Magnesium Isoglycyrrhizinate Ameliorates Fibrosis and Disrupts TGF-β-Mediated SMAD Pathway in Activated Hepatic Stellate Cell Line LX2.

Jie Kai Tee,Bo Yu,Fei Peng,Han Kiat Ho,Yeong Lan Tan

doi:10.3389/fphar.2018.01018

Abstract

Liver fibrosis is a histological change often attributed to the activation of hepatic stellate cells (HSCs) and the excessive formation of scar tissues in the liver. Advanced stages of the disease frequently lead to cirrhosis. Magnesium isoglycyrrhizinate (MgIG) has been accepted as a hepatoprotective drug with the potential of alleviating inflammatory conditions and thus promote liver recovery from viral- or drug-induced injury. While MgIG has been empirically integrated into the clinics to treat some liver diseases, its anti-fibrotic effect and the associated mechanisms remain poorly characterized. Herein, we demonstrated that 1 mg/ml MgIG attenuated the production of αSMA and collagen-1 in activated HSCs using TGF-β1-induced human HSCs LX2 as the fibrotic cell model. We found that MgIG exerts an inhibitory effect on the TGF-β-SMAD signaling pathway by arresting the binding of downstream transcription factors SMAD2/3 and SMAD4. Furthermore, MgIG was shown to suppress proliferation and induce senescence of activated LX2 cells. Protein expression of p27 and enzymatic activity of senescence-associated β-galactosidase were elevated upon exposure to MgIG. In addition, we observed that exposure of activated LX2 cells to MgIG reduces TGF-β-induced apoptosis. Interestingly, a lower toxicity profile was observed when human fetal hepatocytes LO2 were exposed to the same concentration and duration of the drug, suggesting the specificity of MgIG effect toward activated HSCs. Overall, hepatoprotective concentrations of MgIG is shown to exert a direct effect on liver fibrosis through inhibiting TGF-β-signaling, in which SMAD2/3 pathway could be one of the mechanisms responsible for the fibrotic response, thereby restoring the surviving cells toward a more quiescent phenotype. This provides critical mechanistic insights to support an otherwise empirical therapy.

Highlights

Liver fibrosis is a disease characterized by the activation of hepatic stellate cells (HSCs) and the deposition of extracellular matrix (ECM) such as collagen in response to injury (Friedman, 2004; Pellicoro et al, 2014)
The following antibodies were used: Anti-collagen-1 was purchased from Abcam (Cambridge, United Kingdom), anti-αSMA from Agilent Dako (Santa Clara, CA, United States); anti-GAPDH, anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt, anti-phospho-JNK, anti-JNK, anti-SMAD2/3, anti-SMAD4, secondary anti-mouse and anti-rabbit were purchased from Cell Signaling Technology (Danvers, MA, United States); anti-phospho-p38, anti-p38 and anti-p27 were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). 3-(4,5-Dimethylthiazol-2-yl)2,5-Diphenyltetrazolium Bromide (MTT) was purchased from Duchefa Biochemie (Haarlem, Netherlands) and propidium iodide (PI) was purchased from Sigma-Aldrich
Cells treated with co-treatment showed significant reduction in both αSMA and collagen-1 mRNA levels at 24 h compared to TGF-β treatment alone (Figure 1A)

Summary

Introduction

Liver fibrosis is a disease characterized by the activation of hepatic stellate cells (HSCs) and the deposition of extracellular matrix (ECM) such as collagen in response to injury (Friedman, 2004; Pellicoro et al, 2014). This condition is further aggravated by the loss of fenestrae in sinusoidal endothelial cells and loss of hepatocyte microvilli, thereby deteriorating the overall hepatic function (Friedman, 2000; Dechêne et al, 2010). The perisinusoidal HSCs which are responsible for the deposition of ECM have been the primary focus in treating liver fibrosis (Higashi et al, 2017). Liver transplantation remains the only treatment option for patients with advanced liver fibrosis (Durand and Francoz, 2017)

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