Induction of HO-1 by 5, 8-Dihydroxy-4',7-Dimethoxyflavone via Activation of ROS/p38 MAPK/Nrf2 Attenuates Thrombin-Induced Connective Tissue Growth Factor Expression in Human Cardiac Fibroblasts.

Chien-Chung Yang,Hui-Ching Tseng,Hsin-Hui Lin,Yann-Lii Leu,Jiro Hasegawa Situmorang,Li-Der Hsiao,Chuen-Mao Yang,Tullia Maraldi

doi:10.1155/2020/1080168

Chien-Chung Yang, Hui-Ching Tseng + Show 6 more

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https://doi.org/10.1155/2020/1080168

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Abstract

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 Reevesia formosana. 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.

Highlights

Growing evidence has indicated that oxidative stress participates in several cardiovascular diseases
In human cardiac fibroblasts (HCFs), we found that DDF could induce reactive oxygen species (ROS) generation which was attenuated by pretreatment with N-acetyl cysteine (NAC) (ROS scavenger), but not with diphenyleneiodonium chloride (DPI) (Nox inhibitor) and MitoTempol
We found that DDF treatment decreased intracellular GSH/GSSG ratio and increased ROS levels leading to Heme oxygenase-1 (HO-1) expression in HCFs

Summary

Introduction

Growing evidence has indicated that oxidative stress participates in several cardiovascular diseases. Cardiac fibrosis is characterized by excessive deposition of fibrotic extracellular matrix (ECM) and synthesis and deposition of collagen which directly cause left ventricular (LV) dysfunction, alter electrical conduction, and increase coronary resistance [1]. The changes in these functions might be related to the generation of reactive oxygen species (ROS) through the induction of inflammatory mediators via the activation of several signaling components in the cardiac fibroblasts [2, 3]. The development of an effective antioxidant strategy to target these components could provide a beneficial intervention for the management of cardiovascular diseases

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