Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats

ORIGINAL RESEARCH published: 06 June 2018 doi: 10.3389/fphar.2018.00540 Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats Shao-Ai Cai 1,2† , Ning Hou 3† , Gan-Jian Zhao 4† , Xia-Wen Liu 3 , Ying-Yan He 1 , Hai-Lin Liu 1 , Yong-Quan Hua 1 , Li-Rong Li 4 , Yin Huang 4 , Cai-Wen Ou 1 , Cheng-Feng Luo 4* and Min-Sheng Chen 1* 1 Department of Cardiology, Guangdong Provincial Biomedical Engineering Technology Research Center for Cardiovascular Disease, Sino-Japanese Cooperation Platform for Translational Research in Heart Failure, Zhujiang Hospital, Southern Medical University, Guangzhou, China, 2 The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China, 3 School of Pharmaceutical Sciences and The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China, 4 Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Edited by: Cheorl-Ho Kim, Sungkyunkwan University, South Korea Reviewed by: Ki-Tae Ha, Pusan National University, South Korea Songxiao Xu, Artron BioResearch Inc., Canada *Correspondence: Min-Sheng Chen Cheng-Feng Luo † These authors have contributed equally to this work. Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 22 January 2018 Accepted: 04 May 2018 Published: 06 June 2018 Citation: Cai S-A, Hou N, Zhao G-J, Liu X-W, He Y-Y, Liu H-L, Hua Y-Q, Li L-R, Huang Y, Ou C-W, Luo C-F and Chen M-S (2018) Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats. Front. Pharmacol. 9:540. doi: 10.3389/fphar.2018.00540 Puerarin is an isoflavone isolated from Radix puerariae. Emerging evidence shown that puerarin possesses therapeutic benefits that aid in the prevention of cardiovascular diseases. In this study, we evaluated the effects of puerarin on oxidative stress and cardiac fibrosis induced by abdominal aortic banding (AB) and angiotensin II (AngII). We also investigated the mechanisms underlying this phenomenon. The results of histopathological analysis, as well as measurements of collagen expression and cardiac fibroblast proliferation indicated that puerarin administration significantly inhibited cardiac fibrosis induced by AB and AngII. These effects of puerarin may reflect activation of Nrf2/ROS pathway. This hypothesis is supported by observed decreases of reactive oxygen species (ROS), decreases Keap 1, increases Nrf2 expression and nuclear translocation, and decreases of collagen expressions in cardiac fibroblasts treated with a combination of puerarin and AngII. Inhibition of Nrf2 with specific Nrf2 siRNA or Nrf2 inhibitor brusatol attenuated anti-fibrotic and anti-oxidant effects of puerarin. The metabolic effects of puerarin were mediated by Nrf2 through upregulation of UDP-glucuronosyltransferase (UGT) 1A1. The Nrf2 agonist tBHQ upregulated protein expression of UGT1A1 over time in cardiac fibroblasts. Treatment with Nrf2 siRNA or brusatol dramatically decreased UGT1A1 expression in puerarin-treated fibroblasts. The results of chromatin immunoprecipitation–qPCR further confirmed that puerarin significantly increased binding of Nrf2 to the promoter region of Ugt1a1. Western blot analysis showed that puerarin significantly inhibited AngII-induced phosphorylation of p38-MAPK. A specific inhibitor of p38-MAPK, SB203580, decreased collagen expression, and ROS generation induced by AngII in cardiac fibroblast. Together, these results suggest that puerarin prevents cardiac fibrosis via activation of Nrf2 and inactivation of p38-MAPK. Nrf2 is the key regulator of anti-fibrotic effects and upregulates metabolic enzymes UGT1A1. Autoregulatory circuits between puerarin and Nrf2-regulated UGT1A1 attenuates side effects associated with treatment, but it does not weaken puerarin’s pharmacological effects. Keywords: puerarin, cardiac fibrosis, Nrf2, UGT1A1, metabolic feedback loop Frontiers in Pharmacology | www.frontiersin.org 1 June 2018 | Volume 9 | Article 540 Cai et al. Puerarin Attenuate Cardiac Fibrosis and redox status, contributing to antioxidant response elementregulated physiologic expression of numerous genes (Wang et al., 2012). Nrf2 plays an essential role in preventing fibrosis. In mice subjected to transverse aortic constriction surgery, Nrf2 deficiency exacerbated left ventricular fibrosis. Conversely, Nrf2 overexpression inhibits proliferation of cardiac fibroblasts (Li et al., 2009). In this study, we explored the roles of Nrf2 in puerarin’s preventive effect against cardiac fibrosis, as well as regulation of UGT1A1 and its pathway, in neonatal rat cardiac fibroblasts (NRCF) induced by AngII and a mouse model of cardiac fibrosis induced by abdominal aortic banding (AB). INTRODUCTION The heart manifests robust plasticity in the context of heart disease. This process is donated as pathological remodeling (Burchfield et al., 2013). Pathological myocardial remodeling is characterized by excessive accumulation of extracellular matrix, through a process called cardiac fibrosis (Kong et al., 2014; Travers et al., 2016). Physiologically, extracellular matrix provides a structural scaffold of cardiomyocytes, distributes mechanical forces throughout cardiac tissue, and mediates conduction of electrical impulses (Camelliti et al., 2005; Porter and Turner, 2009; Souders et al., 2009). Cardiac fibrosis is a final common pathway of many heart diseases. Extensive cardiac fibrosis increases myocardial stiffness, worsens diastolic function, and eventually results in progression to heart failure (Kong et al., 2014; Travers et al., 2016). Some drugs, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone antagonists, and β-blockers, reduce morbidity and mortality in patients with chronic systolic heart failure (Fonarow et al., 2011). However, the progression of heart failure cannot be completely suspended. New pharmacological therapies need to be discovered. Puerarin (7,40 -dihydroxyisoflavone-8β-glucopyranoside) is a major active ingredient in the Chinese medicine Pueraria radix which is extracted from the kudzu root [Pueraria lobata (wild) Howe]. Puerarin has been widely prescribed to treat cardiovascular diseases, including hypertension (Tan et al., 2017), coronary heart disease (Xie et al., 2003) and heart failure (Duan et al., 2000). Supporting findings published previously, our laboratory has reported that puerarin may prevent cardiac fibrosis induced by pressure overload (Yuan et al., 2014; Liu et al., 2015; Tan et al., 2017). In a mouse model of cardiac fibrosis, the inhibition of myocardial fibrosis by puerarin involved transforming growth factor (TGF)-β1, monocyte chemoattractant protein (MCP)-1, and peroxisome proliferator-activated receptor (PPAR) α/γ (Chen R. et al., 2012; Tao et al., 2016). Jin (...truncated)