Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 21, 2016; 22(19): 4695-4706
Published online May 21, 2016. doi: 10.3748/wjg.v22.i19.4695
Qinggan Huoxue Recipe suppresses epithelial-to-mesenchymal transition in alcoholic liver fibrosis through TGF-β1/Smad signaling pathway
Tao Wu, Jun-Ming Chen, Tie-Gang Xiao, Xiang-Bing Shu, Han-Chen Xu, Li-Li Yang, Lian-Jun Xing, Pei-Yong Zheng, Guang Ji
Tao Wu, Center of Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Tao Wu, Xiang-Bing Shu, Han-Chen Xu, Li-Li Yang, Lian-Jun Xing, Pei-Yong Zheng, Guang Ji, Institute of Digestive Disease, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
Jun-Ming Chen, Tie-Gang Xiao, Department of Traditional Chinese Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
Guang Ji, E-institute of Shanghai Municipal Education Committee, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Author contributions: Wu T performed the majority of the experiments and analyzed the data; Chen JM, Xiao TG and Shu XB participated in the treatment of animals; Xu HC and Yang LL performed the histological experiments; Xing LJ, Zheng PY and Ji G designed and coordinated the research; Wu T and Ji G wrote and revised the paper.
Supported by National Natural Science Foundation of China, No. 81202979; and Shanghai Rising-Star Program, No. 15QA1403500.
Institutional review board statement: This study was reviewed and approved by the Institute of Digestive Disease, Longhua Hospital, Shanghai University of Traditional Chinese Medicine Institutional Review Board.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Shanghai University of Traditional Chinese Medicine (No. 2013031).
Conflict-of-interest statement: The authors declare that there are no conflicts of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Guang Ji, MD, PhD, Professor, Director, Institute of Digestive Disease, China-Canada Center of Research for Digestive Diseases (ccCRDD), Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 Wanping Road, Shanghai 200032, China. jiliver@vip.sina.com
Telephone: +86-21-64286261 Fax: +86-21-64286261
Received: January 9, 2016
Peer-review started: January 9, 2016
First decision: February 18, 2016
Revised: March 3, 2016
Accepted: April 7, 2016
Article in press: April 7, 2016
Published online: May 21, 2016
Abstract

AIM: To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF).

METHODS: A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-β1/Smad signaling pathway, including TGF-β1, Smad3, snail, occludin, ZO-1 and claudin, was also examined.

RESULTS: Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 ± 0.05 vs control 1.00 ± 0.08; protein: ALF 0.09 ± 0.05 vs control 0.70 ± 0.17, P < 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 ± 0.39 vs 1.00 ± 0.19 and 9.91 ± 0.34 vs 1.00 ± 0.44, respectively, P < 0.01; protein: 1.13 ± 0.42 vs 0.09 ± 0.03 and 1.16 ± 0.43 vs 0.09 ± 0.00, respectively, P < 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-β1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-β1 and snail levels (mRNA: 1.76 ± 0.12 vs 1.00 ± 0.05 and 6.98 ± 0.41 vs 1.00 ± 0.10, respectively, P < 0.01; protein: 1.43 ± 0.05 vs 0.12 ± 0.03 and 1.07 ± 0.29 vs 0.07 ± 0.02, respectively, P < 0.01) and the decrease in Smad3 levels (mRNA: 0.05 ± 0.01 vs 1.00 ± 0.12, P < 0.01; protein: 0.06 ± 0.05 vs 0.89 ± 0.12, P < 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 ± 0.09 vs 1.00 ± 0.12, 0.11 ± 0.00 vs 1.00 ± 0.12 and 0.60 ± 0.01 vs 1.00 ± 0.08, respectively, P < 0.01; protein: 0.05 ± 0.01 vs 0.87 ± 0.40, 0.09 ± 0.05 vs 0.89 ± 0.18 and 0.04 ± 0.03 vs 0.95 ± 0.21, respectively, P < 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 ± 0.04 vs ALF model 0.16 ± 0.05, P < 0.01; protein: QGHXR 0.66 ± 0.21 vs ALF model 0.09 ± 0.05, P < 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 ± 1.05 vs 11.43 ± 0.39 and 1.45 ± 1.51 vs 9.91 ± 0.34, respectively, P < 0.01; protein: 0.09 ± 0.03 vs 1.13 ± 0.42 and 0.10 ± 0.01 vs 1.16 ± 0.43, respectively, P < 0.01). In addition, QGHXR inhibited the expression of TGF-β1 and increased the expression of Smad3 (mRNA: 1.03 ± 0.11 vs 1.76 ± 0.12, 0.70 ± 0.10 vs 0.05 ± 0.01, respectively, P < 0.05 and P < 0.01; protein: 0.12 ± 0.03 vs 1.43 ± 0.05 and 0.88 ± 0.20 vs 0.06 ± 0.05, respectively, P < 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 ± 0.33 vs 6.98 ± 0.41, P < 0.01; protein: 0.08 ± 0.02 vs 1.07 ± 0.29, P < 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 ± 0.05 vs 0.60 ± 0.09, 0.57 ± 0.04 vs 0.11 ± 0.00 and 0.68 ± 0.03 vs 0.60 ± 0.01, respectively, P < 0.01, P < 0.01 and P < 0.05; protein: 0.92 ± 0.50 vs 0.05 ± 0.01, 0.94 ± 0.22 vs 0.09 ± 0.05 and 0.94 ± 0.29 vs 0.04 ± 0.03, respectively, P < 0.01). The effects of QGR and HXR on the TGF-β1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-β1 mRNA levels were not significant.

CONCLUSION: Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-β1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.

Keywords: Alcoholic liver fibrosis, QGHXR, Epithelial-to-mesenchymal transition, Snail, Transforming growth factor-β1/Smad

Core tip: Epithelial-to-mesenchymal transition (EMT) is a dynamic process by which mature epithelial cells lose their distinct characteristics and acquire a mesenchymal phenotype. EMT is characterized by loss of the expression of the epithelial marker E-cadherin and up-regulation of the mesenchymal markers α-SMA, collagen I, vimentin and fibronectin. Our study provided evidence that QGHXR inhibits EMT in alcoholic liver fibrosis by regulating the transforming growth factor-β1/Smad signaling pathway and that QGHXR-mediated inhibition of EMT might be a promising approach to ameliorating alcoholic liver injury.