury in H9c2 cells by promoting cell viability and reducing cell apoptosis. miR-138 was down-regulated by hypoxia treatment but up-regulated by emodin. Up-regulation of miR-138 alleviated hypoxia-induced cell injury. Down-regulation of miR-138 attenuated the growth-promoting effect of emodin on hypoxia-induced injury, whereas up-regulation of miR-138 enhanced the growth-promoting effects of emodin. The underlying mechanism might be by inactivating Sirt1/AKT and Wnt/β-catenin pathways. MLK3 was negatively regulated by miR-138 expression and inactivated Sirt1/AKT and Wnt/β-catenin pathways. Emodin alleviated hypoxia-induced injury in H9c2 cells via up-regulation of miR-138 modulated by MLK3, as well as by activating Sirt1/AKT and Wnt/β-catenin pathways.

心肌梗死（Myocardial infarction, MI）是缺血性心脏病的常见临床表现，而缺血性心脏病是全球主要的致死性病因。大黄素是一种被用于多种疾病治疗的中药蒽醌类成分，但目前其在心肌细胞缺氧损伤中的作用尚未被明确阐明。本研究旨在阐明大黄素对大鼠心肌细胞H9c2缺氧损伤的调控功能，并探讨其潜在分子机制。本研究分别采用细胞计数试剂盒-8（Cell counting kit-8, CCK-8）实验与流式细胞术，检测大黄素对细胞活力与细胞凋亡的影响；通过蛋白质印迹法（western blot）检测细胞增殖与凋亡相关蛋白的表达水平；采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）测定微小RNA-138（miR-138）的相对表达量；通过细胞转染技术调控miR-138与混合谱系激酶3（MLK3）的表达水平；利用双荧光素酶报告基因实验验证miR-138的靶基因；采用蛋白质印迹法检测沉默信息调节因子1/蛋白激酶B（Sirt1/AKT）及Wnt/β-连环蛋白通路相关因子的磷酸化水平。实验结果显示，大黄素可通过提升H9c2细胞活力、减少细胞凋亡，抑制缺氧诱导的细胞损伤。缺氧处理可下调miR-138的表达，而大黄素则可上调其表达。上调miR-138可缓解缺氧诱导的细胞损伤；下调miR-138则会削弱大黄素对缺氧损伤的保护作用，反之上调miR-138可增强大黄素的该保护效应。其潜在机制可能为：MLK3可抑制Sirt1/AKT及Wnt/β-连环蛋白通路的活化，而miR-138可负向调控MLK3的表达；大黄素通过MLK3调控的miR-138上调，以及激活Sirt1/AKT及Wnt/β-连环蛋白通路，从而缓解H9c2细胞的缺氧损伤。