Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan 33302, Taiwan.
School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33302, Taiwan.
Oxid Med Cell Longev. 2020 Dec 3;2020:1080168. doi: 10.1155/2020/1080168. eCollection 2020.
Heme oxygenase-1 (HO-1) has been shown to exert as an antioxidant and anti-inflammatory enzyme in cardiovascular inflammatory diseases. Flavonoids have been demonstrated to display anti-inflammatory and antioxidant effects through the induction of HO-1. 5,8-Dihydroxy-4',7-dimethoxyflavone (DDF), one of the flavonoid compounds, is isolated from . Whether DDF induced HO-1 expression on human cardiac fibroblasts (HCFs) remained unknown. Here, we found that DDF time- and concentration-dependently induced HO-1 protein and mRNA expression, which was attenuated by pretreatment with reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) in HCFs. DDF-enhanced ROS generation was attenuated by NAC, but not by either diphenyleneiodonium chloride (DPI, Nox inhibitor) or MitoTempol (mitochondrial ROS scavenger). Interestingly, pretreatment with glutathione (GSH) inhibited DDF-induced HO-1 expression. The ratio of GSH/GSSG was time-dependently decreased in DDF-treated HCFs. DDF-induced HO-1 expression was attenuated by an inhibitor of p38 MAPK (p38i VIII) or siRNA, but not by MEK1/2 (PD98059) or JNK1/2 (SP600125). DDF-stimulated p38 MAPK phosphorylation was inhibited by GSH or p38i VIII. Moreover, DDF-induced HO-1 expression was mediated through Nrf2 phosphorylation and translocation into the nucleus which was attenuated by NAC or p38 siRNA. DDF also stimulated antioxidant response element (ARE) promoter activity which was inhibited by NAC, GSH, or p38i VIII. Interaction between Nrf2 and the ARE-binding sites on the HO-1 promoter was revealed by chromatin immunoprecipitation assay, which was attenuated by NAC, GSH, or p38i VIII. We further evaluated the functional effect of HO-1 expression on the thrombin-induced fibrotic responses. Our result indicated that the induction of HO-1 by DDF can attenuate the thrombin-induced connective tissue growth factor expression. These results suggested that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway in HCFs. Thus, the upregulation of HO-1 by DDF could be a candidate for the treatment of heart fibrosis.
血红素加氧酶-1（HO-1）已被证明在心血管炎症性疾病中作为一种抗氧化和抗炎酶发挥作用。黄酮类化合物已被证明通过诱导 HO-1 发挥抗炎和抗氧化作用。5,8-二羟基-4',7-二甲氧基黄酮（DDF）是一种黄酮类化合物，从 中分离得到。DDF 是否诱导人心房成纤维细胞（HCFs）中 HO-1 的表达尚不清楚。在这里，我们发现 DDF 时间和浓度依赖性地诱导 HO-1 蛋白和 mRNA 的表达，而在 HCFs 中用活性氧（ROS）清除剂 N-乙酰半胱氨酸（NAC）预处理则减弱了这一表达。DDF 增强的 ROS 生成被 NAC 减弱，但不是被二苯碘（DPI，Nox 抑制剂）或 MitoTempol（线粒体 ROS 清除剂）减弱。有趣的是，用谷胱甘肽（GSH）预处理抑制了 DDF 诱导的 HO-1 表达。在 DDF 处理的 HCFs 中，GSH/GSSG 的比值随时间呈下降趋势。DDF 诱导的 HO-1 表达被 p38 MAPK 抑制剂（p38i VIII）或 siRNA 减弱，但不受 MEK1/2（PD98059）或 JNK1/2（SP600125）的影响。DDF 刺激的 p38 MAPK 磷酸化被 GSH 或 p38i VIII 抑制。此外，DDF 诱导的 HO-1 表达是通过 Nrf2 磷酸化和转位到细胞核来介导的，这一过程被 NAC 或 p38 siRNA 减弱。DDF 还刺激抗氧化反应元件（ARE）启动子活性，该活性被 NAC、GSH 或 p38i VIII 抑制。染色质免疫沉淀分析显示，DDF 刺激的 HO-1 启动子上的 Nrf2 和 ARE 结合位点之间的相互作用被 NAC、GSH 或 p38i VIII 减弱。我们进一步评估了 HO-1 表达对凝血酶诱导的纤维化反应的功能影响。我们的结果表明，DDF 诱导的 HO-1 表达可以减弱凝血酶诱导的结缔组织生长因子表达。这些结果表明，DDF 诱导的 HO-1 表达至少是通过 ROS 依赖性 p38 MAPK/Nrf2 信号通路在 HCFs 中激活的。因此，DDF 上调 HO-1 表达可能是治疗心脏纤维化的候选药物。
