Elsevier

Biomedicine & Pharmacotherapy

Volume 106, October 2018, Pages 83-91
Biomedicine & Pharmacotherapy

The inhibition of Hippo/Yap signaling pathway is required for magnesium isoglycyrrhizinate to ameliorate hepatic stellate cell inflammation and activation

https://doi.org/10.1016/j.biopha.2018.06.102Get rights and content

Highlights

  • MgIG alleviated CCl4-induced liver fibrosis and inflammation in vivo.

  • MgIG inhibited PDGF-BB-induced HSC activation and inflammation in vitro.

  • MgIG improved liver fibrosis and inflammation by inhibiting Hippo/Yap pathway.

Abstract

Liver fibrosis is a reversible pathological process accompanied by abnormal inflammation, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide. This study was to investigate the effect of Magnesium isoglycyrrhizinate (MgIG) on liver fibrosis and inflammation, and to further clarify molecular mechanism. We found that MgIG treatment significantly alleviated carbon tetrachloride (CCl4)-induced liver fibrosis and HSC activation by regulating TGF-β signaling and MMP/TIMP systems. In addition, MgIG treatment significantly inhibited the inflammatory response of liver fibrosis in mice characterized by reduced pro-inflammatory factors expression and increased anti-inflammatory factors expression. Interestingly, experiments in vitro also showed that MgIG treatment significantly reduced the expression of hepatic stellate cell (HSC) activation markers. Besides, MgIG treatment not only inhibited the expression of pro-inflammatory factors, but also promoted the production of anti-inflammatory factors in activated HSCs. Importantly, treatment with MgIG inhibited Hippo/Yap signaling pathway, which was a potential mechanism for MgIG-induced anti-inflammatory effects. The overexpression of Hippo/Yap signaling effector YAP completely impaired MgIG-induced anti-inflammatory and anti-fibrotic effects. Taken together, these results provide novel implications to reveal the molecular mechanism of the anti-inflammatory properties induced by MgIG, by which points to the possibility of using MgIG to treat liver fibrosis.

Introduction

Liver fibrosis is an invertible pathophysiological process associated with intense repair and cicatrization mechanisms [1]. As the pathogenesis progresses without effective management, advanced liver fibrosis can seriously damage the normal function of the liver and give raise to many serious complications, ultimately resulting in hepatocellular carcinoma and liver cirrhosis [2]. Attractively, early cirrhosis and liver fibrosis are dynamic and reversible, thus intervention of fibrogenesis process is necessary for preventive treatment of cirrhosis and hepatic failure [3]. In recent years, natural products have been widely accepted as realistic options for the treatment of liver fibrosis [4]. Novel anti-fibrosis compounds from herbal components represent an attractive alternative for drug development. [4]. Magnesium isoglycyrrhizinate (MgIG), a safe and natural product, shows a good deal of pharmacological activities, including anti-apoptosis, anti-tumor, and anti-oxidant properties [[5], [6], [7]]. However, whether MgIG can ameliorate the inflammatory microenvironment to improve liver fibrosis remains unclear. In the present study, we aimed to assess the effect of MgIG on inflammation of liver fibrosis and further determine the underlying mechanisms.

Inflammation is a central feature of liver fibrosis as suggested by its role in activation of hepatic stellate cells (HSCs) leading to extracellular matrix deposition [8]. Following liver damage, an accumulation of recruited inflammatory cells occurs at the site of injury [9]. A wide repertoire of pro-inflammatory factors and anti-inflammatory compounds, which encompasses cytokines, chemokines, growth factors, and products of oxidative stress, mediates the inflammatory response of immune cells during the fibrosis process [10]. HSCs also take part actively in the inflammation process through interaction with diverse types of immune cells [11]. Furthermore, inflammatory mediators, such as transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α), may induce HSC conversion from a quiescent to an activated state characterized by a myofibroblast-like phenotype responsible for proliferation and excessive extracellular matrix deposition [12]. Therefore, inflammation may be the target of reducing liver fibrosis. Noteworthy, Bian M et al. recently showed that MgIG promoted HSC apoptosis via endoplasmic reticulum stress and ameliorated fibrogenesis in vitro and in vivo [13]. Attractively, whether MgIG can ameliorate liver fibrosis by controlling inflammatory microenvironment is worth to further research.

Several lines of evidence indicated that the Hippo/Yap signaling played a critical role in fibrous diseases [14,15]. The core components and downstream effectors of the Hippo/Yap signaling are very conservative in mammals and include LATS1/2, MST1/2, MOB1, SAV1, TAZ and YAP. MST1/2 composes a heterodimer by the adaptor protein Salvador 1 (SAV1), which reinforces MST1/2 activity of kinase and boosts MST-LATS interaction [16]. Afterwards, Mob1 homolog (MOB1) and LATS1/2 were phosphorylated by MST1/2 [16]. Phosphorylated MOB1 combines with the autoinhibitory domain of LATS1/2, being capable of the phosphorylation and activation of LATS1/2 [16]. The transcription of the co-factors TAZ and YAP were phosphorylated and inhibited by activated LATS1/2 [16]. Interestingly, Hippo/Yap signaling was related to the development of liver fibrosis, and targeting regulation of YAP can inhibit HSC activation [14,15]. Moreover, mutations in YAP that uncouple its ability to apperceive cell tension result in disorganized tissue and organ development, therefrom offering a paradigm by which YAP activity can coordinate liver size [17]. Importantly, determining the function of Hippo/Yap signaling in pathological conditions will furnish a brand new perspective to open out the pathological mechanism and new ideas about the effective diagnostic signs and therapeutic targets in inflammatory diseases.

In the current study, we first determined the effect of MgIG on inflammation of liver fibrosis, and to further examine the underlying mechanisms in this molecular setting. We showed that MgIG inhibited HSC inflammation and activation by modulating Hippo/Yap signaling. Our results suggest that MgIG as an impactful drug is the remedy for liver fibrosis.

Section snippets

Reagents and antibodies

MgIG (99.7% pure, #12050617) was bought from the Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Nanjing, China). Recombinant mouse platelet derived growth factor-BB (PDGF-BB, #CRP138D) was bought from Cell Sciences (Canton, MA). Dimethyl sulfoxide (DMSO, #156914), Carbon tetrachloride (CCl4, #488488), and Phosphate buffered saline (PBS, #P5368) were bought from Sigma-Aldrich (St Louis, MO). Fetal bovine serum (FBS), Opti MEM medium, trypsin-EDTA, and Dulbecco’s modified essential medium

MgIG prevents the mouse liver from CCl4-induced injury and fibrogenesis

In the present study, a classical mouse model of hepatic fibrosis caused by injecting with CCl4 for 8 weeks was established [[18], [19], [20]]. Then we evaluated the effect of MgIG on liver injury and fibrogenesis. As expected, gross examination revealed that pathological changes of liver fibrosis appeared in the model group compared with the control group, whereas MgIG treatment significantly improved morphologic lesion of livers (Fig. 1A). Moreover, MgIG treatment also remarkably reduced the

Discussion

Liver fibrosis, a complex pathophysiological process, is an intermediate link of various chronic liver diseases [[1], [2], [3]]. Liver transplantation is the only treatment available for patients with advanced stages of liver fibrosis [29]. Therefore, it is urgent to find novel therapeutic strategies for anti-fibrotic therapy [3]. In the present study, MgIG was identified as a potential anti-fibrotic agent. In other studies, MgIG was reported to protect hepatic LO2 cells from

Conflict of interest

The authors declare no conflict of interest.

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