OBJECTIVE: Tanshinone IIA has a wide range of myocardial protective effects. AK003290 is a long noncoding RNA (lncRNA) that is highly expressed in myocardial tissue, and its expression is down-regulated when myocardial injury occurs. This study aims to explore the mechanism for tanshinone IIA in alleviating myocardial cell damage induced by oxygen glucose deprivation (OGD).
METHODS: OGD model was established in rat H9C2 cardiomyocytes. siRNA was transfected to reduce AK003290 expression. H9C2 cells were divided into 6 groups: A control group, a tanshinone IIA (TAN) group, an OGD group, a tanshinone IIA+OGD (TAN+OGD) group, a scrambled siRNA transfection+tanshinone IIA+OGD (scrambled siRNA+TAN+OGD) group, and a AK003290 siRNA transfection+tanshinone IIA+OGD (AK003290 siRNA+TAN+OGD) group. H9C2 cells in the TAN group were treated with 40 μmol/L tanshinone IIA for 12 h. The TAN+OGD group was treated with 40 μmol/L tanshinone IIA for 12 h, followed by OGD treatment for 12 h. The scrambled siRNA+TAN+OGD group and AK003290 siRNA+TAN+OGD group were transfected with the scrambled siRNA or AK003290 siRNA. Twenty-four hours later, the cells were treated with tanshinone IIA and OGD. Real-time RT-PCR was used to detect the expression of AK003290. Spectrophotometry was used to detect the content of lactate dehydrogenase (LDH) in cell culture medium to reflect LDH leakage rate, and enzyme-linked immunosorbent assay (ELISA) was used to detect the content of interleukin-1β (IL-1β) and interleukin-18 (IL-18). Western blotting was used to detect the protein expression of phospho-nuclear factor- κB (p-NF-κB).
RESULTS: Compared with the control group, the leakage rate of LDH, the content of IL-1β and IL-18 in culture medium, and the protein expression level of p-NF-κB were increased in the OGD group (P<0.01 or P<0.001). Compared with the OGD group, the leakage rate of LDH, the content of IL-1β and IL-18 in culture medium, and the protein expression level of p-NF-κB were decreased in the TAN+OGD group (P<0.05 or P<0.01). Compared with the control group, the AK003290 expression was increased in the TAN group (P<0.01) and it was decreased in the OGD group (P<0.05). Compared with the OGD group, the AK003290 expression was increased in the TAN+OGD group (P<0.05). Compared with the scrambled siRNA+TAN+OGD group, the leakage rate of LDH, the content of IL-1β and IL-18 in culture medium, and the protein expression level of p-NF-κB were increased in the AK003290 siRNA+TAN+OGD group (P<0.05 or P<0.01).
CONCLUSIONS: Tanshinone IIA inhibits NF-κB activity and attenuates OGD-induced inflammatory injury of cardiomyocytes through up-regulating AK003290.
目的: 丹参酮ⅡA具有广泛的心肌保护作用。AK003290是1种在心肌组织中高表达的长链非编码RNA(long noncoding RNA，lncRNA)，在心肌发生损伤时表达下调。本研究旨在探讨丹参酮ⅡA减轻氧糖剥夺(oxygen glucose deprivation，OGD)诱导心肌细胞损伤的机制。方法: 以H9C2大鼠心肌细胞为研究对象，构建OGD损伤模型。采用转染siRNA的方法敲低AK003290的表达。将H9C2细胞分为6组：对照(control)组、丹参酮ⅡA(TAN)组、OGD组、丹参酮ⅡA+OGD(TAN+OGD)组、scrambled siRNA转染+丹参酮ⅡA+OGD(scrambled siRNA+TAN+OGD)组、AK003290 siRNA转染+丹参酮ⅡA+OGD(AK003290 siRNA+TAN+OGD)组。TAN组只用40 μmol/L的丹参酮ⅡA预处理细胞12 h。OGD组只进行OGD处理12 h。TAN+OGD组先用40 μmol/L的丹参酮ⅡA预处理12 h，再行OGD处理12 h。Scrambled siRNA+TAN+OGD组和AK003290 siRNA+TAN+OGD组在转染相应siRNA 24 h后进行后续处理。采用实时反转录PCR(real-time reverse transcription PCR，real-time RT-PCR)检测AK003290的表达，分光光度法检测细胞培养液中乳酸脱氢酶(lactate dehydrogenase，LDH)的水平以反映LDH漏出率，酶联免疫吸附测定(enzyme-linked immunosorbent assay，ELISA)检测细胞培养液中白细胞介素-1β(interleukin-1β，IL-1β)和白细胞介素-18(interleukin-18，IL-18)的含量，蛋白质印迹法检测磷酸化的核因子κB(phospho-nuclear factor-κB，p-NF-κB)的蛋白质表达水平。结果: 与control组比较，OGD组LDH漏出率及细胞培养液中IL-1β和IL-18含量增加，p-NF-κB的蛋白质表达水平上调，差异均有统计学意义(P<0.01或P<0.001)；与OGD组相比，TAN+OGD组LDH漏出率及细胞培养液中IL-1β和IL-18含量减少，p-NF-κB的蛋白质表达水平下调，差异均有统计学意义(P<0.05或P<0.01)。与control组比较，TAN组细胞中AK003290的表达水平明显上调(P<0.01)，OGD组细胞中AK003290的表达水平明显下调(P<0.05)；与OGD组相比，TAN+OGD组细胞中AK003290的表达水平明显上调(P<0.05)。与scrambled siRNA+TAN+OGD组相比，AK003290 siRNA+TAN+OGD组LDH漏出率及细胞培养液中IL-1β和IL-18含量增加，p-NF-κB的蛋白质表达水平上调，差异均有统计学意义(P<0.05或P<0.01)。结论: 丹参酮ⅡA通过上调AK003290抑制NF-κB活性，从而减轻OGD导致的心肌细胞炎症损伤。.

Acute myocardial infarction is the leading cause of death and disability worldwide. Reperfusion is the main treatment method. However, ischemia-reperfusion (I/R) injury aggravates tissue and cell damage. In this study, we aim to find a strategy to reduce I/R injury and promote cardiac function recovery. The expression of AK003290 was downregulated in I/R injury both in vitro and in vivo. Overexpression of AK003290 reduced infarction area, oxidative stress, cell apoptosis, and promoted cardiac function recovery. AK003290 was observed to sponge miR-539-3p. Moreover, the expression of miR-539-3p was upregulated in I/R injury. Overexpression of miR-539-3p reversed the beneficial role of AK003290 in I/R injury. The target gene of miR-539-3p was proved to be ErbB4, as identified by database prediction, dual-luciferase reporter assay, and pull-down assay. The expression of ErbB4 was negatively correlated with the expression of miR-539-3p, but positively correlated with the expression of AK003290. Subsequently, the key downstream proteins were determined. AK003290 promoted p-AKT and bcl-2 expression and inhibited p-ERK1/2, Bax, cytoplasmic cyto-c, and c-caspase-3 expression. The application of ErbB4 siRNA significantly reversed the effect of AK003290 on the expression of these proteins. These results suggest that ErbB4 is the key downstream gene, which regulates myocardial cell apoptosis by influencing the miR-539-3p expression. To the best of knowledge, this study is the first to demonstrate that the AK003290/miR-539-3p/ErbB4 axis regulates myocardial cell apoptosis. These findings provide a potential novel target for the treatment of myocardial I/R injury.

BACKGROUND: Acute myocardial infarction (AMI) is the leading cause of death globally and has thus placed a heavy burden on healthcare. Tanshinone IIA (TSA) is a major active compound, extracted from Salvia miltiorrhiza Bunge, that possesses various pharmacological activities. The aim of the present study was to investigate the role of TSA in AMI and its underlying mechanism of action.
RESULTS: We have shown that TSA decreased the apoptosis rate, the amount of LDH, MDA as well as ROS of cardiomyocytes. Meantime, it elevated mitochondrial membrane potential (MMP) which was decreased by H/R treatment. It was also determined that miR-124-5p targets AK003290 directly. TSA up-regulated the expression of AK003290 and its function can be reversed by knock down of AK003290 as well as miR-124-5p overexpression.
CONCLUSIONS: TSA exerts the protective role against H/R induced apoptosis, oxidative and MMP loss of cardiomyocytes via regulating AK003290 and miR-124-5p signaling.