RESEARCH ARTICLE
QiShenYiQi Attenuates Renal Interstitial Fibrosis by Blocking the Activation of β-Catenin Zhanmei Zhou1☯, Zheng Hu1☯, Mei Li1, Fengxin Zhu1, Hao Zhang2, Jing Nie1, Jun Ai1* 1 State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, P.R. China, 2 Tasly R&D Institute, Tianjin, P.R. China ☯ These authors contributed equally to this work. *
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Abstract
OPEN ACCESS Citation: Zhou Z, Hu Z, Li M, Zhu F, Zhang H, Nie J, et al. (2016) QiShenYiQi Attenuates Renal Interstitial Fibrosis by Blocking the Activation of β-Catenin. PLoS ONE 11(9): e0162873. doi:10.1371/journal. pone.0162873 Editor: Utpal Sen, University of Louisville, UNITED STATES
Chronic kidney disease (CKD) is becoming a worldwide problem. However, current treatment options are limited. In the current study we showed that QiShenYiQi (QSYQ), a waterethanol extract from several Chinese medicines, is a potent inhibitor of renal interstitial fibrosis. QSYQ inhibited transforming growth factor-β1 (TGF-β1)-responsive α-smooth muscle actin (α-SMA), collagen I, and fibronectin up-regulation in obstructive nephropathy and cultured cells. Administration of QSYQ also inhibited the established renal interstitial fibrosis in obstructive nephropathy. Interestingly, QSYQ selectively inhibited TGF-β1-induced β-catenin up-regulation and downstream gene transcription. Taken together, our study suggests that QSYQ selectively inhibits TGF-β1-induced β-catenin up-regulation and might have significant therapeutic potential for the treatment of renal fibrosis.
Received: August 6, 2015 Accepted: August 26, 2016 Published: September 16, 2016 Copyright: © 2016 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Nanfang Hospital Fundation, Southern Medical University (2012C08) to Dr. Ai. Tasly R&D Institute provided support in the form of salaries for author HZ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.
Introduction Chronic kidney disease (CKD) has a high prevalence and mortality rate, and is thus becoming a worldwide problem. [1–3] However, there are still few clinical treatment options which can block the progression of CKD. Renal fibrosis is recognized as a final common pathway of progressive CKD [4–6]. Inhibition of renal fibrosis may be a key factor to develop new clinical treatment options. Transforming growth factor-β1 (TGF-β1), via downstream signaling molecules, such as Smad2/3, p38, PI3K, and ERK, plays a critical role in the pathogenesis of renal fibrosis [7–12]. However, β-catenin, a key protein in Wnt signaling, also plays a great role in renal interstitial fibrosis [13,14]. It is now clear that all these pathways play a critical role in a wide variety of fibrotic CKDs, such as obstructive nephropathy [15], diabetic nephropathy [16], and drug toxicity-induced nephropathy[17]. Thus, these molecules might be a potential target for therapeutic intervention of fibrotic CKD. QiShenYiQi (QSYQ) is a water-ethanol extract from Radix astragali, Salvia miltiorrhiza, Panaxnotoginseng, and rosewood. Qishenyiqi dropping pills, produced by Tasly
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QSYQ Attenuates Renal Fibrosis
Competing Interests: We have the following interests. Hao Zhang is employed by the Tasly R&D Institute. QiShenYiQi pills are developed and sold by Tasly Pharmaceutical Co., Ltd that is a commercial company. Tasly R&D Institute is a R&D department affiliated to Tasly Pharmaceutical Co., Ltd and owns a large group of Departments of scientific research management and service departments, like the Modern TCM Department, Chemical Medicine Department, Pharmacology and Toxicology Department, Medical Analysis Department, and so on. As an employee of Tasly R&D Institute, Zhang Hao made a significant contribution to drug supply in this study. Tasly has made extensive cooperation with scientific research institutes and universities like Peking University, Tianjin Institute of Pharmaceutical Research and Shenyang Pharmaceutical University. They independently or jointly published many reports. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Pharmaceutical Co. Ltd., was approved for clinical use for coronary heart diseases and myocardial infarction rehabilitation by the State Food and Drug Administration of China in 2003. Recently, QSYQ has been reported to be an inhibitor of hepatic fibrosis and myocardial inflammation and fibrosis [18–21]. Thus, we determined whether or not this compound derived from Chinese medicines can inhibit renal interstitial fibrosis, and if so, the underlying mechanisms by which QSYQ exert a renal reparative effect.
Materials and Methods QSYQ Preparation QSYQ (Batch number 20090122) was a gift from Tasly Pharmaceutical Co., Ltd. (Tianjin, P.R. China). QSYQ was prepared from water-ethanol extracts of R. astragali, S. miltiorrhiza, P. notoginseng, and rosewood according to the guidelines of Good Manufacturing and Laboratory Practices, and verified by a Chinese government agency.
Animal Model and Treatment Experimental animals. Male Sprague-Dawley rats with a body weight of 200–250 g were purchased from the Animal Experiment Committee of Southern Medical University (Guangzhou, China). Rats were maintained under standard room temperature (22 ± 2°C) and relative humidity (60% ± 10%) with a 12 h light/dark cycle. Rats were allowed free access to food and water throughout the acclimatization and experimental period. Ethical statement. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Nanfang Hospital (Permit number NFYY-2013-046). All efforts were made to minimize suffering as follows. Rats were anaesthetized with an intra-peritoneal injection of sodium pentobarbital at a concentration of 3%. Operations involving the unilateral ureteral obstruction (UUO) and tissue collection were performed only after the rats had been anaesthetized. Rats were sacrificed with deep anesthesia after tissue collection or when we needed to stop the experiments or severe side effects occurred. Experimental procedures. UUO was performed using an established protocol [22,23]. Treatment with QSYQ at a dose of 500 mg/kg/d attenuated myocardial fibrosis in the previous study [18,19]. Therefore, two doses of QSYQ (250 and 500 mg/kg/d) was used in the following experiments. Experient 1: To evaluate the effect of QSYQ on renal fibrosis, 24 rats were randomized into 4 groups (n = 6 in each group), as follows: (1) sham operated; (2) UUO; (3) UUO+QSYQ (intra-gastric, 250 mg/kg/d, 8h after UUO procedure); and (4) UUO+QSYQ (intra-gastric, 500 mg/kg/d, 8h after UUO procedure). Experient 2: To determine the effect of delaying administration of QSYQ, 18 rats were randomized into 3 groups (n = 6 in each group), as follows: (1) sham operated; (2) UUO; and (3) UUO+QSYQ (intra-gastric, 500 mg/kg/d) 7 d after the UUO procedure. QSYQ was dissolved in normal saline, and were administered by oral gavage once per day for two weeks. The body weight, limbs mobility, hair color and appetite, were observed every day during the experiment period. All the rats were sacrificed 14 d after the UUO procedure. Experimental outcomes. The primary experimental outcome was change in rat kidney interstitial fibrosis. Sections of paraffin-embedded kidney tissues were subjected to Masson trichrome and hematoxylin-eosin (HE) staining to analyze the morphologic changes. Kidney tissues were subjected to real-time PCR, immunohistochemical staining, and Western blot for αSMA, collagen I, and fibronectin to analyze the changes in fibrotic genes.
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Cell Culture and Treatment Normal kidney proximal tubular (NRK52E) and renal fibroblast cells (NRK49F) were a kindly gift from Doctor HuiyaoLan. These cells were cultured in DMEM-Ham’s medium (Gibco, Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies). Cells were serum-starved for 12 h when approximately 50% confluence was reached and pre-treated with the indicated amount of QSYQ for 1 h, and followed by incubation with recombinant TGF-β1 (10ng/ml; R&D Systems, Minneapolis, MN, USA) for the indicated time period. QSYQ was dissolved in PBS to the desired concentration and filtered (0.22 μm).
Morphologic and Immunohistochemical Analysis Masson trichrome or HE staining was performed using commercial kits (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocols on 2-μm sections of paraffin-embedded kidney tissues. To assess renal tubulointerstitial injury, The HE-stained sections were evaluated semi-quantitatively as described previously. Tubulointerstitial damage was graded on a scale from 0 to 3 (0, no changes; 1, changes affecting 50% of the section) [24]. To further assess the degree of tubulointerstitial collagen deposition, Masson trichrome-stained sections were graded on a scale from 0 to 4 (0, no staining; 1,
