Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-β1/Smad2-Mediated Endothelial-to-Mesenchymal Transition

Hindawi PPAR Research Volume 2017, Article ID 2647129, 14 pages https://doi.org/10.1155/2017/2647129 Research Article Puerarin Protects against Cardiac Fibrosis Associated with the Inhibition of TGF-𝛽1/Smad2-Mediated Endothelial-to-Mesenchymal Transition Ya-Ge Jin,1,2,3 Yuan Yuan,1,2,3 Qing-Qing Wu,1,2,3 Ning Zhang,1,2,3 Di Fan,1,2,3 Yan Che,1,2,3 Zhao-Peng Wang,1,2,3 Yang Xiao,1,2,3 Sha-Sha Wang,2,3 and Qi-Zhu Tang1,2,3 1 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China Cardiovascular Research Institute of Wuhan University, Wuhan, China 3 Hubei Key Laboratory of Cardiology, Wuhan, China 2 Correspondence should be addressed to Qi-Zhu Tang; Received 13 February 2017; Accepted 23 April 2017; Published 30 May 2017 Academic Editor: Qinglin Yang Copyright © 2017 Ya-Ge Jin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Puerarin is a kind of flavonoids and is extracted from Chinese herb Kudzu root. Puerarin is widely used as an adjuvant therapy in Chinese clinics. But little is known about its effects on regulating cardiac fibrosis. Methods. Mice were subjected to transverse aorta constriction (TAC) for 8 weeks; meanwhile puerarin was given 1 week after TAC. Cardiac fibrosis was assessed by pathological staining. The mRNA and protein changes of CD31 and vimentin in both animal and human umbilical vein endothelial cells (HUVECs) models were detected. Immunofluorescence colocalization of CD31 and vimentin and scratch test were carried out to examine TGF-𝛽1-induced changes in HUVECs. The agonist and antagonist of peroxisome proliferator-activated receptor-𝛾 (PPAR-𝛾) were used to explore the underlying mechanism. Results. Puerarin mitigated TAC-induced cardiac fibrosis, accompanied with suppressed endothelial-to-mesenchymal transition (EndMT). The consistent results were achieved in HUVECs model. TGF-𝛽1/Smad2 signaling pathway was blunted and PPAR-𝛾 expression was upregulated in puerarin-treated mice and HUVECs. Pioglitazone could reproduce the protective effect in HUVECs, while GW9662 reversed this effect imposed by puerarin. Conclusion. Puerarin protected against TAC-induced cardiac fibrosis, and this protective effect may be attributed to the upregulation of PPAR-𝛾 and the inhibition of TGF-𝛽1/Smad2-mediated EndMT. 1. Introduction Cardiac fibrosis is a final common pathway of a wide spectrum of heart diseases, including myocardial infarction [1], ischemia-reperfusion injury [2], hypertrophic cardiomyopathy [3], and diabetic cardiomyopathy [4]. Cardiac fibrosis is characterized by uncontrolled and excessive accumulation of extracellular matrix (ECM) and collagen, which increase the ventricular stiffness, deteriorate the diastolic function, and eventually lead to heart failure. Myofibroblasts, expressing 𝛼smooth muscle actin (𝛼-SMA) and exhibiting huge capability of producing ECM and collagen (types I and III), as well as inhibiting the activity of matrix metalloproteinases (MMPs), are known as the main effector cells responsible for cardiac fibrosis. Myofibroblasts can originate from quiescent fibroblasts in situ, which can be activated and transdifferentiate into myofibroblasts rapidly after heart injuries [5]; bone marrowderived circulating fibrocytes, attracted by different kinds of cytokines and chemokines, migrate and accumulate in myocardium, representing another important source of myofibroblasts [6]; recent studies have reported another source of myofibroblasts: to cope with different kinds of stress, epithelial/endothelial cells lose their intrinsic characteristics and acquire mesenchymal cell characteristics, a process known as epithelial/endothelial-to-mesenchymal transition (EMT/EndMT) [7, 8]. First proposed by Karasek [9], EMT/ EndMT has been continuously shown to play an important role in fibrosis and should be given enough attention. 2 Puerarin (7,4󸀠 -dihydroxy-8-𝛽-D-glucosylisoflavone, C21 H20 O9 ) (Pue) [10] is a kind of flavonoids, which is extracted from Chinese herb Kudzu root and widely used in Chinese clinics as an adjuvant therapy for the treatment of angina pectoris, diabetes [11], and ischemic cerebrovascular diseases [12]. Our previous [13] and others’ studies [14] have demonstrated that puerarin attenuated pressure or angiotensin IIinduced cardiac hypertrophy in mice, and puerarin could also inhibit inflammation and apoptosis in LPS-stimulated cardiomyocytes [15]. However, there is a lack of data on puerarin’s effects on cardiac fibrosis. Our present study is aimed at elucidating the protective effect of puerarin on cardiac fibrosis induced by transverse aorta constriction (TAC). 2. Materials and Methods 2.1. Chemicals and Reagents. Puerarin (98% purity as detected by high-performance liquid chromatography analysis) was acquired from Shanghai Winherb Medical S&T Development Co. Ltd. (Shanghai, China). TGF-𝛽1 was purchased from PEPROTECH (100-21C). GW9662 was purchased from Sigma (M6191) and pioglitazone was also purchased from Sigma (CDS021593). 2.2. Animals. All animal procedures used in this study were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (protocol number: 00013274) and conformed to the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication number 85-23, revised 1996). 8-week-old, male C57/BL6 mice weighing 23.5–27.5 g were purchased from the Institute of Laboratory Animal Science, CAMS & PUMC (Beijing, China) and kept in specific pathogen free animal lab with 12 h light-dark cycle and constant temperature and humidity. After 1 week of adaptation, the mice were randomly assigned to four groups, as sham + vehicle, sham + pue, TAC + vehicle, and TAC + pue, among which vehicle or puerarin were given 1 week after TAC. The dose (65 mg/kg body weight/day) and administration route (premixed in daily feed) of puerarin were determined by referring to our previous study [13]. All the mice had free access to drinking water and feed. Food consumption was monitored once a week and no difference was found between the four groups. The chosen dose of puerarin had no adverse effects on their growth or the food and water consumption. 8 weeks after sham or TAC, the mice were sacrificed and their hearts were taken for further studies. 2.3. Cell Culture. Human umbilical vein endothelial cell (HUVEC) line 12 was purchased from YRGene (NC006) and cultured in Gibco RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), endothelial cell growth supplement (ECGS, ScienCell, 1052, 10 𝜇l/ml), and heparin (0.1 mg/ml) in a humidified 5% CO2 incubator at 37∘ C. Cells between the fourth and sixth passages growing to 70–80% confluence were used. PPAR Research After 12 hours of starvation for synchronization, HUVECs were preincubated with different concentratio (...truncated)